International Journal of Pharmaceutics 436 (2012) 359–378

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics

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

Review

Proteins, polysaccharides, and their complexes used as stabilizers for emulsions:

Alternatives to synthetic surfactants in the pharmaceutical field?
Eléonore Bouyer a,b , Ghozlene Mekhloufi a,b , Véronique Rosilio a,b , Jean-Louis Grossiord a,b ,
Florence Agnely a,b,∗
a
Univ Paris Sud, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92296, Châtenay-Malabry Cedex, France
b
CNRS, UMR 8612, Institut Galien Paris-Sud, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France

a r t i c l e i n f o a b s t r a c t

Article history: Emulsions are widely used in pharmaceutics for the encapsulation, solubilization, entrapment, and con-
Received 1 August 2011 trolled delivery of active ingredients. In order to answer the increasing demand for clean label excipients,
Received in revised form 22 June 2012 natural polymers can replace the potentially irritative synthetic surfactants used in emulsion formula-
Accepted 22 June 2012
tion. Indeed, biopolymers are currently used in the food industry to stabilize emulsions, and they appear
Available online 1 July 2012
as promising candidates in the pharmaceutical field too. All proteins and some polysaccharides are able to
adsorb at a globule surface, thus decreasing the interfacial tension and enhancing the interfacial elasticity.
Keywords:
However, most polysaccharides stabilize emulsions simply by increasing the viscosity of the continuous
Emulsion
Stabilization
phase. Proteins and polysaccharides may also be associated either through covalent bonding or electro-
Biopolymer static interactions. The combination of the properties of these biopolymers under appropriate conditions
Drug delivery systems leads to increased emulsion stability. Alternative layers of oppositely charged biopolymers can also be
Layer-by-layer formed around the globules to obtain multi-layered “membranes”. These layers can provide electro-
static and steric stabilization thus improving thermal stability and resistance to external treatment. The
novel biopolymer-stabilized emulsions have a great potential in the pharmaceutical field for encapsula-
tion, controlled digestion, and targeted release although several challenging issues such as storage and
bacteriological concerns still need to be addressed.
© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
2. Emulsion stability and characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
2.1. Emulsion classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
2.2. Destabilization mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
2.3. Stabilization methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
2.4. Emulsion characterization and stability assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
2.4.1. Emulsion type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
2.4.2. Emulsion granulometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
2.4.3. Emulsion stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
2.4.4. Rheological properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
2.4.5. Zeta potential measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
2.4.6. Determination of biopolymer adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
2.4.7. Interfacial tension measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
2.4.8. Interfacial rheology measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
2.4.9. Interfacial film thickness and refractive index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

∗ Corresponding author at: Institut Galien Paris-Sud, UMR CNRS 8612, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92296, Châtenay-Malabry Cedex, France.
Tel.: +33 1 46 83 56 26; fax: +33 1 46 83 58 82.
E-mail address: florence.agnely@u-psud.fr (F. Agnely).

0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2012.06.052
360 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378

3. Stabilizing emulsions with biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

3.1. Protein-stabilized emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
3.1.1. Caseins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
3.1.2. Whey proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
3.1.3. Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
3.1.4. Pea proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
3.2. Polysaccharide-stabilized emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
3.2.1. Non-adsorbing polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
3.2.2. Adsorbing polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
3.3. Protein–polysaccharide stabilized emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
3.3.1. Covalent complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
3.3.2. Non-covalent complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
3.3.3. Without complexation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
4.1. Comparison of biopolymers and small-molecule surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
4.1.1. Comparison of the interfacial behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
4.1.2. Comparison of emulsifying properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
4.1.3. Comparison of emulsion stabilizing properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
4.2. Promising applications of biopolymers for the stabilization of pharmaceutical emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
4.3. Challenging issues raised by biopolymers in the field of pharmaceutical emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

1. Introduction tolerated molecules would thus be a major breakthrough. It is sur-

prising to note that up to now, pharmaceutical emulsions have not
evolved much compared to food ones. In the food industry, the
In the pharmaceutical field, emulsions are widely used as drug introduction of natural polymers such as proteins and polysaccha-
delivery systems for parenteral, oral, and topical (skin and eye) rides as emulsifying and/or stabilizing agents has been a successful
routes (Becher, 1983). They consist of mixtures of at least one approach for the formulation of highly stable emulsions. This was
liquid dispersed in another one in the form of droplets, both liquids allowed thanks to a better understanding of the physicochem-
being immiscible, or poorly miscible one with the other (Tadros istry of emulsion stability, which in turn was made possible by the
and Vincent, 1983). The main interest of emulsions is to encapsu- introduction of new characterization techniques such as interfacial
late a hydrophilic or lipophilic active molecule inside the dispersed rheology.
phase, thus ensuring its protection against environmental stress This review aims at summarizing the contribution of these
and degradation (oxygen, light, enzymes, acidity etc.), and allow- natural stabilizing agents especially in food emulsions, and to dis-
ing its controlled delivery. Emulsions can also mask an unpleasant cuss how this knowledge could be implemented to formulate a new
smell or taste (Ley, 2008), reduce a drug toxicity (Brime et al., 2002), generation of emulsions for pharmaceutical applications that will
enhance its penetration through the skin (Kogan and Garti, 2006) answer the increasing demand for natural excipients and sustain-
or behave as detoxifying systems to entrap toxic molecules (pollu- able development. The first part of this review gives a survey of
tants, pesticides, etc.) in the inner phase (Grossiord and Stambouli, the most recent research on emulsion stability on a physicochem-
2008). In addition, the formation of emulsions can be the inital ical standpoint, and presents the most advanced methods used to
step to obtain nano- and micro-particle systems for drug targeting assess this stability. The second part of this article reviews ongo-
(Grigoriev et al., 2008). ing research on emulsion stabilization by biopolymers (proteins,
The main challenge raised by the use of emulsions is their polysaccharides, and their mixtures). In the last part, a compari-
thermodynamic instability. Indeed, they are prone to destabiliza- son is drawn between small-molecule surfactants and biopolymers,
tion and phase separation (Tadros, 2009; Tadros and Vincent, and the potential pharmaceutical applications of biopolymer emul-
1983). Addition of emulsifiers with interfacial and/or thickening sion stabilizers are described and discussed.
properties allows for the emulsion formation and stabilization.
Pharmaceutical emulsions are mostly stabilized by synthetic sur-
factants (Scherlunda et al., 1998). However, the use of these 2. Emulsion stability and characterization
emulsifiers directly or indirectly raises toxicity and environment
issues (Cserháti et al., 2002; Liwarska-Bizukojc et al., 2005). Syn- 2.1. Emulsion classification
thetic surfactants can be intrinsically toxic or may alter the
distribution and elimination of co-administered drugs (Buggins Emulsions can be classified into two groups: simple emulsions
et al., 2007; Cserháti et al., 2002). Most surfactants induce irri- and multiple emulsions. Two classes of simple emulsions can be
tant skin reactions and may cause toxic symptoms in animals defined, namely oil-in-water (O/W), and water-in-oil (W/O) (Fig. 1).
and humans. For example, the possible binding of anionic sur- Multiple emulsions constitute a more sophisticated system. The
factants to proteins, enzymes, and phospholipid membranes may simplest ones are oil-in-water-in-oil (O/W/O) or water-in-oil-in-
result in biochemical changes such as a modification of protein water (W/O/W) double emulsions (Fig. 1). In the latter for example,
structure and dysfunction of enzymes and phospholipid mem- the emulsion is composed of aqueous droplets, which are dispersed
branes (Cserháti et al., 2002; Effendy and Maibach, 1995). According inside oily drops, these oily drops being themselves dispersed in
to cytotoxicity tests, nonionic surfactants have a lower toxic an external aqueous phase (Grossiord and Sellier, 2001). Emul-
effect than cationic, anionic, and amphoteric ones, the toxicity of sions can also be classified according to their droplet size into three
cationic surfactants being the highest (Effendy and Maibach, 1995; categories: macroemulsions, microemulsions, and nanoemulsions.
Vlachy et al., 2009). Replacing synthetic surfactants by much better Macroemulsions are emulsion systems with droplet sizes ranging
 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
a b
simple emulsions
O rily to microemulsions, nanoemulsions are not thermodynamically
W
W O
c d
multiple emulsions
W energy is therefore significantly increased. In accordance with
O
O
W global energy minimum state, emulsions rapidly tend to phase
W O
Fig. 1. Schematic representation of (a) oil-in-water, (b) water-in-oil, (c) water-in-
oil-in-water, and (d) oil-in-water-in-oil emulsions.
from 0.1 to 100 !m. These sizes allow light scattering and give their
white color to these systems. Microemulsions generally contain
both a surfactant and co-surfactant that induce spontaneous for-
mation of the system. Microemulsions are often transparent to the
eye, with low viscosity, and are thermodynamically stable (Friberg
and Venable, 1983). This stability is due to their very low interfa-
cial tension (enthalpy), typically 10−1 to 10−2 mN/m (Capek, 2004),
and to the low enough droplet size (entropy). Indeed, their droplet
size varies from 100 Å to 100 nm (Solans et al., 1997). The last
category, nanoemulsions, refers to emulsions with droplet sizes in
the nanometric scale, i.e. with a mean diameter of 20–200 nm. The
coalescence
flocculation
O
W
Fig. 2. Schematic representation of the various destabilization mechanisms for an oil-in-water emulsion.
361
relatively small size of these droplets with regards to the optical
wavelengths of the visible spectrum implies that many nanoemul-
sions appear optically transparent, even at large droplet volume
fraction and for large refractive index contrast (Mason et al., 2006;
Solans et al., 2005). However, nanoemulsions tend to be slightly
turbid if droplet diameter approaches 80 nm (McClements, 2011;
Wooster et al., 2008). Above this size, still in the submicron range,
they appear white due to significant multiple scattering. Contra-
stable.
2.2. Destabilization mechanisms
During emulsification, the interfacial area between the con-
tinuous and the dispersed phases is considerably augmented
compared to the interface before dispersion. The interfacial free
the thermodynamic dictum that all systems evolve towards their
separate in order to minimize the interfacial contact area and
free energy. This instability manifests itself by various mecha-
nisms: flocculation, coalescence, creaming or sedimentation, and
Ostwald ripening (Fig. 2). Flocculation and coalescence are the
major destabilization mechanisms (Walstra, 1987). Flocculation is
the result of attractive forces between the droplets and leads to the
formation of flocs of dispersed phase. It may occur in biopolymer-
stabilized emulsions, either by depletion or bridging mechanisms
(Fig. 3) (Blijdenstein et al., 2004). Depletion flocculation arises
when biopolymer concentration exceeds a critical value. Indeed,
the presence of non-adsorbing or excess biopolymer in the contin-
uous phase increases the attractive force between the droplets by
osmotic effect and provokes the exclusion of the biopolymer chains
from the narrow region surrounding two droplets (McClements,
2000). This depletion mechanism is not specific to biopolymers.
Indeed, it can also be induced by non-adsorbed small surfactant
coalescence
and
phase
separation
362 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
a b
O O
W W
Fig. 3. Schematic representation of (a) depletion and (b) bridging flocculation in
presence of polymer.
micelles, and by inorganic or organic nanoparticules. Bridging floc-
culation occurs when a single biopolymer molecule adsorbs at
the surface of more than one emulsion droplet. It thus acts as
a polymeric link and promotes bridging flocculation (Guzey and
McClements, 2006a). The formed flocs are the first step to droplet
sedimentation (in W/O emulsions) or creaming (in O/W emulsions),
and coalescence.
In some cases, the flocculation mechanism is reversible and
simple magnetic stirring or hand shaking can break the flocs.
However, in some other cases, it is irreversible and droplets coa-
lesce. Small droplets fuse together and progressively form larger
globules. Emulsions can also undergo creaming or sedimentation,
which consist in the migration of globules to the top or bottom
of the dispersion, depending on the relative densities of the two
liquid phases. Finally, Ostwald ripening is a diffusion mechanism
of dispersed phase molecules through the continuous phase, with-
out any contact between droplets, related to the solubility of the
dispersed phase in the external phase. This diffusion takes place
from small droplets towards larger ones, driven by a higher Laplace
pressure existing in the former. Over time, the droplet size distribu-
tion shifts toward larger values. Ultimately, these mechanisms can
lead to the oiling off and the emulsion breaks up into separated
oil and water phases. Flocculation, sedimentation and cream-
ing are reversible mechanisms of droplets migration, whereas
Ostwald ripening and coalescence are non-reversible mechanisms
of droplets size increase. For an in-depth understanding of these
destabilization mechanisms, a very good description can be found
in the review authored by Damodaran (2005).
In the pharmaceutical field, it is necessary to control these
destabilization mechanisms. Depending on emulsions application,
it may be important to slow them down or to determine the exact
environmental conditions (shear rate, pH, etc.) allowing emulsion
destabilization and consequent release of the encapsulated active
pharmaceutical ingredient at the right time, at the targeted site.
2.3. Stabilization methods
Emulsifiers and stabilizers are commonly used to kinetically
stabilize emulsions. They are classified in four categories (Myers,
1999). The first class is composed of non-surfactant ions that can
adsorb at the droplet surface without affecting the interfacial ten-
sion or facilitating the emulsification process. However, some may,
under appropriate conditions, impose a slight electrostatic barrier
between approaching droplets thus contributing to emulsion sta-
bilization. The second class is composed of small non-surfactant
colloidal solids (i.e. silica or clay particles of Pickering emulsions)
that adsorb and form a physical barrier between droplets, thereby
delaying or preventing coalescence. The third class is constituted
of classical monomeric surfactants such as sodium dodecyl sul-
phate. They decrease interfacial tension and increase emulsion
stability but can be quite irritative and potentially toxic towards the
environment as previously discussed. Finally, the fourth class com-
prises polymer surfactants. In addition to their general interfacial
tension lowering properties, polymers induce steric or electro-
static interactions, changes in the interface viscosity or elasticity,
or changes in the bulk viscosity of the system thus improving emul-
sion stability.
Biopolymers (proteins and polysaccharides) belong to this
last class. They prevent flocculation and coalescence by com-
bined mechanisms. Biopolymer gels and non-adsorbing thickening
polysaccharides reduce droplets movements and encounters in
emulsions by increasing the viscosity of the continuous phase.
Adsorbing biopolymers act on inter-droplet interactions. Indeed,
flocculation and coalescence can be avoided by inducing repulsive
electrostatic interactions or steric hindrance between droplets.
Emulsion stabilization or destabilization by a biopolymer
depends on various parameters, such as polymer nature, poly-
mer concentration, pH, ionic strength, etc. When formulating
a biopolymer-stabilized emulsion, it is thus necessary to con-
trol them. The emulsifying process is also a key parameter that
influences emulsion microstructure (droplet size). Emulsions can
be formed by various protocols, involving rotor-stator systems,
ultrasound, high-pressure systems, phase inversion temperature,
micropores, etc. (Schubert and Engel, 2004). However, it is out of the
scope of this review to give a detailed description of these processes.
2.4. Emulsion characterization and stability assessment
This section gives a general overview of the most common and
advanced techniques used for the characterization of emulsions
and for the elucidation of their stabilization mechanisms. However,
the following list is not exhaustive.
2.4.1. Emulsion type
The continuous phase of an emulsion can be water (in O/W
emulsion) or oil (in W/O emulsion). However, the general aspect
of both systems is often very similar, even if some sensorial tech-
niques rapidly allow the identification of one or the other emulsion
type (shining aspect, oily touch, dilution in water, dissolution of
methylene blue) (Groeneweg et al., 1993). Measurement of the
external phase conductivity is more accurate. Indeed, an aque-
ous continuous phase has a higher conductivity than an oily one
(Suttiprasit et al., 1993). Most biopolymers are water soluble, and
thus tend to form O/W emulsions.
2.4.2. Emulsion granulometry
The granulometry of an emulsion is a very important parameter,
which is related to emulsion process. The droplet size distribu-
tion and polydispersity can be measured by means of various
experimental techniques, such as laser diffraction (Gharsallaoui
et al., 2010a; Li et al., 2010), dynamic light scattering (Guzey and
McClements, 2006b; Ogawa et al., 2004), and optical microscopy
(Guzey and McClements, 2006b). The most popular definitions
available to obtain the droplet-size are (Brittain, 2001):
!
ni di3
the volume − surface(or Sauter) mean diameter : d32 =
i
!
ni di2
i
(1)
!
ni di4
i
the volume − moment mean diameter : d43 = ! (2)
ni di3
i
 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
where ni and di are the number and diameter of the globule popu-
lation i, respectively;
kB T
and the hydrodynamic radius : Rh =
6!"D
where kB is the Boltzmann constant, T the temperature, " the vis-
cosity of the continuous phase, and D the diffusion coefficient of a
droplet.
2.4.3. Emulsion stability
Emulsion stability is a result of physical stability (no phase
separation), chemical stability (no chemical reaction), and micro-
biological stability (no germ development). The physical stability
can be evaluated by two accelerated aging methods, which mimic
conditions that emulsions could undergo during transport and stor-
age. The first one consists in evaluating samples stored at different
temperatures (from 0 to 40 ◦ C) and for various periods of time (from
24 h to 15 days). It may also include repeated hot–cold cycles. The
second method enhances by centrifugation the sedimentation or
creaming processes that can lead to coalescence (Tcholakova et al.,
2005). Accelerated aging studies by centrifugation allow determi-
nation of the emulsion stability index (ES) following Eq. (4):
remaining emulsion height
ES(%) = × 100
initial emulsion height
However, one must be careful when drawing conclusions from
results obtained by these methods, as they do not perfectly mimic
the natural aging process.
Another relatively new approach is to evaluate emulsion sta-
bility by using a Turbiscan (Formulaction® , L’Union, France). This
apparatus is composed of a reading head consisting of a pulsed near
infrared light source (# = 850 nm) and two synchronous detectors
that scan the entire length of an emulsion filled tube. The trans-
mission detector measures the light flux transmitted (at 0◦ angle)
through the emulsion, and the backscattering detector receives
the light backscattered (at 135◦ angle) by the emulsion. Diffuse
reflectance and transmittance versus sample height and time are
obtained, allowing detection of flocculation and coalescence pro-
cesses even at an early stage (invisible to the eye) (Lemarchand
et al., 2003; Mengual et al., 1999).
2.4.4. Rheological properties
The flow characteristics of emulsions are influenced by various
interaction forces that occur in the system during sedimenta-
tion or creaming, flocculation, coalescence, and Ostwald ripening.
Several factors affect emulsion rheology such as volume fraction
and viscosity of the dispersed phase, droplet granulometry, viscos-
ity of the continuous phase as well as its chemical composition
(polarity, pH), nature and electrolyte concentration, and inter-
facial rheology (Tadros, 1994). Numerous methods are available
to investigate emulsion flow properties. It is possible to perform
flow, creep, and oscillatory experiments. In oscillatory measure-
ments, a viscoelastic material is subjected to a sinusoidally varying
strain (or stress). The response in stress (respectively strain) is then
monitored. From these measurements, it is possible to determine
emulsion viscoelastic parameters such as storage (G% ) and loss mod-
uli (G%% ). G% modulus represents the elastically stored energy in the
emulsion, and G%% modulus the energy dissipated in viscous flow
(Tadros, 1994). Depending on their properties, emulsions can either
maintain the necessary fluidity for their transport and distribu-
tion or exhibit a yield behavior inhibiting flow under small shear
stress. The shear-thinning behavior is probably the most encoun-
tered rheological behavior in the field of emulsions: the apparent
viscosity decreases when increasing the shear rate. From the varia-
tion of the different rheological parameters, it is possible to infer the
363
stability of emulsions, their texture, syringeability, and condition-
ing possibilities for future applications (Tadros, 1994).
(3) 2.4.5. Zeta potential measurements
Zeta potential measurements may help predict flocculation and
coalescence because emulsion stability can be influenced by the
interfacial electric charges (Gharsallaoui et al., 2010a; Guzey and
McClements, 2006b; Li et al., 2010; Ogawa et al., 2004). The effec-
tive charge of a droplet differs from its actual charge because of
the presence of counter-ions in the solution. The closest counter-
ions are attached by electrostatic attractions. They are in immediate
contact with the droplet surface and form the Stern layer. They thus
decrease the droplet actual charge. Their concentration then rapidly
decreases when deviating from the droplet surfaces till reaching
their equilibrium solution concentration. In an electrical field, the
adsorbed counter-ions of the Stern layer move jointly with the
droplet. The electrophoretic mobility ($ep ) is first calculated using
the von Smoluchowski theory modified by Henry (Hunter, 1986)
(Eq. 5), with v, the droplets rate and E, the applied electrical field.
v
$ep = (5)
E
The zeta (%) potential can then be calculated applying Henry’s
relation (Eq. 6):
(4)
2ε%f (ka)
$ep = (6)
3"
where ε is the liquid dielectric constant, " its viscosity, and f(ka)
is Henry’s function being equal to 1.5 when approximating with
Smoluchowski. The %-potential corresponds to the potential mea-
sured at the Stern layer. A high absolute value of the %-potential
avoids flocculation. Conversely, a small value (close to zero) allows
the droplets to come in closer contact with each other and floccu-
late.
The ionic strength is of importance in emulsion stability as high
ionic strength can induce droplet flocculation if the electrolyte con-
centration is high enough to electrostatically screen droplet charge
(Laplante et al., 2005). This screening can easily be detected by
%-potential measurement because its value gets closer to zero.
2.4.6. Determination of biopolymer adsorption
It is possible to plot adsorption isotherms of a biopolymer at the
oil–water interface. The graph represents the amount of adsorbed
polymer versus its bulk concentration (Graham and Phillips, 1979;
Jenkins and Ralston, 1998). The shape of the curve indicates when
the interface is entirely covered by ending as a plateau. It also gives
information about the adsorbed molecule conformation and about
competitive adsorption (Chatelier and Minton, 1996).
In the case of proteins, their interfacial concentration (' ) can
also be calculated from the specific surface area of the oil droplets,
and from the difference between the amount of protein used to pre-
pare the emulsion and that measured in the aqueous phase after
the emulsification process. After a centrifugation step, the follow-
ing mass balance (Eq. 7) is frequently used (Euston et al., 2000;
Tcholakova et al., 2005, 2006; Ye, 2010):
VC (CINI − CSER ) (1 − ˚)d32
' = = (CINI − CSER ) (7)
SVOIL 6˚
where S is the specific surface area of the droplets, VC and VOIL are
volumes of the continuous and oil phases, respectively, ˚ is the oil
volume fraction, CINI is the initial protein concentration in the solu-
tion, and CSER is the protein concentration in the aqueous phase (in
the serum) after emulsification determined by the Bradford method
(Bradford, 1976). It is important to note that to perform such a
characterization, the equilibrium between the adsorbed and the
364 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
non-adsorbed chains should not be modified by the centrifugation
step.
2.4.7. Interfacial tension measurements
Three techniques can allow interfacial tension measurements:
the drop method, based on the analysis of the shape of a drop, the
Wilhelmy plate and the Du Noüy ring methods consisting in mea-
suring the force that can pull out a plate or ring from the studied
liquid surface. The pendant (or rising) drop and Wilhelmy plate
techniques allow the determination of the adsorption kinetics of
an amphiphilic molecule at the interface, which is of importance
for the emulsification process (Ward and Tordai, 1946; Graham and
Phillips, 1979; Demé et al., 1995; Rosilio et al., 1997).
2.4.8. Interfacial rheology measurements
The interfacial rheology technique is based on surface modifi-
cation induced by mechanical forces, such as shear or dilational
stresses, giving the functional relationship between the deforma-
tion of the interface (gas/liquid or liquid/liquid interfaces), the
stresses applied in and on it, and the resulting flows in the adja-
cent fluid phases (Ivanov et al., 2005; Krägel and Derkatch, 2010;
Ravera et al., 2010). It is suitable in a wide range of technical
applications involving multiphase systems with a high specific
area such as emulsions and foams. This technique is relatively
new and still not developed enough. Few or no studies of emul-
sification and emulsion stability, wetting or two-phase flow have
been compared to interfacial shear rheological properties. How-
ever, interfacial rheological techniques give very useful information
on inter- and intramolecular interactions at interfaces (Krägel and
Derkatch, 2010).
Stress/strain relationships occurring at interfaces of bi-
dimensional phases such as surfactants or biopolymers single-
/multi-layers in an emulsion can be studied by interfacial rheology.
Elastic and viscous moduli are obtained for each interfacial strain.
The drop method can be used for dilational rheological measure-
ments. The volume of a pendant (or a rising) drop is submitted to
sinusoidal variations, thus resulting in expanding or compressing
the surface of the drop. The interfacial tension is determined by
analyzing the profile of the drop according to the Laplace equation,
by means of a Charge Coupled Device camera connected to a com-
puter. Another technique consists in using a rheometer equipped
with a bi-conical disk. This disk is placed at the interface between
the two liquids and rotates to determine the viscous and elastic
shear rheological parameters of the studied interface. A complex
modulus (E*) can be defined for both techniques as follows (Eq. 8):
E ∗ = E % + iE %%
where E% is the elastic modulus describing the elastic properties of
the interface, and E%% the viscous modulus correlated to its viscous
resistance. The phase angle (ı) is related to E% and E%% moduli by Eq.
(9):
E %%
tan ı =
E%
where ı values below 45◦ reveal a predominantly elastic behavior, a
more favourable condition for emulsion stability. Correspondingly,
a high elastic modulus compared to the viscous one is correlated
to good emulsion stability. Bos and Vliet (2001) consider that dila-
tional elasticity and viscosity are very important indicators of film
rupture, since this rupture can mainly be seen as a dilational defor-
mation. If we consider emulsion stability, this means that when two
droplets of the emulsion encounter, the elastic interface resists to
the strain stress created by the deformation of the droplets, thus
preventing coalescence and maintaining emulsion stability (Laza-
Knoerr et al., 2011). Emulsions containing adsorbing biopolymers
are very well stabilized because of the formation of viscoelastic
adsorbed layers (Bos and Vliet, 2001; McClements, 2004).
2.4.9. Interfacial film thickness and refractive index
The thickness of an interfacial film can be determined by
ellipsometry. This technique can be applied to the adsorption of
surfactants molecules at the air-liquid interface (Binks et al., 2000;
Castellani et al., 2010) as well as the oil–water interface (Binks
et al., 2003; Bylaite et al., 2001). The principle of the measure-
ments is based on the change in the state of polarization of the
reflected polarized light against the studied interface. This change
is expressed in amplitude ratio (( ) and phase shift ()). These two
parameters give the refractive index of the two materials provided
that the interface is clean. In the case of an adsorbed interfacial film,
they depend on its thickness and refractive index. It is then possible
to determine the adsorbed layer thickness, and its mean refractive
index, knowing these parameters (( , )) for several surrounding
media. The monitoring of ( and ) allows studying the process of
interfacial layer formation upon adsorption of an emulsifying agent
(Bylaite et al., 2001; Russev et al., 2000). For example, Russev et al.
(2000) obtained a thickness of 1.8 nm for the first "-casein layer
adsorbed at the oil–water interface, and 5.4 nm for the second one.
It is thus possible to apply this technique to a multi-layered inter-
facial film. It is also possible to show evidence of the destabilization
of an interface, for example upon dilution (Henni et al., 2005).
3. Stabilizing emulsions with biopolymers
Biopolymers such as proteins and polysaccharides are good
examples of natural emulsifiers/stabilizers currently encountered
in the food industry and could be used to stabilize pharma-
ceutical emulsions. In the pharmaceutical field, these natural
polymers are already used for other applications such as cap-
sule formation (gelatin), tablet binder (gum arabic, chitosan,
hypromellose), suspending agent (gum arabic, hypromellose),
mucoadhesive formulations (chitosan), matrix for extended-
release tablets (hypromellose), etc. (Rowe et al., 2006). Many
proteins can act as emulsifiers because of their ability to adsorb
at the oil–water interface (Möbius and Miller, 1998) and to
increase emulsion stability (McClements, 2004). Most polysac-
charides behave as emulsion stabilizers by forming an extended
network in the continuous phase which thus becomes highly vis-
cous (Dickinson, 2009) and can even form a gel (Benna-Zayani
et al., 2008; Weiss et al., 2005). Only few polysaccharide deriva-
tives possess surface properties that enable their adsorption at the
(8) oil–water interface (Dickinson, 2003; Garti and Reichman, 1993).
Combining the properties of proteins and polysaccharides under
appropriate conditions (concentration, protein-to-polysaccharide
ratio, pH, ionic strength, and temperature) may be a valuable
strategy for improving emulsion stability (Dickinson, 1995,
2008a,b; Guzey and McClements, 2006a). Indeed, proteins and
polysaccharides can form complexes through covalent binding or
(9) attractive electrostatic interactions. In the latter case, alternative
layers of oppositely charged biopolymers can be formed around
the oil globules to obtain multi-layered “membranes”. These layers
provide both electrostatic and steric stabilizations thus improving
thermal stability and resistance to external treatment (i.e. ther-
mal processing, chilling, and freezing) (Gu et al., 2007; Guzey and
McClements, 2006a; Thanasukarn et al., 2006).
The most recent studies on emulsion formulation and stabiliza-
tion by proteins, polysaccharides, and their mixtures in the food
area are presented below. These biopolymers are classified accord-
ing to their main emulsifying/stabilizing mechanism, and to the
nature of the interactions in the case of protein–polysaccharide
mixtures. The examples of biopolymers presented below are
 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
mainly those already used in pharmaceutical applications other
than emulsion formulation. Their use in pharmaceutical emulsions
should therefore be more easily transferable.
3.1. Protein-stabilized emulsions
Many proteins are able to adsorb at the interface and facili-
tate droplets disruption by lowering the interfacial tension (Norde,
2003). However, at equivalent interfacial concentration small syn-
thetic surfactants are generally more effective than proteins in
reducing the interfacial tension. Typically, most proteins decrease
the oil–water tension by about 15–20 mN/m at saturated mono-
layer coverage compared to 30–40 mN/m for small synthetic
surfactants (Razumovsky and Damodaran, 1999). However, pro-
teins lead to thermodynamically and kinetically more stable
emulsions (Bos and Vliet, 2001; Damodaran, 2005). In their primary
structure, proteins contain hydrophilic and hydrophobic residues
randomly spread all over the structure. In the tertiary folded con-
formation, some of these residues exist as segregated patches. At
the oil–water or air–water interface, protein adsorption proceeds
in three main stages (Graham and Phillips, 1979): diffusion from
the bulk to the vicinity of the interface, actual adsorption, and
reorganization of the adsorbed protein. During adsorption, only a
fraction of the protein hydrophobic groups is embedded into the
lipophilic phase and most of the protein structure remains into
the aqueous one. Proteins then undergo conformational changes
in order to maximize the number of favorable interactions and
minimize the number of unfavorable ones in their environment.
Intermolecular interactions between adsorbed protein molecules
can lead to the formation of a highly viscoelastic film that prevents
coalescence (Dickinson, 1999). The remaining protein residues in
the aqueous phase also provide steric stabilization against floccu-
lation and coalescence (Wilde et al., 2004). It is important to note
that all proteins possess various amounts of hydrophobic groups
and exhibit different flexibility. Consequently, they exert diverse
interfacial properties.
Typically, 1–3 wt% protein concentration is used to stabilize
emulsions (Damodaran, 2005). In this concentration range, protein
adsorption rate is not a limiting factor to emulsion formation. The
efficiency of proteins in emulsion stabilization can be improved
through physical, enzymatic, and genetic modifications. For exam-
ple, partial denaturation or unfolding of proteins can be obtained
under controlled heating and shearing conditions (Demitriades
et al., 1997; Mitidieri and Wagner, 2002). These modifications allow
them to expose more hydrophobic groups towards the air or oil
phase.
Among others, caseins, whey proteins, gelatins, and pea proteins
are commonly used in the food industry. We describe them in more
details in the following sections.
3.1.1. Caseins
Caseins are the main protein components of mammalian milk. In
emulsion formulation, sodium caseinate is generally used. It mainly
consists of a partially aggregated mixture of four individual flexi-
ble caseins (#s1 , #s2 , ", $) (19,000–25,000 g/mol) (Dickinson and
Davies, 1999). In milk, caseins are intimately associated with cal-
cium phosphate in the form of colloidal particles called “casein
micelles” (Dickinson, 2006). The two major individual caseins, #s1
and ", are extremely effective emulsifying agents. They are able
to decrease the interfacial tension during emulsification, and to
protect newly formed fine droplets against flocculation and coa-
lescence by a combination of electrostatic and steric repulsions
(Dickinson and Davies, 1999). Moreover, adsorbed "-casein lay-
ers at the air–water interface display a viscoelastic behavior as
evidenced by magnetic rod interfacial shear rheometry (Bantchev
and Schwartz, 2003) and electrocapillary wave diffraction (Gau
365
et al., 1994). Casein-stabilized emulsions do, however, destabi-
lize by a flocculation mechanism in presence of calcium salts, as
observed by Dickinson and Davies (1999) by droplet-size mea-
surements. Dalgleish and others (Agboola and Dalgleish, 1995;
Dalgleish, 1997) explained this phenomenon by the strong spe-
cific calcium ion binding to the negatively charged phosphoserine
groups of casein, which reduces the net negative charge and thick-
ness of the adsorbed caseins layer. To our knowledge, no marketed
pharmaceutical emulsion containing caseins exists to this date, but
these proteins can be used in some nutritional supplementation
formulations.
3.1.2. Whey proteins
Milk globular whey proteins are a mixture of #-lactalbumin, "-
lactoglobulin, and several other minor proteins (immunoglobulins,
serum albumin) that are less flexible than caseins. They are
currently used with success in food emulsions, especially "-
lactoglobulin (18,300 g/mol) and #-lactalbumin (14,200 g/mol)
(Dickinson, 1997; Tcholakova et al., 2006). For example, Kim et al.
(2002) have shown that it was possible to stabilize emulsions
with "-lactoglobulin at pH 7, as this protein bears a net nega-
tive charge and induces relatively strong electrostatic repulsion
between droplets. Indeed, the pI for this protein is 5.2. However,
these authors also observed by particle size measurements that "-
lactoglobulin-stabilized emulsions were rather heat sensitive and
flocculated during heat treatments (Kim et al., 2002). This finding
is in agreement with Ye’s results (2010). Temperature should not
exceed a certain critical value (∼70 ◦ C) otherwise proteins unfold
and expose reactive groups that increase attractive interactions
between them (Kim et al., 2002), leading to extensive droplet
flocculation (McClements, 2004). Beside its stabilizing effect by
electrostatical interactions, "-lactoglobulin is also able to decrease
the air water or oil–water interfacial tension (Paulsson and Dejmek,
1992; Wüstnek et al., 1999). Moreover, Wüstnek et al. (1999) have
performed interfacial dilational rheology measurements using the
pendant drop method and reported an elastic behavior for this pro-
tein. They described that at low protein concentrations, when the
first biopolymer molecules reach the interface in optimal condi-
tions, they can fully adsorb at any locus and occupy a maximum
interface area, because the interface is still free of molecules. How-
ever, at higher concentrations, abundant lateral protein–protein
interactions at the interface induce a more compact conformation
of the protein. At interfacial saturation, elastic and viscous moduli
reach a maximum and for higher interfacial concentrations, both
moduli decrease. In a previous work, we also observed this behav-
ior in the oil–"-lactoglobulin solution system (Bouyer et al., 2011).
Bovine serum albumin (66,338 g/mol (Peter, 1975)) can also form
interfacial elastic films, as inferred from pendant drop and sur-
face pressure-area measurements (Burgess and Ozlen Sahin, 1997;
Serrien et al., 1992; Ward and Regan, 1980).
To this day, there is no example of "-lactoglobulin and #-
lactalbumin applications in the pharmaceutical industry. Human
serum albumin, however, can be used in hypovolemia to increase
intravascular volume by increasing plasma colloid oncotic pres-
sure. The increased intravascular pressure is the consequence of
increased protein concentration in the intravascular spaces upon
albumin infusion (Copelin, 1998).
3.1.3. Gelatin
Gelatin is a relatively high molecular weight protein derived
from animal collagen, e.g. pig, cow or fish (Ledward, 1986).
The latter is often preferred because there is no concern about
contamination with bovine spongiform encephalitis, and because
ethnic groups that do not consume pig or cow can eat it. Collagen
is hydrolyzed by boiling in the presence of acid (Type A gelatin,
pI ∼ 7–9) or alkaline (Type B gelatin, pI ∼ 5) (Taherian et al., 2011).
366 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
Gelatin is slightly surface active and can act as an emulsifier in oil-
in-water emulsions (Lobo, 2002). Surh et al. (2006) have confirmed
that physically stable oil in water emulsions could be obtained with
fish gelatin at concentrations equal to or above 4.0 wt%. However,
these authors observed a small fraction of relatively large droplets
(>10 !m). They explained it by the relatively low surface activity
of fish gelatin compared to "-lactoglobulin, for example. Gelatin is
currently used as a binder in emulsions containing light sensitive
silver halide salts for photographic films (Lourenço and Sampaio,
2009; Sheppard, 1929). In the pharmaceutical field, gelatin remains
mostly used in the manufacture of capsules (Jones, 2004).
3.1.4. Pea proteins
The two major protein components of pea proteins are the
storage globulins: 7S (vicilin, 150,000–180,000 g/mol) and 11S
(legumin, 360,000 g/mol) (Gueguen et al., 1988). They have a regu-
lar quaternary structure, trimeric for vicilin proteins and hexameric
for legumin ones. It has been shown that pea proteins are able
to decrease the oil–water interfacial tension and contribute to
emulsions stabilization by the formation of a rigid membrane at
the interface (Ducel et al., 2004a,b). However, pea proteins sta-
bilized emulsions can be very sensitive to environmental factors
such as pH and ionic strength. For this reason, they are usually
combined with another emulsifier/stabilizer such as high methoxyl
pectin (Gharsallaoui et al., 2010a,b). To the authors’ knowledge,
no marketed pharmaceutical product containing pea proteins
exists.
3.2. Polysaccharide-stabilized emulsions
Polysaccharides are known for their water-holding and thick-
ening properties because of their hydrophilic character and high
molecular weight. They can be classified in two categories for their
use in stabilizing emulsions droplets. Most common polysaccha-
rides do not have much of a tendency to adsorb at fluid interfaces.
Non-adsorbing polysaccharides have no or limited surface activity
and enhance the emulsion stability by gelling or modifying the vis-
cosity of the aqueous continuous phase, which slows down droplet
movement and encounters (Paraskevopoulou et al., 2004). Some
other polysaccharides, such as gum arabic, chemically modified
starch or cellulose derivatives, naturally occurring galactoman-
nan hydrocolloids (guar gum, fenugreek gum), acetylated pectin
from sugar beet, etc. (Dickinson, 2003) display surface/interfacial
activity. They first stabilize emulsions by adsorption at the oil
droplet surface and then prevent droplet flocculation and coales-
cence through electrostatic and/or steric repulsive forces. For gum
arabic and galactomannans, the surface activity results mostly from
the presence of a protein fraction in their structure. It also seems
that the protein associated with the pectin plays an important
role in stabilizing emulsion. Conversely, for cellulose derivatives,
the surface activity is due to the combination of hydrophobic and
hydrophilic groups along the cellulose backbone. In the follow-
ing subsections, polysaccharides are classified in non-adsorbing
polysaccharides if they do not exhibit (or only limited) surface
activity and adsorbing ones if they can stabilize emulsions through
their adsorption at the oil–water interface by lowering the interfa-
cial tension.
3.2.1. Non-adsorbing polysaccharides
3.2.1.1. Xanthan gum. Xanthan gum is an anionic polysaccharide
produced by the bacterium Xanthomonas campestris. Its structure
is composed of a "-(1-4)-d-glucose main chain, and side chains
each one out of two glucose residues. Side chains are constituted
of a #-d-mannose, "-d-glucuronic acid, and "-d-mannose as ter-
minal residues. Pyruvic acid and acetate groups can also be found
on terminal mannose residues and non-terminal ones, respectively
(Alderman, 1984). Casas et al. (2000) have reported that depending
on fermentation conditions, the content in acetate and pyruvate
groups varies, as well as the average molecular weight of the gum
(from 1.5 × 106 to 5 × 106 g/mol), influencing the viscosity of its
solutions. In water, the stiff polymer chain may exist as a single,
double or triple helix that interacts with another chain to form
a complex, loosely bound network (Jansson et al., 1975; Melton
et al., 1976). This particular structure gives the gum its unusual
thickening properties, with a yield stress and shear-thinning and
thixotropic behaviors (Benmouffok-Benbelkacem et al., 2010). Due
to these unique rheological properties, xanthan gum is recognized
as an excellent emulsion stabilizer (Hennock et al., 1984; Sun et al.,
2007). Under appropriate concentration conditions, the gum is used
to prevent flocculation and creaming in emulsions. Several authors
have calculated the minimum yield stress value (* 0 ) that an emul-
sion should have at a given droplet radius in order to inhibit gravity
effects such as creaming. A general way of estimating * 0 is reported
in Eq. (10) (Benna-Zayani et al., 2008; Chhabra, 1993; Jossic and
Magnin, 2005).
*0 ≥ Y)+gR (10)
with Y a dimensionless number describing the yield stress
effects to gravity (6 × 10−2 to 7 × 10−2 ), )+ the density difference
between the two phases, g the gravity acceleration, and R the mean
radius of the droplets. Knowing the mean radius allows for the
determination of the necessary minimum yield stress value for sta-
bilizing emulsions. When xanthan gum is mixed with guar gum or
magnesium aluminium, some synergistic rheological effects on the
viscosity occur (increased viscosity or gelation) (Kovacs, 1973), as
well as when mixed with locust bean gum (Benna-Zayani et al.,
2008).
In the pharmaceutical field, xanthan gum is used as a suspend-
ing and stabilizing agent in oral and topical formulations (Singh,
2006a), for the production of sustained-release matrix tablets (Billa
et al., 2000; Ceulemans et al., 2002; El-Gazayerly, 2003; Peh and
Wong, 2000; Phaechamud and Ritthidej, 2007; Yeole et al., 2006)
or for its mucoadhesive properties (Gacesa, 1988).
3.2.1.2. Alginates. Alginates are hydrophilic colloidal carbohy-
drates extracted with dilute alkali from various species of
brown seaweeds (Phaeophyceae), especially Laminaria hyper-
borean, Macrocystis pyrifera, and Ascophyllum nodosum. The
chemical composition of alginates is directly dependent on the
biological source, growth, and environmental conditions. The struc-
ture of alginate is mainly composed by alternating blocks of
#-(1-4)-l-guluronic acid (G), "-(1-4)-d-mannuronic acid (M), and
mixed GM blocks (Gacesa, 1988). The pKa values of G and M
monomers have been reported to be 3.38 and 3.65, respectively, in
0.1 M NaCl (Haug, 1964). Several authors showed that the stiffness
of the three blocks decreased in the order GG > MM > GM (Mackie
et al., 1980; Smidsrød, 1970). Sodium alginate can give highly vis-
cous solutions. It has unique colloidal properties, which include
thickening, stabilizing, suspending, film forming, gel producing,
and emulsion stabilizing (Fabra et al., 2008; Moe et al., 1995). Diva-
lent ions (such as Ca2+ ) form ionic bridges with glucuronic acid
units belonging to different chains (Gomez-Diaz and Navata, 2004).
This kind of ionic association, so-called “the egg-box model”, leads
to the formation of physical gels (Morris et al., 1978). Hambleton
et al. (2009a) have reported the very promising results obtained
with alginate emulsion-based edible films to protect microencap-
sulated aroma compounds. In the marketed pharmaceutical field,
alginates are mainly used as thickeners and gelifiers, in capsules or
tablets.
3.2.1.3. Carrageenans. Carrageenans are key natural gelling
polysaccharides extracted from the cell wall and intracellular
 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
matrix of numerous species of seaweeds (Eucheuma, Chondrus,
and Gigartina). They are a family of sulfated linear polysaccharides
(Therkelsen, 1993). There are three major types of carrageenans:
kappa ($), iota (%), and lambda (&)-carrageenans. Their primary
structure is composed of alternating disaccharide repeating
sequences of "-d-galactose linked at position 3, and #-d-galactose
linked at position 4. They differ in the number/position of sulphate
groups and the content of 3,6-anhydrogalactosyl rings per disac-
charide (Glickman, 1982). Carrageenans are able to form gels on
cooling. The gelation process starts by the transition of disordered
coils to coaxial double helices (McKinnon et al., 1969). Helix–helix
aggregation promoted by cations (such as K+ or Ca2+ ) gives rise to
thermal hysteresis between sol–gel and gel–sol transitions (Morris
et al., 1980). Kappa and iota are the main carrageenans able to
form gels due to their anhydride bridges allowing conversion of
coils in double helix structures. Conversely, &-carrageenan has
no anhydride bridge and is therefore non-gelling (Stanley, 1990).
It is possible to insert an anhydride bridge by alkali modification
(Ciancia et al., 1993) and obtain a theta carrageenan. However, the
latter cannot form a gel either, due to sulfation at O(2) of 3-linked
residues preventing the formation of the double-helix structure.
Doyle et al. (2010) have recently confirmed the very low viscosity
of theta carrageenan solutions.
Carrageenans can be used to influence the rheology and skin
permeation of microemulsion formulations. Valenta and Schultz
(2004) showed that the presence of carrageenans in the con-
tinueous phase of microemulsions increased the porcine skin
permeation of sodium fluorescein by improving the required con-
sistency for a better application on large skin areas. Like alginates,
$- and %-carrageenans can be used to encapsulate aroma com-
pounds (Hambleton et al., 2009b; Marcuzzo et al., 2010). They are
often studied in aqueous solutions in combination with another
biopolymer such as "-lactoglobulin (Ould Eleya and Turgeon,
2000), pea proteins (Musampa et al., 2007), or locust bean gum
(Dunstan et al., 2001). They are also combined with other biopoly-
mers for emulsion stabilization (with "-lactoglobulin for example)
(Dickinson and Pawlowsky, 1998; Gu et al., 2005, 2007). Stable
emulsions can be obtained with only 0.1–0.5 w/v % carrageenan
concentrations (Singh, 2006b). Carrageenans are used in many
nonparenteral dosage forms such as suspensions, emulsions, gels,
lotions, eye drops, suppositories, tablets, and capsules (Singh,
2006b).
3.2.1.4. Hyaluronan. Hyaluronan is an anionic linear biopolymer
composed of alternating disaccharide units of d-glucuronic acid
and N-acetyl-d-glucosamine with "(1-3) and "(1-4) linkages
(Ambrosio et al., 1999). It is ubiquitous in the human body
(extracellular matrix, synovial fluid, umbilical cord) with excellent
physicochemical and biological properties including biodegrad-
ability, biocompatiblity, non-toxicity, non-immunogenicity, spe-
cific viscoelasticity, hydration, and lubrification (Garg and Hales,
2004; Kuo, 2006). Its molecular weight ranges from 1 × 103 to
1 × 107 g/mol (Oh et al., 2010). In solution, hyaluronan has a
stiffened random coil structure with little tendency to form cross-
linked gels (Oh et al., 2010) and displays a shear thinning behavior
(Pisárčik et al., 1995). It has been reported that many proteins can
bind to hyaluronan (hyaladherins) (Day and Prestwich, 2002), influ-
encing its conformation (Day and Sheehan, 2001). Hyaluronan is
used in osteoarthritis (by intra-articular injections, known as vis-
cosupplementation), ocular surgery (as, for example, a vitreous
replacement), plastic surgery (generally used as a matrix which
is injected to treat facial wrinkles and folds, and to enhance the
appearance of the lips), tissue engineering (for tissue regeneration,
restoration, and repair, mainly because of its high biocompatibil-
ity), etc. (Oh et al., 2010). It is also increasingly studied for targeted
specific and long-acting delivery of various biopharmaceutics such
367
as protein, peptide, and nucleotide therapeutics (Oh et al., 2010).
Hyaluronic acid is often used in emulsions as the first step of hydro-
gel particles or microspheres synthesis. Drug delivery systems can
then be obtained with these hydrogel particles (Dulong et al., 2004;
Iglin et al., 2010; Yun et al., 2004). Recently, a hyaluronic acid gel
that contained vasoactive intestinal peptide-loaded liposomes was
developed (Lajavardi et al., 2009). Interactions between hyaluronic
acid chains and liposomes increased the viscosity of the gel and
enhanced its elasticity. This resulted in a delayed release of the
vasoactive intestinal peptide for the treatment of uveitis.
3.2.1.5. Chitosan. Chitosan is a naturally occurring polysaccharide,
commonly obtained from the alkali or enzymatic deacetylation of
chitin. Chitin is found in the cuticles of crustacean, insects, molluscs,
and cell walls of microorganisms. Chitosan structure is composed
of glucosamine and N-acetylglucosamine copolymers. It is posi-
tively charged in acidic aqueous solution due to the protonation
of its amino residues, which have a pKa value of 6.2–7.0 (Claesson
and Ninham, 1992). Various molecular weights can be found:
(50–2000) × 103 g/mol (Baldrick, 2010). Chitosan is widely investi-
gated for many different applications, including drug delivery sys-
tems, due to its biodegradability, biocompatibility, bioadhesivity,
antimicrobial activity, and film forming properties (No et al., 2007;
Shahidi and Abuzaytoun, 2005; Varum and Smidsrød, 2006). Its
cationic nature enables chitosan to strongly interact with the
oppositely charged lipids of biomembranes. It is an amphiphilic
polyelectrolyte that combines both electrosteric and viscosifying
stabilization mechanisms in emulsions (Rodriguez et al., 2002).
Chitosan is a very good viscosity-enhancing agent in an acidic envi-
ronment, owing to its high molecular weight and linear unbranched
structure. It has a shear thinning behavior (Singla and Chawla,
2001). Skaugrud (1991) reported that the viscosity of chitosan solu-
tions increased with its concentration and degree of deacetylation,
and decreased with the temperature. Payet and Terentjev (2008)
showed that an aqueous chitosan solution was able to emulsify
paraffin oil. They observed chitosan adsorption at the air–water
and oil–water interfaces with limited interfacial tension decrease,
and formation of a dense network of polyelectrolytic brushes in
the continuous phase. Chitosan chains reduced the oil droplet dif-
fusion by forming a rigid elastic network in the aqueous phase
thus improving emulsion stability. Its low surface properties com-
pared to its viscosifying ones made us classify chitosan in the
non-adsorbing polysaccharide category. Chitosan can be used in
controlled drug delivery systems (Jones and Mawhinney, 2006;
Sezer and Akbuğa, 1995). Vasconcellos et al. (2011) for example,
successfully produced papain-loaded chitosan microparticles for
controlled release applications. Chitosan was also proposed as a
component of mucoadhesive dosage forms and in gene delivery
systems (Hejazi and Amiji, 2003). It allowed, on its own or in com-
bination with a surfactant, the stabilization of simple and multiple
emulsions (Chuah et al., 2009; Hino et al., 2000; Moschakis et al.,
2010; Schulz et al., 1998). However, chitosan is not in any marketed
drugs because its safety as a pharmaceutical excipient has not been
assessed yet (Baldrick, 2010). Nevertheless, chitosan is currently
used in dietary preparations for obesity, and has been recognized
as safe in the US for use in foods (Baldrick, 2010).
3.2.2. Adsorbing polysaccharides
3.2.2.1. Gum arabic. Gum arabic is the most well known of all
polysaccharide emulsifiers in food industry. It is widely used
in soft drinks for its emulsifying properties (Chanamai and
McClements, 2002; Dickinson, 2003; Garti, 1999a; McNamee et al.,
1998). It is exudated from the Acacia senegal tree (Randall et al.,
1989). Gum arabic is a hetero-polysaccharide containing both
protein and polysaccharide subunits. It is composed of three
fractions depending on the relative protein-to-polysaccharide
368 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
content: the arabinogalactan (AG) (80–90 wt% of the total gum),
the glycoprotein (GP) (2–4 wt% of the total gum), and the
arabinogalactan-protein (AGP) (10–20 wt% of the total gum) frac-
tions (Anderson et al., 1983). The molar mass of the entire gum
can vary from 3.0 × 105 to 5.8 × 105 g/mol depending on its origin
and tree age (Idris et al., 1998). Aqueous solutions of gum arabic
have a rather low viscosity (100 mPa s for a 30% w/v solution at
20 ◦ C (Kibbe, 2006a)) compared to those of other polysaccharides
having similar molecular masses. Its rheological behavior is consid-
ered as Newtonian up to 20–30 wt% gum concentrations (Sanchez
et al., 2002). The AGP fraction is considered to be responsible for the
emulsifying properties of gum arabic (Erni et al., 2007; Fauconnier
et al., 2000; Picton et al., 2000; Randall et al., 1988). The association
of polysaccharidic blocks along the peptidic chain gives a “wat-
tle blossom”-type structure and confers to the macromolecule an
amphiphilic character favoring its adsorption at the air–water and
oil–water interfaces (Dickinson, 2003; Idris et al., 1998). Indeed, the
proteinaceous fractions of the gum would embed in the air or oil
phase, while the carbohydrate component would extend out from
the surface into the aqueous phase (Chanamai and McClements,
2002; Picton et al., 2000). In emulsion systems, the adsorption of
the gum at the oil–water interface allows steric hindrance pre-
venting the droplets to come in closer contact. Moreover, the gum
being negatively charged above pH 2 (Kravtchenko, 1997) allows
for electrostatic stabilization of emulsions. Although its surface
activity is considered as rather low when compared to typical pro-
tein emulsifiers (McNamee et al., 1998), we recently showed that
after sufficient adsorption time, gum arabic lowered the interfacial
tension to the same extent as "–lactoglobulin (Bouyer et al., 2011).
In order to generate stable sub-micron-sized droplets, it is neces-
sary to use a rather high gum-to-oil weight ratio, i.e. approximately
1:1 (McNamee et al., 1998).
Gum arabic is used in various delivery systems. Recently, Avadi
et al. (2010) obtained promising results in the development of
insulin-loaded nanoparticles based on ionic gelation of chitosan
and gum arabic for oral delivery. In another study, gum arabic
was used to encapsulate and control the release of endoglucanase
(Ramakrishnan et al., 2007). Lu et al. (2003) also used the gum as
an osmotic, suspending, and expanding agent in an osmotic tablet
system. It is reported that optimal concentrations of gum arabic
are of 10–20 wt% when used as an emulsifying agent, 10–30 wt% as
a pastille base, 5–10 wt% as a suspending agent, and 1–5 wt% as a
tablet binder (Kibbe, 2006a).
3.2.2.2. Hydroxypropylmethylcellulose (HPMC). HPMC, also named
Hypromellose, is part of an interesting group of surface active
cellulose derivatives which have a strong tendency to accumu-
late at the oil–water interface causing a reduction of the surface
tension (Nahringbauer, 1995). It is a partly O-methylated and O-
(2-hydroxypropylated) cellulose. HPMC finds various applications
in pharmaceutical and food industries where it is used for con-
trolled drug release, control of texture and rheological properties
of dispersions, emulsification, etc. The use of HPMC as an emulsifier
for oil in water emulsions was first described by Daniels and Barta
(1993). The emulsion stabilizing properties of cellulose derivatives
could be associated with their structural features. Indeed, the sur-
face activity of these macromolecules is due to their hydrophobic
(rich in methoxylgroups) and hydrophilic (rich in hydroxypropyl
groups) regions distributed along the cellulose backbone, allow-
ing for their adsorption at fluid interfaces and the lowering of
the interfacial tension (Camino et al., 2009; Ochoa-Machiste and
Buckton, 1996; Wollenweber et al., 2000). HPMCs exhibit differ-
ent surface activities with varying the methoxyl/hydroxypropyl
ratio. When adsorbed, the conformation of this biopolymer may
be described according to the train-loop-tail model: hydropho-
bic segments along the polymer chain attach to the interface and
form trains which are separated by hydrophilic loops and tails
that extend into the aqueous phase (Wollenweber et al., 2000).
Nahringbauer (1995) proposed that, similarly to proteins, adsorp-
tion was followed by changes in molecular conformation. Initially,
in bulk solution, the macromolecules are coiled and after adsorp-
tion the polymer backbone starts to unfold. Pharmaceutical use of
HPMC (0.45–1 w/w %) has been reported in vehicles for eye drops
and artificial tear solutions (Harwood, 2006). In oral pharmaceu-
tical products, it is primarily used as a tablet binder (Chowhan,
1980), film-coating (Banker et al., 1981; Rowe, 1980), or matrix for
extended-release tablets (Dahl et al., 1990; Hogan, 1989). In addi-
tion, HPMC is an alternative to gelatin in the manufacturing of hard
capsules (Harwood, 2006).
3.2.2.3. Galactomannans. Galactomannans are abundant plant
polysaccharides that occur as energy reserves in certain legumi-
nous seeds. They are rather rigid hydrophilic biopolymers with
a polymannose backbone ("-(1-4)-d-mannose) and grafted #-
(1-6)-d-galactose units. The mannose to galactose (M/G) ratio
is dependent on the galactomannan source and environmental
growth conditions. The M/G ratios of fenugreek gum, guar gum,
tara gum, and locust bean gum are of about 1, 2, 3, and 4, respec-
tively (Dea and Morrison, 1975; Wu et al., 2009). They can form
highly viscous solutions even at low concentrations (for instance,
a viscosity of 4.86 Pa s for a 1 w/v % guar gum dispersion) (Kibbe,
2006b). Galactomannans are thus generally used as thickening,
water-holding, and stabilizing agents. The rheological behavior of
these solutions is usually non-Newtonian. Their shear-thinning
behavior has been described as following the Cross model (Eq. 11)
(Cross, 1965; Sittikijyothin et al., 2005).
" #
"0 − "∞
* = "∞ + p ,̇ (11)
1 + (ˇ,̇)
with * the shear stress, ,̇ the shear rate, "0 and "∞ the zero and
infinite shear viscosities, ˇ and p the time and rate constants.
Several groups (Garti, 1999b; Garti and Reichman, 1994;
Mikkonen et al., 2009; Wu et al., 2009) have proposed some of
these galactomannans for emulsion stabilization. Their interfacial
tension lowering properties are assumed to be most probably due
to the presence of a small fraction of protein closely associated with
the polysaccharide structure, as in gum arabic (Brummer et al.,
2003; Dickinson, 2003; Youssef et al., 2009). However, Garti and
Reichman (1994) showed that the protein fraction of galactoman-
nans played no significant role in the gum activity. They suggested
that the strong interfacial activity of galactomannans was due to
a “salting-out” effect, which helped precipitation of a gum film
onto the oil droplets. This contradiction between results obtained
by the different groups is due to the method used for separat-
ing the protein fraction from the whole galactomannan molecule.
Indeed, Garti’s method was a physical separation (ultracentrifu-
gation), whereas Brummer et al. (2003) used enzyme hydrolysis
to remove the proteins. Brummer et al. (2003) showed that the
purified gum was less surface active than the unpurified one. The
proteins closely associated with galactomannans could not be eas-
ily separated by physical methods, but were susceptible to enzyme
hydrolysis.
In the pharmaceutical field, galactomannans are binders and
disintegrants in solid-dosage forms. Coviello et al. (2007), for exam-
ple, formed a tablet matrix from guar and locust bean gums, and
studied the release of model molecules. They showed that it was
possible to modulate the release of the molecules from this galac-
tomannan matrix.
3.2.2.4. Pectin. Pectin is a complex polysaccharide extracted from
plant cell walls, especially citrus peels, apple pomace, and sugar
 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
beet pulps. Its structure is mainly composed of esterified d-
galacturonic acid residues in an #-(1-4) chain, and depends on
the pectin source and extraction process. This backbone is then
regularly branched with rhamnogalacturonan segments. Pectin is
mainly used for its gelling and dispersion stabilizing properties.
In addition, it has emulsifying properties, and as early as 1927,
was proposed for the preparation of vegetable oils emulsions and
mayonnaise (Rooker, 1927). Pectin is able to produce fine and stable
emulsions (Leroux et al., 2003) in the same manner as gum arabic
but at much lower dosage (Leroux et al., 2003; Siew and Williams,
2008). Since it contains protein residues, the model of its associa-
tion to oil droplets may be similar to that of gum arabic (Dickinson,
2003; Leroux et al., 2003). Thus, pectin emulsification properties
have been mainly attributed to its protein content, but also to
its acetyl groups (Leroux et al., 2003; Siew and Williams, 2008).
Indeed, the presence of acetyl groups in pectins (4–5%) enhances
the hydrophobic nature of the polysaccharide. However, the pro-
tein associated with pectin plays the key role, and the presence of
acetyl groups is not an absolute requirement with respect to the
emulsifying efficiency (Leroux et al., 2003). According to Siew and
Williams (2008), apart from protein content, other factors, such as
the molecular weight, the ferulic acid, acetic acid, and methyl ester
contents, and their accessibility to the surface of the oil droplets,
contribute to the emulsification properties of pectins. Pharmaceu-
tical applications of pectin include formulations for the treatment
of diarrhea, constipation, and obesity (Cook, 2006). Pectin has
also been studied in gel formulations for oral sustained release of
ambroxol (Kubo et al., 2004), and in gel beads for controlled drug
delivery within the gastrointestinal tract (Murata et al., 2004).
3.3. Protein–polysaccharide stabilized emulsions
The combination in the same system of the advantages of pro-
teins (fast adsorption compared to polysaccharides) and polysac-
charides (steric repulsion or viscosity enhancement) to stabilize
emulsions is increasingly studied. Indeed, proteins and polysaccha-
rides both contribute by their emulsifying/stabilizing properties to
create novel emulsions with improved stability and functionality.
The development of surface active protein–polysaccharide com-
plexes can be achieved either by covalent bonding or electrostatic
interactions. In the latter case, it is important to wisely choose
the polymers and physicochemical conditions so that the protein
and polysaccharide carry opposite charges. Another way to stabi-
lize emulsions is to combine protein and polysaccharide properties
without developing any attractive interactions between the two
biopolymers.
3.3.1. Covalent complexes
An extreme type of protein–polysaccharide interaction occurs
when a covalent linkage is formed between the two biopoly-
mers, creating a new amphiphilic biopolymer with improved
surface properties. Protein–polysaccharide conjugates can be
formed without using any chemicals, generally by linking pro-
tein '-amino-groups to the reducing end carbonyl groups of
polysaccharides through controlled Maillard reaction (slow dry
heating) (Kato et al., 1990). The newly formed conjugate exhibits
substantially improved solubility and emulsifying properties
compared to the protein (i.e. "-lactoglobulin + dextran (Dickinson
and Galaska, 1991; Wooster and Ann Augustin, 2006), whey
protein isolate + maltodextrin (Akhtar and Dickinson, 2007),
sodium caseinate + maltodextrin (O’Regan and Mulvihill, 2010))
and polysaccharide alone. Furthermore, the adsorbed protein layer
seems to be protected against destabilization under unfavorable
environmental conditions (i.e. heating, freezing, high electrolyte
concentrations, etc.). O’Regan and Mulvihill (2010), for example,
have reported improved emulsion stability after seven-day storage
369
at 45 ◦ C when emulsions were stabilized by a sodium caseinate-
maltodextrin conjugate, compared to sodium caseinate alone.
The large size of the bound polysaccharide generates long-range
steric repulsion between emulsion droplets surfaces. The differ-
ent experimental studies of protein–polysaccharide conjugates
published over the 2004–2007 period have been reviewed by
Dickinson (2009).
3.3.2. Non-covalent complexes
More often protein–polysaccharide complexation arises from
non-covalent association, mainly driven by attractive electrostatic
interactions. Numerous studies have shown improved emulsion
stability, attributable to the presence of associative interfacial inter-
actions between the protein and polysaccharide. The main studies
based on this stabilization mechanism are gathered in Table 1. Two
alternative procedures for emulsion formation can be used. The first
one consists in preparing a mixed solution of the biopolymers, and
using the resulting protein–polysaccharide complex for the emul-
sification (Laplante et al., 2006; Lutz et al., 2009). The second one,
named the layer-by-layer (LBL) electrostatic deposition technique,
consists in forming a primary protein-stabilized emulsion and then
in adding the polysaccharide. Complexation occurs directly at the
droplet surface through attractive electrostatic interactions. This
leads to the formation of a second layer and allows surface charge
reversal (Khalloufi et al., 2009a). This last step can be repeated
using oppositely charged biopolymers to increase the number of
layers coating the oil droplets. It seems important to start by a
protein layer because of its faster adsorption resulting in a rapid
lowering of the interfacial tension (Klein et al., 2010). Under cer-
tain physicochemical conditions, this multilayer coating displays a
better stability to environmental stresses such as pH, ionic strength,
heating, chilling, freezing, and dehydration, than a single interfa-
cial layer (Aoki et al., 2005; Gu et al., 2007; Guzey and McClements,
2006b). The detailed principles of this LBL method can be found
in the review of Guzey and McClements (2006a). The LBL depo-
sition of biopolymers onto the oil droplets surface can be easily
adapted to high-scale production conditions because of the fast-
ness, simplicity, and relative cheapness of this method (Grigoriev
and Miller, 2009). However, in order to achieve stable and homo-
geneous multilayered stabilized emulsions, it is necessary to fully
control the composition of the system and the conditions of prepa-
ration (protein:polysaccharide ratio, pH, ionic strength, biopolymer
concentration, etc.). For example, the presence of free biopolymer
molecules in the aqueous phase during addition of the oppositely
charged coating biopolymer may lead to the formation of com-
plexes in the solution bulk. These complexes often provide bridging
flocculation and strongly reduce emulsion stability (Surh et al.,
2005). They can also lead to depletion flocculation, due to an excess
in polysaccharide. Emulsion stability also depends on the protein
interfacial coverage: if protein molecules do not saturate the inter-
face, the added polysaccharide can anchor at the globule surface.
This co-adsorption modulates the interfacial stability.
3.3.3. Without complexation
This last stabilization mechanism consists in adsorbing first a
biopolymer (generally a protein), and then adding a polysaccharide
that does not interact with the adsorbed biopolymer but increases
the viscosity of the continuous aqueous phase. The protein allows
the formation of small droplets during the emulsification process,
and the polysaccharide generates an extended thickening network,
which induces high viscosity at low shear rate, thus slowing down
droplet motion. The improved stability can also be explained by
a limited thermodynamic compatibility between the two biopoly-
mers, resulting in enhanced interfacial protein adsorption. Many
examples of such protein–polysaccharide associations can be
found in the literature, among which sodium caseinate + xanthan
370 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378

Table 1
Main published experimental studies on protein–polysaccharide electrostatic combination to stabilize emulsions.

Protein1 Protein2 or Protein3 or Experimental conditionsa (pH; References

polysaccharide1 polysaccharide2 concentrations; pr:ps ratio; incorporation
order)

Bilayer stabilization Whey protein isolate Flaxseed gum pH 3.5; 0.6 w/v % pr, 0–0.33 w/v % ps; pr1 Khalloufi et al. (2009a)
–
then ps1
pH 3.5, 7; 0.6 w/v % pr, 0–0.33 w/v % ps; pr1 Khalloufi et al. (2009b)
then ps1
Chitosan – pH 5.5, 6; 0.1% ps; 0:1, 2.5:1, 5:1, 7.5:1, Laplante et al. (2006)
10:1 ratios; pr1 and ps1 premix
Pectin – pH 2–8; 4:0.5 w/w % solution; pr1 and ps1 Lutz et al. (2009)
premix
Xanthan gum – pH 7; 0.5–6 wt% pr, 0.5 wt% ps; pr1 then ps1 Benichou et al. (2007a)
Galactomannans – pH 7; 5 wt% pr; 4:0.5 (wt/wt) ratio; pr1 Benichou et al. (2007a)
then ps1
$-carrageenan – pH 4.5–7.5; 0.5, 3 w/w % pr, 0–0.4 w/w % Singh et al. (2003)
ps; pr1 and ps1 premix
Gum arabic – pH 7; 4:1, 3:1, 2:1, 1:1 (1–10 wt%) ratios; Klein et al. (2010)
pr1 and ps1 premix and pr1 then ps1
Gelatin – pH 3.4, 6.8; 1 wt% pr; 1:1; pr1 then pr2 Ledward (1986)

"-lactoglobulin Pectin – pH 7 → 4; 0.225 wt% pr, 0.2 wt% ps; pr1 Guzey and McClements (2006b)
then ps1
pH 3, 7; 0.225 wt% pr, 0.2 wt% ps; pr1 then Guzey et al. (2004)
ps1
pH 3; 0.45 wt% pr, 0–0.22 wt% ps; pr1 then Moreau et al. (2003)
ps1
Carrageenans – pH 3.5, 6; 0.5 wt% pr, 0–0.15 wt% ps; pr1 Gu et al. (2005)
then ps1
%-carrageenan – pH 3.4, 4, 6; 3:7–3:2 (0.5 wt%) ratios; pr1 Ru et al. (2009)
then ps1
Alginate – pH 3–7; 0.45 wt%; 0–0.5 wt%; pr1 then ps1 Pongsawatmanit et al. (2006)
Gum arabic – pH 4.2; 0.5, 2.5 w/w %; 2:1, 1:2; pr1 and ps1 Bouyer et al. (2011)
premix, pr1 then ps1 , ps1 then pr1
Sodium caseinate Pectin – pH 7; 0.5% pr; 0–0.2% ps; pr1 then ps1 Bonnet et al. (2005)
Gellan gum – pH 5.4; 1% pr; 0.03, 0.05% ps; pr1 and ps1 Sosa-Herrera et al. (2008)
premix
Dextran sulfate – pH 6; 0.5 wt% pr, 0.1–1 wt% ps; pr1 and ps1 Jourdain et al. (2008)
premix and pr1 then ps1
Pea protein Pectin – pH 2.4; 0.25 wt% pr, 0.2 wt% ps; pr1 then Gharsallaoui et al. (2010a)
ps1
Trilayer stabilization "-lactoglobulin %-carrageenan Gelatin pH 6; 0.5 w/w % pr1 , 0.1 w/w % ps1 , 0.2 Gu et al. (2007)
w/w % pr2 ; pr1 then ps1 then pr2
Pectin Chitosan pH 4; 0.1125% pr1 , 0.1% ps1 , 0.15% ps2 ; pr1 Guzey and McClements (2006b)
then ps1 then ps2
Chitosan Pectin pH 3–7; 0.05–0.1 wt% pr1 , 0–0.1 wt% ps1 , Li et al. (2010)
0–0.25 wt% ps2 ; pr1 then ps1 then ps2
Chitosan Alginate pH 3–7; 0.05–0.1 wt% pr1 , 0–0.1 wt% ps1 , Li et al. (2010)
0–0.25 wt% ps2 ; pr1 then ps1 then ps2
a
pr: protein; ps: polysaccharide; pr and ps premix: pr and ps were mixed in solution prior to emulsion formulation; pr then ps: ps was added after emulsion formulation
with pr; ps then pr: pr was added after emulsion formulation with ps. 1 stands for the first pr or ps, 2 stands for the second one, and 3 for the third one.

gum (Hemar et al., 2001; Moschakis et al., 2005), whey protein
isolate + xanthan gum (Sun and Gunasekaran, 2009), whey pro-
tein isolate + flaxseed gum (Khalloufi et al., 2008), and sodium
caseinate + locust bean gum (Perrechil and Cunha, 2010).
4. Discussion
4.1. Comparison of biopolymers and small-molecule surfactants
The aim of this section is to compare the interfacial behavior
of small-molecule surfactants and biopolymers and to discuss the
implications of their differences on emulsion formation and stabi-
lization.
4.1.1. Comparison of the interfacial behavior
Because of their smaller size and lower molecular weight,
surfactants diffuse, adsorb, and stabilize an interface more
rapidly than biopolymers. With small-molecule surfactants
true thermodynamic equilibrium between bulk and interface is
reached within seconds. Conversely, owing to their higher size,
their structural complexity, and the cooperative nature of their
adsorption, adsorption of biopolymers is a much slower process:
the steady state is reached only after hours or days – or even not at
all (Dickinson, 2011). Whereas the adsorption of small surfactants
is reversible, the adsorption of biopolymers is usually considered
as practically irreversible (from a kinetic and not a thermodynamic
standpoint) due to a very low desorption rate (Bos and Vliet, 2001).
Theoretical desorption rates of proteins are 104 –108 times slower
than for usual surfactants. As a consequence, experiments per-
formed on timescales of 104 s are unable to discriminate between
reversibility and irreversibility of adsorbed biopolymers (Ferri
et al., 2010). The adsorption of biopolymers also involves consid-
erable changes in their three-dimensional structure, unlike low
molecular weight surfactants for which such structural changes
and conformational rearrangements are very limited. Indeed, pro-
teins and adsorbing polysaccharides do not display clearly defined
hydrophilic head and hydrophobic tail as found in low molecular
weight surfactant structure. Thus, the structural and dynamic
properties of the adsorbed layers at liquid interface are mostly
determined by the degree of unfolding of the biopolymer before
 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
and after adsorption. Proteins and polysaccharides can adsorb at
the interface with several segments (trains), and then change their
conformation and orientation to allow more segments to adsorb
over time. Owing to their higher molecular weight, polysaccharides
usually adsorb more slowly at the interface than proteins (Bouyer
et al., 2011). Although the adsorption process is different for low
molecular weight surfactants and biopolymers, it is interesting to
note that the same kinetic approach is used in the literature to
describe the behavior of the two kinds of amphiphilic molecules. At
low surface pressures (low bulk concentration and short adsorp-
tion times), diffusion is the rate-determining step and a modified
Ward and Tordai equation can be used (Ward and Tordai, 1946):
$ Dt %1/2
˘ = ,(t) − ,0 = C0 kB T
! flexibility of the macromolecule backbone, molecular weight, etc.).
where ˘ is the surface pressure, ,(t) and , 0 are the surface tension
at time t and at time t = 0 respectively, C0 is the bulk concentration
of the amphiphilic molecule in the aqueous phase, kB is the
Boltzmann constant, T the absolute temperature, D the diffusion
coefficient of the surface active molecule, and t the adsorption
time. If diffusion controls the adsorption process the plot of
surface pressure versus t1/2 is linear, and the diffusion coefficient
can be calculated from its slope (Demé et al., 1995). At higher
interfacial pressures (high concentrations or semi-dilute regime),
solute–solute interactions occur and the plot is no longer linear.
To analyse the adsorption, unfolding and rearrangements of
molecules occurring at higher surface pressures, the approach pro-
posed by Graham and Phillips (1979) can be used, where the rates
of the different proccesses are analysed by first-order equations of
the following form:
˘f − ˘t
ln = −ki t
˘f − ˘0
In this equation, ˘ f , ˘ 0 , and ˘ t are the surface pressures at
the end of the adsorption process, at time t = 0, and at time t,
respectively, and ki the first-order rate constant. When experi-
mental data are plotted according to Eq. (13), two or more linear
regions can be observed. The first one corresponds to the adsorp-
tion, with the first-order constant of adsorption kads . The slope
of the second linear region is taken as the first-order constant of
rearrangement (kr ), occuring among a more or less constant num-
ber of adsorbed molecules. For low molecular weight surfactants
this rearrangement process is negligible. The two above described
models have been recently applied to the determination of the
kinetics of adsorption of various biopolymers: a mixture of "-
lactoglobulin and low methoxyl pectin (Ganzevles et al., 2006),
whey protein concentrate in the presence of sodium alginate or
&-carrageenan (Perez et al., 2009), "-lactoglobulin in the pres-
ence of sodium alginate or &-carrageenan (Perez et al., 2012),
hydroxypropylmethylcellulose (Camino et al., 2009), anionic or
ionic surfactants and ovalbumin (Seta et al., 2012).
The different adsorption mechanisms observed for low
molecular weight surfactants and biopolymers also lead to dif-
ferent molecular organization within the interfacial film. Small
surfactants can pack together more closely at the interface than
biopolymers. The concentration of adsorbed surfactant molecules
is typically 10−5 mol/m2 , which corresponds to adsorbed amounts
ranging between 1 and 2 mg/m2 (Bos and Vliet, 2001). For proteins,
the interfacial concentration is much lower: about 10−7 mol/m2 ,
with an adsorbed amount ranging between 2 and 3 mg/m2 (Bos and
Vliet, 2001). For polysaccharides, owing to their higher molecular
weight, the concentration of adsorbed molecules is even lower
(about 10−8 mol/m2 ) whereas the adsorbed amount is higher
(2–14 mg/m2 ) (Buffo et al., 2001; Padala et al., 2009; Siew and
Williams, 2008).
371
The thickness of the interfacial layer of monomeric amphiphilic
molecules does not vary to a great extent with surface coverage and
remains close to the size of a surfactant molecule. Conversely for
biopolymers, this parameter strongly depends on the surface cover-
age (Camino et al., 2009; Miller et al., 2000; Perez et al., 2006). At low
biopolymer interfacial concentration, the protein or the polysac-
charide can adopt a flat and extended conformation at the interface.
When the biopolymer concentration is increased, the molecular
area occupied by one adsorbed molecule decreases and the biopoly-
mer conformation is more condensed. The number and length of the
biopolymer loops and tails protruding into the aqueous phase thus
increase, leading to larger values of the layer thickness. The extent
of thickness variations depends on the nature of biopolymer (num-
ber of effective hydrophobic groups and distance between them,
(12)
At the highest biopolymer bulk concentrations, a collapsed struc-
ture with multilayer formation can be observed (Bouyer et al., 2011;
Bos and Vliet, 2001; Camino et al., 2009).
The resulting mechanical properties of the interfacial films are
also very different. The interfacial shear viscosities of biopolymers
are much higher than those of small-molecule surfactants. This
was attributed to lateral interactions between biopolymers due
to hydrogen bonding, covalent bonding, hydrophobic and electro-
static interactions (Bos and Vliet, 2001).
Many studies have attempted to combine the different inter-
facial mechanisms and properties of low molecular weight
surfactants and biopolymers in order to improve emulsion sta-
bilization. However, the interfacial mechanisms involved in such
mixed systems are far more complex than for single molecules.
The competitive displacement of proteins from the interface
by small-molecule surfactants has been studied and computer
simulated (Courthaudon et al., 1991; Dickinson et al., 1990; Mackie
(13) et al., 2000; Seta et al., 2012). At monolayer saturation coverage,
only about one-third of the accessible surface area is usually occu-
pied by train segments of a protein. Thus, the numerous small gaps
in the adsorbed biopolymer layer are readily accessible to small
molecules such as surfactants (Dickinson and Matsumara, 1994).
The observed mechanisms depend on the surfactant type, i.e. ionic
or non-ionic, and on its concentration. Non-ionic surfactants ini-
tially adsorb into the void spaces at the interface, which raises the
local surface pressure. As the nucleated domains grow with time,
the surrounding protein film is compressed, and the local surface
pressure increases more. At sufficiently high surface pressures (i.e.
high surfactant concentrations), the protein film looses its integrity
and the protein desorbs in the bulk phase. Complete removal
of the protein occurs at high surfactant to protein ratios. This
mechanism is called the orogenic displacement (Mackie et al., 2000)
or replacement mechanism (Bos and Vliet, 2001). Courthaudon
et al. (1991) showed that the addition of a water-soluble non-
ionic surfactant to an oil-in-water emulsion stabilized by "-casein
led to total displacement of the protein from the oil–water inter-
face at the surfactant-to-protein molar ratio of 17 ± 1. Conversely,
ionic surfactants tend to complex with the adsorbed protein. With
increasing surfactant concentrations, electrostatic interactions are
preponderant until all the protein charges are compensated by sur-
factant charges. The complex is then globally neutral and displays
a higher surface activity. Upon further addition of surfactant, an
increasing number of surfactant molecules interact with the com-
plex through hydrophobic interactions, making step-by-step the
complex more hydrophilic, and less surface active. Due to the com-
petition in the adsorbed layer, the soluble complex is progressively
replaced by surfactant molecules, so that typically when the free
surfactant concentration in the bulk reaches the critical micellar
concentration the adsorption layer is mainly formed by the small-
surfactant molecules (solubilisation mechanism) (Bos and Vliet,
2001; Maldonado-Vaderrama and Rodriguez Patino, 2010; Miller
372 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
et al., 2000). Compared to non-ionic surfactants, a much higher
concentration of ionic surfactant is required for complete displace-
ment of a protein from the interface. This is attributable to the
relatively low surface activity of the charged surfactant and to
the strong interfacial surfactant–protein interactions (Dickinson,
2001). Few studies are devoted to the interfacial behavior of mixed
polysaccharide–surfactant systems and these systems also display
a complex behavior (Garti et al., 1999; Mezdour et al., 2008; Ropers
et al., 2008).
4.1.2. Comparison of emulsifying properties
During the emulsification process, the adsorption kinetics of an
amphiphilic molecule is influenced by its molecular weight: a small
monomeric surfactant adsorbs at the interface very quickly, which
contributes to the production of small droplets. Small-molecule
surfactants such as Tween 20 and sodium dodecyl sulphate are
more effective at producing small droplets in nanoemulsions than
caseinate and "-lactoglobulin under similar homogenization con-
ditions because they adsorb to the droplet surfaces more rapidly
(Qian and McClements, 2011). Moreover, the small surfactant size
allows a higher droplet curvature (reduced steric hindrance) and
globules are generally smaller than with biopolymers. Biopolymer-
stabilized emulsions generally have droplet sizes in the order
of 1 !m or more and formation of nanoemulsions with them
is thus more difficult. However very recently, McClement and
co-workers have obtained nanoemulsions with "-lactoglobulin
(Li et al., 2012; Qian and McClements, 2011; Qian et al., 2012),
sodium caseinate (Qian and McClements, 2011), and whey pro-
tein isolate (Lee et al., 2011). The emulsification process used often
limits the choice of emulsifiers that can be used in nanoemul-
sion formulation. For example formation of nanoemulsions by
phase inversion temperature or spontaneous emulsification meth-
ods is not possible with proteins or polysaccharides (McClements,
2011). As for microemulsions, to our knowledge, no successful
formulation study has been reported with biopolymers, without
addition of small-molecule surfactants. Actually, only low mole-
cular weight surfactants allow reaching the ultra low interfacial
tensions at specific monolayer curvature required for the obtention
of a microemulsion (McClements, 2012). When comparing biopoly-
mers to small surfactants, it is also important to note that due to
their solubility in water, biopolymers are only used to produce
O/W emulsions when used alone. For instance, water-in-silicon oil
emulsions cannot be stabilized by human serum albumin by itself
and it is necessary to add an oil-soluble and end-functionalized
silicon polymer (Bartzoka et al., 2000; Zelisko et al., 2008). Con-
versely, small surfactants, depending on their HLB (hydrophilic
lipophilic balance), can be applied to the formulation of O/W or W/O
emulsions. This also explains why double emulsions (W1 /O/W2 )
formulated with biopolymers contain an oil-soluble surfactant to
prepare the primary W1 /O emulsion. The oil-soluble surfactant
commonly used in food emulsions is PGPR (polyglycerol ester of
polyricinoleic acid). The biopolymer can be incorporated in the pri-
mary emulsion, either to gelify the aqueous internal phase W1 as
with gelatine (Sapei et al., 2012) or to decrease the concentra-
tion of the oil-soluble surfactants as with sodium caseinate (Su
et al., 2006). Biopolymers are also used as external emulsifiers:
sodium caseinate (Su et al., 2006), sodium caseinate–dextran con-
jugates (Fechner et al., 2007), a modified gum arabic (Su et al.,
2008), xanthan + whey protein isolate (Benichou et al., 2007a)
carboxymethylcellulose + whey protein isolate (Murillo-Martinez
et al., 2011), low-methyl pectin + whey protein isolate (Murillo-
Martinez et al., 2011), mesquite gum + maltodextrin + whey protein
concentrate (Carillo-Navas et al., 2012), etc. O1 /W/O2 double emul-
sions have been less studied although Benichou et al. (2007b) have
reported that it was possible to stabilize the inner oil droplets
with xanthan–whey–protein isolate hybrids, whereas Abil EM90
(a hydrophobic polyether–polysiloxane block copolymer) was used
as the external emulsifier.
4.1.3. Comparison of emulsion stabilizing properties
Low molecular weight surfactants are more effective than
biopolymers at producing emulsions with small droplets – owing to
kinetic and structural aspects, as previously mentioned. Obtaining
globules with a small diameter and with a narrow size distribu-
tion is an efficient way of reducing destabilization mechanisms
such as Ostwald ripening and creaming. However, the long-term
stability of an emulsion is mostly influenced by flocculation and
coalescence. The biopolymer segments that remain suspended
into the aqueous phase provide an efficient steric stabilization of
the oil droplet. Indeed, the strength and the range of the repul-
sive interactions between two adjacent droplets are important
factors. The minimum interdroplet separation is primarily deter-
mined by the physical space occupied by the adsorbed molecules,
except at very low ionic strength. The relative thickness of the
thin films between closely approaching oil droplets is classified
in ascending order: monomeric surfactant molecules, proteins and
hydrocolloids (Dickinson, 2009). Stability against droplet coales-
cence also depends on the mechanical properties of the interfacial
film. In this respect again, biopolymers are more efficient than
low molecular weight surfactants. For example, some high mole-
cular weight biopolymers that slowly adsorb at the interface and/or
decrease the interfacial tension to a small extent only, still confer
the interface very good viscoelastic properties allowing stabiliz-
ing emulsions (Bouyer et al., 2011). Moreover, the layer-by-layer
deposition method is an efficient way to protect globules against
environmental stresses (heating, ionic strength, pH etc.). However,
the stability of such emulsions is highly dependent on the amounts
of the biopolymers used. Indeed, an incorrect ratio between the
layer components can lead to bridging flocculation (if the amount of
biopolymer does not allow a full coverage of the globules) or deple-
tion flocculation (if there is an excess of unabsorbed biopolymer
or of unabsorbed soluble complexes). When the formulation and
process conditions are carefully controlled, it is possible to obtain
emulsions with improved stability compared to those stabilized
with small surfactants. From an application standpoint, another
interest of biopolymers is that thanks to their viscosifying pro-
perties, they simultaneously stabilize emulsions and control their
texture. It is therefore possible to obtain emulsion gels i.e. soft-solid
materials which are both an emulsion and a gel (Benna-Zayani et al.,
2008; Dickinson, 2012; Weiss et al., 2005).
4.2. Promising applications of biopolymers for the stabilization of
pharmaceutical emulsions
Various biopolymer stabilization mechanisms have been
described, demonstrating the possibility to formulate stable “clean
labelled” emulsions. Indeed, it is possible to completely replace
small-molecule surfactants by biopolymers to formulate O/W
emulsions with increased stability. In double emulsions, the
amount of small-molecule surfactants can only be reduced by the
use of biopolymers, as previously explained. In the modern context
of health and environmental concerns and sustainable develop-
ment, biopolymers such as proteins and polysaccharides could thus
be a good alternative to synthetic surfactants in some pharmaceu-
tical formulations, especially for oral and topical applications.
As previously described, various naturally occurring polymers
can be used either alone or in combination for emulsion stabiliza-
tion, and many of them are already used in the pharmaceutical
field for other applications. A wide variety of structures and tex-
tures can be obtained with biopolymers: O/W macroemulsions,
O/W nanoemulsions, O/W/O or W/O/W double emulsions, and
emulsion gels. Nanoemulsions, double emulsions, and emulsion
 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
gels are particularly interesting systems from a pharmaceutical
standpoint. Indeed, nanoemulsions exhibit some potential advan-
tages over conventional emulsions. They display a higher stability
especially to gravitational separation. They can also increase the
bioavailability of encapsulated lipophilic pharmaceutically active
ingredients (Gao et al., 2011; Prakash and Thiagarajan, 2011). The
obtention of nanoemulsions formulated with biopolymers is thus
a major breakthrough. McClements and co-workers (Qian et al.,
2012) have shown that it is possible to obtain nanoemulsions
encapsulating "-carotene, a component that may reduce the inci-
dence of some diseases (some cancers, cardiovascular diseases,
age-related macular degeneration and cataracts). Interestingly, "-
carotene degradation was considerably slower in "-lactoglobulin
stabilized nanoemulsions than in Tween-20 stabilized nanoemul-
sions although further studies are still required to clearly identify
the mechanisms involved. It would thus be very interesting to study
the encapsulation and the release of pharmaceutically active ingre-
dients from such systems. As regards double emulsions, it was
shown that complexes of whey protein isolate and xanthan gum
could stabilize W/O/W emulsions (as external emulsifiers) and act
as an efficient barrier against the release of vitamin B1 entrapped in
the inner aqueous droplets (Benichou et al., 2007a). In their study of
an O/W/O double emulsion stabilized with whey protein isolate and
xanthan gum, Garti and co-workers (Benichou et al., 2007b) have
shown that the use of these biopolymers to stabilize the primary
emulsion allows for a high yield of encapsulation of flumethrin® (a
veterinary drug model). Moreover, the absence of monomeric sur-
factant in the intermediate aqueous phase of the double emulsion
prevents uncontrolled release of the entrapped compound through
the micellar diffusion mechanism. Recently, Murillo-Martinez et al.
(2011) have obtained edible films from W/O/W double emulsions
stabilized by protein–polysaccharide complexes. The mechanical
properties of these edible films were comparable to those obtained
from hydrophilic edible films of proteins or polysaccharides, but
with vapour permeability comparable to that of lipid-made edi-
ble films. Those systems can encapsulate an active molecule, and
constitute an attractive form that could prevent destabilization
upon long-term storage and microbacterial development (see next
section). Regarding emulsion gels, it should be possible to design
delivery systems with long-term physical stability (entrapment
of the globules preventing coalescence, flocculation and cream-
ing) and with mucoadhesive properties. Indeed, the mucoadhesive
properties of some biopolymers such as chitosan and hyaluronan
are of particular interest for oral and topical routes of administra-
tion, including vaginal and rectal ones.
The association of proteins and polysaccharides properties to
stabilize emulsions is increasingly studied in order to enhance
emulsion stability. Depending on the association method of
biopolymers (covalent, electrostatic, no interaction), the obtained
properties are different and open various promising application
perspectives. Covalent and electrostatic protein–polysaccharide
associations protect emulsions against destabilization under unfa-
vorable environmental conditions (i.e. heating, freezing, high
electrolyte concentrations, lipid oxidation, etc.) (Aoki et al., 2005;
Gu et al., 2005, 2007; Guzey and McClements, 2006a; O’Regan
and Mulvihill, 2010). The most promising stabilization technique
is most probably the layer-by-layer deposition. This method offers
many perspectives regarding the targeted delivery of oil soluble
compounds. A potential benefit of multilayer emulsions as deli-
very systems is that they can be formulated entirely from natural
excipients (oil, proteins, polysaccharides) using simple processing
operations (homogenization, mixing). These biopolymer coatings
can be designed to improve the stability of delivery systems to
environmental stresses, to protect encapsulated compound from
chemical degradation, and to release encapsulated components in
response to specific environmental triggers. For example, Li et al.
373
(2010) have shown that the physical stability of oil droplets coated
by multilayer biopolymers is largely determined by the electrical
characteristics of the outer biopolymer layer. If a delivery system
must remain stable under acid conditions (i.e. in the stomach),
but breakdown at neutral pH (i.e. in the intestine), then a cationic
outer coating of chitosan could be chosen. On another hand, if a
delivery system must remain stable under neutral conditions but
breakdown at acid pH, then an anionic outer layer of alginate or
pectin could be used. This shows that it is possible to achieve a
site-specific delivery of an oil-soluble compound contained in an
emulsion. More recently, Benjamin et al. (2012) have shown that
alternative layers of "-lactoblobulin and pectin around oil droplets
were very efficient to control the release of encapsulated volatile
compounds using pH and salt as release triggers. A review on the
use of structured emulsion-based delivery systems to control the
digestion and release of lipophilic components has recently been
written by McClements and Li (2010). This knowledge can be used
to select the most appropriate emulsion-based delivery system for
specific pharmaceutical applications, such as encapsulation, con-
trolled digestion, and targeted release.
Grigoriev et al. (2008) also used the LBL technique to encap-
sulate an emulsion. They performed a layer-by-layer deposition
on a liquid core using synthetic polymers such as poly(sodium
4-styrenesulfonate and poly(diallyldimethylammonium chloride)
to form loaded micro- and nano-containers. The droplet emul-
sion sizes were in the order of 2 !m. This novel approach is rather
general and could be extended to biopolymers and to the encapsu-
lation of a broad range of pharmaceutical emulsions comprised of
various hydrophobic substances and oil-soluble compounds.
4.3. Challenging issues raised by biopolymers in the field of
pharmaceutical emulsions
It is important to note that the use of natural polymers also raises
some challenging issues that will have to be addressed before their
current use in the pharmaceutical field.
First, the variability of biopolymer composition depending on
its source and origin is of importance and may be a problem for
the reproducibility of the formulations. Indeed, composition vari-
ations may influence the interfacial properties of biopolymers and
their rheological behaviors, among others. However, this issue has
already been addressed for some polymers such as HPMC, pectin,
gelatin, etc., which are already available in pharmaceutical grades.
Second, most of these polymers are currently used in the food
industry and their ingestion is safe. However, the use of some nat-
ural polymers can raise allergenicity concerns. For instance, whey
proteins such as "-lactoglobulin are well known to cause food aller-
gies. The route of administration is important. Indeed, for topical
use "-lactoglobulin might not induce allergic reactions, as it would
remain in the stratum corneum. Complementary studies are thus
required. Moreover, Mouécoucou et al. (2004) have shown that the
protein to polysaccharide association is likely to reduce the IgG/IgE
binding of the protein and thus its allergenicity. In addition, in the
framework of topical administration, the high molecular weight of
the protein–polysaccharide complex could also be an advantage to
prevent its skin permeation. Other polymers, such as chitosan or
hyaluronan, will need to be further studied and evaluated as to oral
ingestion safety. The safety of chitosan for its use in food has already
been recognized in the U.S. (Baldrick, 2010). These safety and risk-
benefit preoccupations will also have to be taken into account for
other delivery routes (dermatologic, ocular, colonic, rectal, etc.)
when these natural biopolymers are part of formulations.
Third, as already mentioned, the obtained droplet sizes seem
generally to be in the order of 1 !m or more with either broad
or narrow distributions. This will necessarily also influence the
delivery route, as it would not be safe to use them for parenteral
374 E. Bouyer et al. / International Journal of Pharmaceutics 436 (2012) 359–378
administration (except for the newly developed nanoemulsions).
For this reason, biopolymer stabilized emulsions seem to be better
designed for oral or topical administration.
Finally, because of their organic origin, there are bacteriological
and storage concerns. The addition of antimicrobial agents may be
necessary. For example, benzoic acid, sodium benzoate, methyl-
and propyl-parabens are currently used in the pharmaceutical field
(Kibbe, 2006a). However, the use of these additives is incompa-
tible with respect to sustainable development concerns. Another
solution consists in lyophilizing (Corveleyn and Remon, 1998; Li
et al., 2008) or spray-drying (Gharsallaoui et al., 2007, 2010a, 2012)
emulsions to avoid their destabilization upon long-term storage.
Starches, gum arabic, whey-protein isolate, gelatin, pectins, and
their associations have shown suitable properties for multi-layered
shell formation by spray-drying (Gharsallaoui et al., 2007). This
process can thus enable the encapsulation of oil-soluble phar-
maceutical compounds. However, heating and evaporation of the
solvent necessary for the sedimentation/crystallization of poly-
mers layers may also cause chemical transformations in the core.
That is why another currently studied method is the formation of
the solid layers around the oil droplets by chemical reactions. Low
methoxy pectin microcapsules with hydrophobic core can thus be
formed by interfacial reaction (Muhiddinov et al., 2004). In this
study, the authors promoted interfacial film formation by using
either anionic sodium dodecyl sulphate (physical cross-linking
interaction) or cationic benzalkonium chloride (electrostatic
interaction). The obtained microcapsules were smaller than 3 !m.
Again, this approach could be extended to biopolymers.
5. Conclusion
The growing interest and demand of consumers in natural pro-
ducts incites industrials to invest in this direction. The present
review reveals the potentialities of biopolymers for the formula-
tion and stabilization of pharmaceutical emulsions. Indeed, due to
the versatility and large number of available biopolymers, protein
and/or polysaccharide stabilized emulsions appear to be a very
promising approach for pharmaceutics in the same way as they
already are in the food industry. However, further research work
is still needed to meet the specific requirements of this new field
of applications. For examples, formulation feasibility, interactions
with active molecules, and possible influences of biopolymers on
drug bioavailability and pharmacokinetics must be studied. It is
worth to note that the formulation and stability control of the
obtained emulsions require a thorough knowledge of the phys-
ical chemistry of these systems. Recent techniques have led to
great progress in understanding the mechanisms of stabilization
and consequently in optimizing the formulation process.
Acknowledgement
We thank Dr. Christine Vauthier for her useful remarks and
advices for the review redaction.
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