the case of cationic emulsions

Abstract
Chemistry and Physics of Lipids 131 (2004) 1–13
Extensive surface studies help to analyse zeta potential data:
the case of cationic emulsions
Laura Rabinovich-Guilatt a,b , Patrick Couvreur a , Gregory Lambert b ,
Danny Goldstein c , Simon Benita c , Catherine Dubernet a,∗
a UMR CNRS 8612, School of Pharmacy, Université Paris Sud, 5 rue JB Clément, Châtenay Malabry Cedex 92296, France
b Novagali SAS, Evry 91000 France
c School of Pharmacy, Hebrew University of Jerusalem, Jerusalem 91120, Israel
Received 31 October 2003; received in revised form 15 March 2004; accepted 19 March 2004
Available online 7 June 2004
The present study is aimed to characterize the electrostatic parameters of oil in water emulsion droplets composed of MCT
(medium chain triglycerides), PL (phospholipids) and Poloxamer and containing increasing concentrations of the cationic lipid
oleylamine (OA), in Hepes 20 mM pH 7.4. The initial ζ-potential data suggesting saturation of the droplet surface at high OA
concentrations were completed by supplementary analysis: the distribution of the oleylamine within the droplet was determined
by reacting the amino groups with the hydrophilic TNBS (trinitrobenzenesulfonic acid), the method being initially standardised
with vesicles. In addition, surface potential and pH at the droplet surface were monitored by the pH-sensitive fluorophore
4-heptadecyl-7-hydroxycoumarin. Our results demonstrate that almost all the OA is localised and fully ionised at the droplet
surface for all concentrations and that the observed plateau in the ζ-potential values obeys the Gouy–Chapman theory of ion
condensation. It is also shown that the slipping plane separation as estimated by the Eversole–Boardman equation is higher that
the expected values of 0.2 nm as a result of the relative position of the fluorophore and the outer boundary of the lipid interface
thickness and the Poloxamer anchored at the interface only plays a minor role.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cationic emulsion; Oleylamine; Poloxamer; Surface pH; Surface potential; ζ-Potential
1. Introduction
Emulsions are widely used in pharmaceutical practice
as they allow to entrap large doses of lipophilic
substances. They are convenient for all routes of
∗ Corresponding author. Tel.: +33-1-46-83-53-86;
fax: +33-1-46-61-93-34.
E-mail address: catherine.dubernet@cep.u-psud.fr (C. Dubernet).
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2004.04.003
administration (Benita, 1999; Klang et al., 1994;
Stevenson et al., 2000), they decrease inter and
intra-individual absorption variations when given
orally (Gershanik and Benita, 2000), they are stable
(Min-Woo et al., 2001), can be easily sterilised
(Groves and Herman, 1993) and have a relatively low
cost of production.
More recently, cationic emulsions have received
increased attention as potential drug deliv-

2 L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13
ery systems as they succeeded to show improved
electrostatically-induced stability and enhanced in
vivo absorption, probably due to the increased interactions
with the biological membranes (Gershanik
et al., 2000; Yang and Benita, 2000). In addition,
they permit to condense negatively charged macromolecules
as DNA or oligonucleotides (Kim et al.,
2003; Teixeira et al., 2001, 2003).
In this context emulsions containing triglycerides
as the oil phase, phospholipids (PL) and Poloxamer
as emulsifiers and a lipophilic cation such as stearylamine
(SA) or oleylamine (OA) are developed as potential
drug carriers (Benita, 1999; Pongcharoenkiat
et al., 2002; Yang and Benita, 2000). The influence
of the amine on the emulsions characteristics is therefore
an essential aspect for this technology, as it was
demonstrated that the surface charge might affect
physical and chemical stability (Grit and Crommelin,
1993; Zuidam and Crommelin, 1995) and future encapsulation
of active molecules (Pongcharoenkiat
et al., 2002).
This is the reason why it was urgent to examine
the effects of increasing amounts of cationic
lipid on the electrostatic properties of the emulsion
surface. Surprisingly, only a few papers deal with
surface properties in cationic emulsions (Kim et al.,
2003; Min-Woo et al., 2001; Pongcharoenkiat et al.,
2002; Teixeira et al., 2001), most of them limiting
their research to ζ-potential measurements and
none of them presenting a complete analysis of the
issue in respect to the cation concentration. More
sophisticated studies were performed only in liposomes
(Meidan et al., 2000; Zuidam and Barenholz,
1997). While standard ζ-potential measures are usually
satisfactory for industrial purposes, they are far
from being adequate for a full comprehension of the
surface.
The aim of this study was therefore to investigate
and fully evaluate the effect of increasing amounts
of the cationic lipid oleylamine in the electrostatic
properties of the emulsion. In order to perform such
investigation, experimental methods existing previously
for liposomes were adapted to our system and
new approaches have been developed. These tools
allowed us to achieve a comprehensive analysis of
the ζ-potential data discriminating between surface
saturation, ionisation saturation and shear plane shift
effects.
2. Experimental procedures
2.1. Materials
Oleylamine was developed conjointly by Novagali
and Sigma. It was synthesised from vegetal oleic acid
and contained at least 85% oleylamine (95% total
primary amines content), in contrast to the commercially
available one which contains 98% total primary
amines but among which only 70% are oleylamine.
Lipoid E-80 was purchased from Lipoid Gmbh
(Ludwigshafen, Germany). According to the manufacturer,
the mixture is composed of 80% phosphatidylcholine
(PC), 8% phosphatidylethanolamine (PE)
and 3% lysophosphatidylcholine (lyso-PC) with an
averaged molecular weight of 707 g/mol. Poloxamer
188 (a triblock copolymer of poly(ethylene oxide)80poly(propylene
oxide)27-poly(ethylene oxide)80) was
purchased from BASF (Ludwigshafen, Germany),
MCT (medium chain triglycerides) from the Société
des Oléagineux (France) and TNBS (trinitrobenzene
sulfonic acid) solution (5%, w/v) was obtained
from Sigma (MO, USA). HC (7-heptadecyl-4hydroxycoumarin)
was bought from Molecular Probes
(OR, USA), dissolved in tetrahydrofuran (50 mM)
and stored in the dark at −20 ◦ C until used.
All other used reagents and solvents were of analytical
grade. Water was purified by reverse osmosis
(MilliQ, Millipore ® , MA, USA).
2.2. Cationic emulsion preparation
The submicron emulsions were prepared according
to a previously described procedure (Klang et al.,
1994). Briefly, the oil and aqueous phases were prepared
separately and heated to 75 ◦ C, then mixed
and stirred with a magnetic stirrer. Final emulsions
were obtained after homogenisation with an IKA
Ultraturrax T-25 (Staufen, Germany) for 5 min at
9500 rpm and a M110S Microfluidizer ® (Microfluidics,
MA, USA) for four cycles at 4 bar pressure.
The formulations consisted of: MCT 149 mM, Lipoid
E-80 28 mM, OA 0–18.7 mM, Poloxamer 2 mM,
-tocopherol 0.5 mM and glycerol 100 mM.
A set of emulsions without Poloxamer was similarly
prepared in order to study the influence of
this compound on the surface characteristics of the
emulsions.

2.3. OA liposomes preparation
L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13 3
Small unilamellar vesicles (SUV) were obtained by
the ethanol injection method as described elsewhere
(New, 1990). 750 L of an ethanolic solution containing
the lipids were rapidly injected into 10 ml of the
continuously stirred aqueous medium through a fine
needle. The ethanol was subsequently removed from
the bulk medium by extensive dialysis. The liposomes
consisted of 2.8 mM Lipoid and 0–0.93 mM OA, i.e.
10-fold more diluted than the emulsions.
2.4. Ionic strength determination
The ionic strength of the solutions used for ζ-potentials
and pH surface measurements was determined by
the freezing point depression method with a cryoscope
Fiske Mark 2 (Advanced Instruments, USA).
2.5. Size and ζ-potential measurements
The mean particle size of the emulsions and liposomes
was determined by quasi-elastic light scattering
after dilution in water at 20 ◦ C using a Nanosizer
Coulter N4 (Beckman Coulter, CA, USA).
The electrophoretic mobility was measured at 25 ◦ C
in a Malvern Zetasizer 4 (Malvern Instruments, UK)
following a 1/400 dilution in buffer Hepes 20 mM pH
7.4. The mobilities were converted into ζ-potential
values through Henry’s equation using f(Ka) values
of 1.43, as calculated according to Ohshima (1994)
(for an ionic strength of 0.013 and a particle radius of
75 nm).
The mean and standard deviation of at least three
different sets of emulsions are presented for each OA
concentration.
2.6. Oleylamine distribution
The evaluation of the distribution of the oleylamine
between the internal core and the surface of the emulsion
droplet (or liposomes) was performed using the
TNBS (trinitrobenzene sulfonic acid) method, modified
from that proposed by Barenholz to localise the
amino groups of phosphatidylethanolamine (PE) in liposomes
leaflets (New, 1990). Essential changes were
introduced in this method concerning the temperature
and pH of the reaction, as discussed later.
2.6.1. Total amine content determination
Fifty microliter of a 1:10 or 1:40 diluted emulsion
(according to the OA content) or 50 L of undiluted
liposome were added to 20 L of water and solubilized
by incubation (50 ◦ C, 30 min) with 40 L of buffer
Hepes 0.8 M (pH 7.4 or 8.5) containing 10% (w/w)
Triton X-100. The samples were cooled to 4 ◦ C before
20 L of TNBS 2.5 mg/ml were added and the reaction
held for 30 min over ice or at room temperature. After
the reaction was stopped by the addition of warm HCl
1.5 M (40 L, 50 ◦ C), the samples were incubated for
further 30 min at 50 ◦ C (in order to respect the same
treatment for all). The absorbance was read at 405 nm
in a microplate reader (Multiskan MS, Labsystems,
USA) after the samples had reached room temperature.
2.6.2. External amine content determination
Twenty microliter of water and 40 L of buffer
Hepes 0.8 M (pH 7.4 or 8.5, at 4 ◦ C) were added to
50 L of a 1:10 or 1:40 diluted emulsion or to 50 L
of undiluted liposome suspension. The reaction was
started by adding 20 L of TNBS 2.5 mg/ml and continued
for 30 min over ice or at room temperature.
Addition of warm HCl 1.5 M (40 L, 50 ◦ C) containing
10% w/w Triton X-100 stopped the reaction and
further incubation at 50 ◦ C (for 30 min) allowed to
solubilize the samples. The samples were analysed as
described above.
The extent of external amine labelling (Ext) and of
total amine labelling (Tot) were assumed to be proportional
to the absorbance increase at 405 nm, measured
after exposure for 30 min to TNBS. The relative
external distribution was calculated from Ext (%) =
Ext/Tot.
The presented results stand for mean and standard
deviation of at least three independent experiments.
2.7. Surface pH
Until now, all published work concerning the surface
pH of liposomes or micelles was mainly done using
the pH-dependent lipophilic probe 4-heptadecyl-7hydroxycoumarin,
HC (Fernandez and Fromherz,
1977; Kraayenhof et al., 1993; Zuidam and Barenholz,
1997). HC has an alkyl chain that anchors into the
phospholipid membrane, leaving the fluorophore hydroxycoumarin
embedded in the lipid headgroup domain.
The molecule has an isosbestic peak at 330 nm

4 L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13
(emitting at 385 nm) and a pH-dependent peak at
380 nm (emitting at 453 nm) (Pal et al., 1985).
2.7.1. HC Incorporation
Before measuring surface pH, preliminary experiments
were done in order to assess effect of the probe
concentration at the membrane. In this view, HC was
incorporated into the cationic emulsions at 1:10, 1:50,
1:200, 1:400 and 1:1000 HC:PL molar ratio by incubation
(2 h at 50 ◦ C) of 50 L of the emulsion with
not more than 2.5 L of the HC in THF solution (Pal
et al., 1985).
Surface pH was finally measured in emulsions
containing similar HC:PL molar ratio as in previously
published liposomes studies, i.e. 1:200 or
1:400 (Kraayenhof et al., 1993, 1996; Zuidam and
Barenholz, 1997).
2.7.2. Fluorescence measurements
The HC-labelled preparations were diluted 1:1000
in 1 ml of buffer Hepes 20 mM at different pH, such
high dilution factor being necessary because of the
turbidity of the emulsions. The fluorescence measurements
were performed at room temperature in a
LS-50B luminescence spectrometer (Perkin Elmer,
MA, USA) by scanning the excitation wavelength
between 300 and 400 nm at an emission wavelength
of 450 nm (slit 5 nm). An emission filter of 430 nm
was added.
The dissociation degree of the HC incorporated
into the phospholipid layer could be monitored by the
ratio of the excitation fluorescence intensities at 380
and 330 nm (380/330) against the pHbulk. To simplify
the comparison between the different curves, the dissociation
degree of HC was calculated assuming that
100% dissociation corresponds to the maximum value
of 380 nm/330 nm as done by Zuidam and Barenholz
(1997).
3. Results
3.1. Size and ζ-potential
The mean droplet diameter of the emulsions was
not affected by the OA content and varied from 140
to160 nm showing an unimodal distribution (polydispersity
index:

O.D.
0.70
0.60
0.50
0.40
0.30
0.20
0.10
50%
L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13 5
0.00
0.00
(A) 0mM OA 3.7mM OA 18.7mM OA (B)
80%
95%
0.70
0.60
0.50
0.40
0.30
0.20
0.10
32%
57%
61%
0mM OA 3.7mM OA 18.7mM OA
Fig. 2. Absorbance o.d. values for the total (grey) and external (white) amino groups within liposomes at (A) room temperature and (B)
4 ◦ C as determined by the modified TNBS method at buffer Hepes pH 8.5. The percentage correspond to the calculated fraction of total
NH2 groups situated at the surface. Mean ± S.D.; n = 3.
cle relative to the total amount (Litman, 1973). Levy
et al. (1994) were the first to adapt this technique
to localise PE in negatively charged emulsions. All
these studies were done at room temperature and at
pH 8.5, conditions at which the amino groups are
unprotonated and fully reactive.
Concerning SUV liposomes containing OA, experiments
performed in the standard conditions (room
temperature, pH 8.5) led to Ext (%) of 80–95%,
whereas when the reaction was held at 4 ◦ C this
value dropped to 57–61%. In non-cationic liposomes,
whether the reaction was held at room temperature or
at 4 ◦ C, 50–32% of the NH2 groups originated from
the phosphatidylethanolamine (PE) were located at
the external leaflet, respectively (Fig. 2).
As for emulsions, the reaction was not only carried
out at pH 8.5 but also at pH 7.4 corresponding to the
bulk pH of the emulsion in physiological conditions.
Fig. 3 shows the evolution of Ext (%) for increasing
OA contents at both pH and 4 ◦ C that was identified
as the optimal temperature limiting the flip-flop of the
interfacial lipids (as will be further discussed). It is
clearly seen that while at pH 7.4 almost all the NH2
were localised facing the water phase for all OA concentrations,
at pH 8.5 their relative amount decreased
to 67% for the highest OA content.
3.3. Surface pH
To our knowledge, pHsurface measurements using
HC has never been done in oil/water submicron
emulsions.
In a preliminary study, the influence of HC on
the emulsion surface properties was evaluated independently
through pHsurface and ζ-potential measurements.
While the ζ-potential failed to show any influence
at all tested HC:PL molar ratios (data not shown),
the HC dissociation degree curve along the pHbulk at
the further applied range suggested a modification of
the profile for the highest probe concentration (HC:PL
1:10). Fig. 4 shows a 9.3 mM OA emulsion as an example
and similar behaviour was observed for other
OA contents. Thus, molar ratios of 1:200 and 1:400
were used indistinctly for further experiments.
The titration curves of the cationic emulsions containing
1:200 or 1:400 HC:PL molar ratio (Fig. 5)
External NH2 (% of total)
120%
100%
80%
60%
40%
20%
0%
0 2 4 6 8 10 12 14 16 18 20
Total OA content (mM)
Fig. 3. Distribution of the amino groups within the emulsion droplet
at 4 ◦ C as determined by the new TNBS method at buffer pH 8.5
() and pH 7.4 (). The external NH2 fraction was calculated
as the ratio between external and total amino content (see Section
2 for details). Mean ± S.D.; n = 3.

6 L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13
HC dissociation degree (%)
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
1:10
1:50
1:200
1:400
2 3 4 5 6 7 8 9 10 11 12 13 14
pH bulk
Fig. 4. Influence of HC:PL molar ratio on the emulsion surface
properties: titration curve of a 9.3 mM OA emulsion containing
HC incorporated at 1:10, 1:50, 1:200 and 1:400 HC:PL molar ratio
(similar profiles were obtained for other OA contents), in 20 mM
Hepes buffer.
exhibited sigmoidal characteristics similar to those
reported for cationic liposomes, although much less
steep (Zuidam and Barenholz, 1997). At a pHbulk of
7.4, the degree of ionisation of HC was found to be
3, 7, 13, 26 and 40% for 0, 1.9, 3.7, 9.3 and 18.7 mM
OA emulsions, respectively. Assuming that the pHbulk
= pHsurface for the neutral emulsion, the titration curve
of the emulsion without OA can be taken as a reference
to establish the correspondence between surface
pH and the degree of ionisation of HC. Hence, it could
be concluded that for a pHbulk of 7.4, the pHsurface was
Table 1
Electric properties of emulsion lipid surfaces diluted in 20 mM Hepes, pH 7.4
OA
(mM)
Ext a
(%)
Ion b
(%)
OAsurf +c
(mM)
pH
surface d
pK HC
app e pK OA
app f σ HCg
(C/m 2 )
ψ HCh
(mV)
ζ i
(mV)
σtheorj 0
(C/m2 )
0 84 – 0 7.4 k 10.6 11.4 l 0 k 0 k 0 0 k 0 k
1.9 96 100 1.8 8.5 9.4 10.2 0.016 73 38 0.024 92
3.7 91 100 3.4 9.0 9.1 10.0 0.022 87 47 0.044 122
9.3 91 97 8.3 9.6 8.1 9.0 0.070 145 64 0.096 162
18.7 87 94 15.3 10.0 7.7 8.6 0.105 167 67 0.162 188
ψtheorg 0
(mV)
a Percentage of external NH2 groups within the droplet, obtained by the TNBS method.
b Percentage of ionised OA at the droplet surface, calculated from Eq. (4) exploiting pHsurface results and data from Ptak et al. (1980).
c External OA ionised at the surface, obtained from the combination of footnotes a and b.
d From HC dissociation curves.
e From HC dissociation curves and Eq. (1).
f From Eq. (2) and data from Ptak et al. (1980).
g From Eq. (3).
h From Eq. (2).
i Normalized to the non-cationic emulsion value.
j Estimated from OAsurf + and the molecular area of oleic acid in phospholipid monolayer as assessed by Levy et al. (1991).
k Assumed to be neutral at pHbulk 7.4.
l Extrapolated value of pK OA in a neutral lipid membrane.
HC dissociation degree (%)
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0mM OA
1.9mM OA
3.7mM OA
9.3mM OA
18.7mM OA
2 3 4 5 6 7 8 9 10 11 12 13 14
pH bulk
Fig. 5. Titration curve expressing the relative dissociation degree
of HC against the pHbulk for emulsions containing 0, 1.9, 3.7, 9.3
and 18.7 mM total OA concentration, in 20 mM Hepes buffer.
8.5, 9.0, 9.6 and 10.0 for the 1.9, 3.7, 9.3 and 18.7 mM
OA content cationic emulsions, respectively (Table 1).
The apparent pKa of HC at the surface (pKHC app ) could
be calculated from linear regression of the modified
Henderson–Hasselbalch equation (Eq. (1)) (Babcock,
1983):
pHbulk = pK HC
D − Dmin Ia
app + A log
+ log
Dmax − D Ib
(1)
where D represents the dissociation degree of
HC (Dmin and Dmax being minimal and maximal

L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13 7
dissociation values), A the stoechiometry of the probe
protonation (theoretically 1), and Ia and Ib are the fluorescence
intensities at the isosbestic point in acidic
and basic medium, respectively (for a perfect isosbestic
point Ia/Ib = 1). Therefore, A corresponds to
the slope and pKHC app to the intercept of the linear regression.
As will be discussed later, only the bottom
part of the HC dissociation curve was employed for
the calculations.
When correcting for log I1/Ib (which was close to
0 as predicted), the values found for pKHC app were 10.6,
9.4, 9.1, 8.1 and 7.7 for the 0, 1.9, 3.7, 9.3 and 18.7 mM
OA emulsions, respectively (Table 1). The slope A
(representing the stoechiometry of the HC protonation)
ranged between 1.2 and 1.3.
The experimental surface potential (ψHC ) could be
estimated according to Eq. (2), assuming that only
the surface charge was influenced by the presence of
the cation, without any influence on the membrane
polarity (Grit and Crommelin, 1993; Kalmanzon et al.,
1989):
ψ HC =− pKel kT ln 10
e
= (pKcharged app − pKneutral app )kT ln 10
(2)
e
where k is the Boltzmann constant (1.38×10−23 J/K),
T the absolute temperature, e the electron charge (1.6×
10−19 C) and pK charged
app and pKneutral app are the pKapp’s
of HC in charged and neutral emulsions, respectively.
The obtained values were 73, 87, 145 and 167 mV for
the ascending OA concentrations (Table 1).
The surface charge densities (σ) presented in
Table 1 were derived from the Gouy–Chapman theory
(Eq. (3)):
ψ = 2kT
ze arcsinh
zσλ
(3)
2εε0(kT/e)
where z is the valency of the counterions (1), λ the
Debye screening length (1.65×10−9 m), ε0 the relative
permittivity of free space (8.85 × 10−12 C2 /(N m2 ))
and ε is the dielectric constant at the surface (30 as
derived from Cevc (1990)).
3.4. Fraction of OA ionised at the droplet surface
Ptak et al. (1980) found that the pKapp of stearylamine
(pKSA app ) decreased from 10.7 in aqueous solu-
tion to 9.5 in vesicles with a SA:PL ratio of 0.19. Assuming
that stearylamine behaved similarly to oleylamine
(OA differs from SA only by one unsaturated
bond), such a cation:PL ratio would be equivalent to a
5.3 mM OA emulsion. It was calculated that such an
emulsion had a pHsurface of 9.2 and a surface potential
of 114 mV (estimated from data of Table 1). Knowing
the pK charged
app of SA (9.5) and the respective surface
potential, it was possible to calculate the pKneutral app
of the lipoamine for the neutral and different charged
surfaces (Eq. (2)). These pKSA app were supposed to be
equal to the pKOA app for the same cation:PL ratio. In this
way, values of pKOA app of 10.2, 10.0, 9.0 and 8.6 may
be calculated for the 1.9, 3.7, 9.3 and 18.7 mM OA
emulsions, respectively and extrapolated to a pKOA app of
11.4 for OA in a neutral lipid environment (Table 1).
The next step was the calculation of the level of
the surface OA ionisation, which was obtained from
the non-modified form of the Hendersson–Hasselbalch
equation (Eq. (4))
pH = pKa + log [A− ]
(4)
[HA]
where pH is the pHbulk (7.4 in our case) and pKa is
the pKOA app found for each emulsion. Eq. (4) could be
equally solved employing the different pHsurface’s and
the pKOA app found for the neutral surface. As the surface
becomes more alkaline the fraction of ionised OA
decreased from 100 to 94% of the total surface OA
for the 0 and 18.7 mM OA emulsions, respectively.
Combining the results of this section with those obtained
using the TNBS method, the exact amount of
ionised OA at the droplets surface (OAsurf + ) could
be calculated (Table 1).
3.5. Theoretical charge density and surface
potential
The theoretical surface charge density of the emulsions
was calculated employing data published for
mixtures of oleic acid and phospholipids, assuming
that the molecular area of oleic acid and oleylamine
are similar. In the study by Levy et al. (1991), a mixture
of oleic acid and phospholipids (in a molar ratio
PE:PC 1:3, comparable to Lipoid E-80) had an average
molecular area that decreased linearly from 48 Å 2 for
an oleic acid:PL ratio of 0:1 to 39 Å 2 for a 0.67:1 ratio.

8 L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13
Table 2
Size, ζ-potential and surface potentials of cationic emulsions with and without Poloxamer
OA total content (mM) Diameter (nm) ζ a (mV) ψ HC (mV) δ b (nm)
Poloxamer − + − + − + − +
1.9 216 158 44 37 68 73 0.60 0.95
3.7 208 150 61 45 109 87 0.63 0.85
9.3 205 149 71 61 146 145 0.66 0.85
18.7 204 145 77 65 181 167 0.66 0.84
a Normalized to the non-cationic emulsion value, in mV.
b The distance to the shear plane δ was calculated applying Eq. (5).
The theoretical charge density of the membrane
(σtheor 0 ) calculated employing the amount of charged
molecules (OAsurf + , see above) and their estimated
molecular surface ranged between 0.024 and
0.162 C/m2 (Table 1). The theoretical surface potential
(ψtheor 0 ) that was then derived from Eq. (3)
reached 188 mV for the highest OA content (Table 1).
3.6. Shear plane position
The exact position of the shear (or slipping) plane,
separating the mobile and immobile layers during electrokinetic
measures could be calculated according to
Eversole and Boardman (1941), assuming that the fluorophore
is situated at the surface plane (Fig. 6):
zeζ
zeψHC
ln tanh = ln tanh − κδ (5)
4kT
4kT
κ being the reciprocal Debye length (nm −1 ) and δ is
the distance from the surface to the shear plane.
Table 2 shows the average size, surface and ζ-potentials
for both Poloxamer-containing and Poloxamerlacking
emulsions and the assessed distances δ from
the surface plane to the shear plane. While the
Poloxamer-lacking droplets were somehow larger
than the naked ones both the higher ζ-potential and
lower calculated δ evidenced the shift on the shear
plane induced by the presence of the copolymer at
the surface of the studied emulsions (Fig. 6).
4. Discussion
The electrokinetic or ζ-potential is defined as the
average electrostatic potential existing at the hydrodynamic
plane of shear, somewhere between the Stern
plane and the end of the diffuse layer and normally
considered to be 0.2 nm from the surface (Cevc, 1990;
Eisenberg et al., 1979; Kraayenhof et al., 1996). This
concept imposes the definition of the surface plane,
which is still ambiguous in the majority of papers
Interface
Shear plane
Surface
plane
H3N O
N
O
O P O
O
O
O
Fig. 6. Proposed arrangement of the phosphatidylcholine, oleylamine,
4-heptadecyl-7-hydroxycoumarin and Poloxamer 188 in
the emulsion droplet membrane. The fluorophore moiety of the
HC is aligned with the phosphoryl groups as proposed by Kachel
et al. (1998) and Kraayenhof et al. (1993), the plane of the amine
of the OA is below that of the choline group, pushing it out into
the aqueous phase (Scherer and Seelig, 1989) and the hydrophobic
portion of the Poloxamer is inserted into the acyl portion of
the membrance (Kostarelos et al., 1999).
O
O
O
O

L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13 9
dealing with surface properties in lipid model membranes.
Indeed the headgroups of the lipid molecules
create an interface of 0.5–1 nm thick which is a polar
zone separating the medium from the more hydrophobic
regions as the alkyl chains (Cevc, 1990). In this
paper the alignment plane of the phosphoryl groups of
the PC will be considered as the surface plane, with
the amino groups of the OA and the fluorophore moiety
of the HC at the same level (Fig. 6).
Measurement of ζ-potential is currently the simplest
and more straightforward way to characterize the
surface of charged colloids and conclusions are easily
drown from the analysis of its data concerning concentration,
distribution, adsorption, ionisation, exposure
or shielding of charged moieties (Delgado, 2002).
The ζ-potential values of the OA-emulsions (in
20 mM Hepes pH 7.4) as depicted in Fig. 2 were
lower than those reported previously by Klang et al.
(1994) for a 11 mM SA-emulsion. However, these
last measures were done in 1 mM NaCl which probably
accounts for this difference as the decrease of the
ζ-potential with increased salinity is predicted by the
classical electrokinetic theory (Delgado, 2002).
The combination of Gouy–Chapman (Eq. (3)) and
Eversole–Boardman (Eq. (5)) equations shapes the
function f(σ0) = ζ into an inverse hyperbolic sinusoidal
curve, with a linear part for low charge
density σ0 values and an apparent plateau for higher
ones. However, in order to confine the behaviour of a
charged colloid into the linear or plateau section, the
charge densities should be known. If a plateau-like
shape is found at low cation concentrations, then phenomena
as surface or ionisation saturation should be
suspected.
Thus, in this case, the plateau observed at OA content
higher than 9.3 mM could stand for these potential
situations: either it was the expected shape or it
evidenced saturation, which could be further differentiated
into surface saturation (with the excess amine
starting to solubilize into the oil core of the droplet)
or ionisation saturation (where even if all the OA
molecules are at the interface, their ionisation is incomplete).
To test these hypotheses, the proportion of amine
localised at the droplet interface has been determined.
For this purpose, a modified TNBS method which derives
from the one used for liposomal characterisation
(New, 1990) was developed and employed to evaluate
the distribution of the cationic lipid within a whole
concentration range of OA-emulsions. It should be
noted, however, that utilising the TNBS to localise
amines such OA introduced new parameters that
needed to be considered. While it is widely known
that flip-flop of phospholipid molecules such PE
across a PL vesicles does not occur rapidly (Homan
and Pownall, 1988), OA presents a less bulky polar
head and a more fluid alkyl chain that facilitates its
general mobility. The flip-flop of fatty acids across
PC bilayers was demonstrated to be extremely fast
with a t1/2 of less than 1 s (Kamp et al., 1995; Zhang
et al., 1996). The flip-flop movement of OA across
the phospholipid bilayer (at pH 8.5 and room temperature),
allowed all the NH2 groups to react with
TNBS independently of their location as demonstrated
for OA-liposomes since an apparent distribution of
80–95% Ext was observed, in contradiction to what
expected (Fig. 2A). Performing the reaction at 4 ◦ C
instead of at room temperature succeeded in reducing
the OA flip-flop (Fig. 2B), with no particular effect
on the reaction yield as already demonstrated (Gruber
and Schindler, 1994). A second parameter to be considered
was the pH of the reaction. In the traditional
TNBS protocol, the addition of the buffer pH 8.5 to
the reaction medium prevents the NH2 from being
protonated thus enhancing the reactivity of the amino
groups. However, in these conditions, a partial solubilization
of this unprotonated OA into the oil core of
the emulsion, equivalent to the flip-flop in liposomes
could not be excluded. Such effect of the protonation
on the translocation kinetics of N-doxyl stearic acids
across PL bilayers has already been investigated by
Yuann and Morse (1999) finding that it was highly
correlated to the degree of ionisation of the molecule.
Performing the TNBS reaction at pH 7.4 instead of at
pH 8.5 resulted obviously in reduced sensitivity because
of the partial protonation and unresponsiveness
of the NH3 + . Nevertheless, the response was still
linear and it was demonstrated that the application of
the method at this pH was feasible.
When applied to OA-emulsions, this modified
TNBS methodology showed that at pHbulk 7.4, about
90% of the OA molecules in the emulsion were situated
at the water/oil interface with the amino groups
facing the water phase (Fig. 3). Since the experiments
were performed at 4 ◦ C, artefacts resulting from a
dynamic equilibrium of the OA molecules between

10 L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13
PL monolayer and the oily core could be minimised,
as explained before. However, at a pHbulk of 8.5, as
the Ext (%) decreased for the 18.7 mM OA emulsion,
an onset of surface saturation and oil-solubilization
could be suspected. This different behaviour at pH
8.5 was probably due to the higher proportion of
non-ionised molecules, more soluble in the oil phase.
Therefore, the surface saturation hypothesis could
be rejected and it was hypothesised that the local pH
(pHsurface) at the interface of the emulsion droplet
could play a major role in the ζ-potential plateau found
for OA contents higher than 9.3 mM. Indeed, an alkaline
microenvironment at the droplet interface may
lead to a partial unprotonation of OA and therefore to
the cancellation of part of its cationic contribution to
the overall charge.
The incorporation of the probe HC into the cationic
emulsions in order to measure the pHsurface introduced
a challenge: along with the OA:PL molar ratio
variation, both HC:OA and HC:PL molar ratio were
varying too. High HC content might influence surface
properties, especially in low OA-content emulsions
while an extremely diluted probe concentration in the
membrane could fail to accurately sense the charge
(Kalmanzon et al., 1989). This is the reason why the
incorporation of HC at a HC:PL molar ratio of 1:10,
1:50, 1:200, 1:400 and 1:1000, resulting in OA:HC
molar ratios ranging from 0.7:1 (lowest OA, highest
HC) to 666:1 (highest OA, lowest HC) has been
tested. While the ζ-potential of all the emulsions was
unaffected by any of the HC relative amounts (data
not shown), a shift in the lower part of the titration
curve was noticed for the highest probe concentration
(Fig. 4). Thus, the HC:PL ratio was fixed to 1:200
or 1:400 as done in previous studies with liposomes
(Kraayenhof et al., 1993; Zuidam and Barenholz,
1997).
The measured pHsurface of the emulsions was found
to be directly correlated to the oleylamine content,
ranging from 8.5 to 10.0 (Table 1). For comparison,
Zuidam and Barenholz (1997) obtained higher
pHsurface values of 10.9 for DOTAP:DOPE 1:1, 11.2
for DOTAP:DOPC 1:1, and 11.6 for DOTAP liposomes.
Accordingly, while at pHbulk 7.4 the HC in
DOTAP-liposomes was 100% ionised, in the 18.7 mM
OA-emulsions it was only 40% dissociated. It should
be noted, however, that the cationic emulsions used in
our study had a maximum molar lipid ratio OA:PL of
Fig. 7. Theoretical titration curved (cut lines) of HC as calculated
from the established pKHC app (Table 1) for emulsions containing 0
(), 1.9 (), 3.7 (), 9.3 (*) and 18.7 () total OA concentration,
compared to the experimental ones (solid lines), in buffer Hepes
20 mM. The insert shows the deviation of the experiment ionisation
value from the theoretical one, reflecting the protonation of the
OA at the interface.
0.67:1, which could easily explain the lower pHsurface
and ψHC values obtained with OA compared with
DOTAP. In the same cited study, the pKHC app found in
neutral DOPC and DOPC:DOPE liposomes was 10.5
and 10.7, very close to the value of 10.6 obtained in
the present study with the neutral emulsion.
The dissociation curves of the OA-emulsions
differed from previously published ones in their
steepness: while in DOPE or DOPC liposomes the
ionisation of HC was complete within 4 pH units
(Zuidam and Barenholz, 1997), in the emulsions it
took a range of 5–7 pH units to fully ionise the probe
(Fig. 5). Since Lipoid E-80 is a mixture of PL containing
8% of PE and 3% of lyso-PL, it is likely that
these PL became ionised together with the HC along
the pH titration range, resulting in a less steep profile.
Interestingly, as the OA content increased a biphasic
pH-dependent dissociation curve of HC was observed
(Fig. 5), similar to that reported by Zuidam and
Barenholz (1997) for DC-CHOL/DOPE liposomes,
which was explained by the deprotonation of
DC-CHOL. It is suspected that at high OA content
and very alkaline pHbulk values, the OA itself became
unprotonated in our emulsions, shaping the dissociation
curve. In Fig. 7 is depicted the theoretical HC
dissociation curves (as calculated using the pKHC app obtained
from bottom part of the experimental dissociation
curve, as if HC were the sole surface molecule to
be titrated). The discrepancy between the theoretical

L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13 11
and the observed values (as displayed in the insert)
shows that as the OA content increased, the maximal
deviation was found at lower pHbulk values, and that
these values corresponded roughly to the found pK OA
app
(Table 1).
By means of the found pHsurface values and pK OA
app ,
the degree of ionisation could be calculated by Eq. (4),
pKOA app being estimated from previously published
studies on SA (Ptak et al., 1980). At pHbulk of 7.4,
the accurate amount of charged molecules at the surface
(OAsurf + ) was 94% (corresponding to 82% of
the total content) for the highest OA:PL ratio. It is
noteworthy that the pHbulk had a tremendous influence
on the amount of effective cationic molecules
at the surface. Indeed, the same 18.7 mM emulsion
diluted in pHbulk 8.5 will only have 67% of its OA
molecules at the surface (Fig. 3) from which only
57% will be in the ionised state, meaning that only
38% of its total cationic content will contribute to
the charge. These findings highlight the interest of a
more accurate characterisation of the interface.
The σHC values were to some extent lower than the
σtheor 0 values (Table 1). This could be explained by
using in the equation molecular areas determined for
oleic acid and extrapolated to oleylamine with possible
different assembly in the PL monolayer.
The important difference detected between the measured
ζ (measured at the slipping or shear plane) and
ψHC (determined by the fluorophore at the surface
plane) suggested that the former was very distant from
the surface (0.84–0.95 nm, Table 2). The Poloxamer
was suspected to be responsible for this phenomenon
as neutral polymers adsorbed at an interface are known
to shift the position of the plane of shear (Barnes and
Prestidge, 2000; Kostarelos et al., 1999). To evaluate
the role of the Poloxamer in the thickness of the stagnant
layer (δ), emulsions without the copolymer were
prepared.
Table 2 shows that the addition of the Poloxamer
during early emulsification process led to smaller
droplet size with a probable incorporation of the hydrophobic
subunit in the acyl chain area rather than
its adsorption to the surface, as demonstrated for liposomes
(Kostarelos et al., 1997, 1999). The presence of
Poloxamer at the droplet surface and the consequent
shift in the shear plane was revealed by the lower
ζ-potential values and the superior δ for the coated
particles. The naked particles exhibited δ values of
0.60–0.66 nm similar to those reported by Kraayenhof
et al. (1993) for pure PC vesicles but significantly
larger than the more widely adopted distance of about
0.2 nm (Eisenberg et al., 1979). The shift in the shear
plane outwards from the surface produced by the
Poloxamer itself was very inferior to the 2 nm reported
by Washington (1997) for perfluorocarbon emulsions
and can be due to its deep immersion in the thick
interface, to a coiled conformation or to other interactions
of the hydrophilic subunits with the surface
as previously reported (Barnes and Prestidge, 2000).
The origin for the values found for δ should then
be searched in the membrane interface structure, as
depicted in Fig. 6. The effective interfacial width
in lipid membranes is 0.5–1 nm owing to the large
headgroups size and motion (Belaya et al., 1994;
Cevc, 1990). In addition, it was demonstrated that
the presence of small cationic lipids in the membrane
pushes the phosphocholine moiety towards the aqueous
medium with the choline nitrogen away from the
surface (Scherer and Seelig, 1989). As the HC has its
fluorophore embedded in the lipid headgroup domain
aligned with the lipid phosphoryl groups (Kachel
et al., 1998, Kraayenhof et al., 1993), the effective
distance between the fluorophore (measuring ψ HC )
and the outer boundary of the interface accounts for
all the interface thickness as illustrated in Fig. 6.
5. Conclusion
In this paper, new approaches and modified methods
for the characterisation of charged colloids in general
and cationic emulsion surface in particular are
presented. This study demonstrates the complexity in
analysing ζ-potential data and the difficulty in relating
it to the cation concentration since phenomena such as
surface saturation, ionisation decrease, ion condensation
and slipping plane thickness are playing a major
role.
With OA containing emulsions up to a OA:PL ratio
of 0.67, it was demonstrated that at pHbulk 7.4
there was neither saturation in the distribution nor in
the ionisation of the OA and that the plateau observed
in ζ-potential was related to the ion condensation described
by the Gouy–Chapman theory. It was also
found that ζ-potential values were considerably lower
than the surface potential measured with the HC ones

12 L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13
as the interfacial thickness resulted in a slipping plane
far away from the fluorophore position. The presence
of Poloxamer at the surface still pushed away the shear
plane but only to a moderate extent.
The practical use of the ζ-potential, especially in
pharmaceutical development cannot be denied since it
allows to conclude on the sign of the charge of particles
and to predict the conditions required for colloid
stability. However, for quantitative studies, the present
work illustrate that cautions conclusions should be
stated unless a complete characterisation of the surface
has been accomplished.
Acknowledgements
We thank the Association Nationale de la Recherche
Technique (ANRT) for supporting Laura Rabinovich-
Guilatt with a CIFRE convention. The authors acknowledge
professor Yechezkel Barenholz from the
Hebrew University of Jerusalem (Israel), Dr. Franck
Artzner and Prof. Dominique Langevin from the Université
Paris Sud (France) for fructiferous discussions.
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