the case of cationic emulsions

the case of cationic emulsions

the case of cationic emulsions


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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.


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


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


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


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


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 (%) =


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


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











L. Rabinovich-Guilatt et al. / Chemistry and Physics of Lipids 131 (2004) 1–13 5



(A) 0mM OA 3.7mM OA 18.7mM OA (B)













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


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)








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 (%)
















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



Ext a


Ion b


OAsurf +c



surface d


app e pK OA

app f σ HCg

(C/m 2 )

ψ HCh


ζ i


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


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 (%)












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,


pHbulk = pK HC

D − Dmin Ia

app + A log

+ log

Dmax − D Ib


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.,


ψ HC =− pKel kT ln 10


= (pKcharged app − pKneutral app )kT ln 10



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




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− ]



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


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

theor 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

theor 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):



ln tanh = ln tanh − κδ (5)



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


Shear plane










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).





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


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,


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


(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


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


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.


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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