Colloids and Surfaces A: Physicochem. Eng.

Aspects 327 (2008) 95102

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Colloids and Surfaces A: Physicochemical and

Engineering Aspects
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Interactions in a nonionic surfactant and chitosan mixtures

I. Pepic , J. Filipovic-Gr
I. Jalsenjak
cic,
Department of Pharmaceutics, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacica 1, PO Box 156, 10 000 Zagreb, Croatia

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

Article history: Interactions in mixtures of nonionic surfactant, Lutrol F127, and cationic polyelectrolyte, chitosan from
Received 20 July 2007 water solution and acetate buffer (pH 6.0 or 6.5 and ionic strength 0.1 and 0.5) have been investigated at
Received in revised form 4 June 2008 two different temperatures (25 and 34 C). Critical aggregation concentration (cac), critical micellization
Accepted 5 June 2008
concentration (cmc) and the minimum area of Lutrol F127 molecule at the air/solution interface (a), were
Available online 13 June 2008
determined using the surface tension measurements. The addition of chitosan to Lutrol F127 solutions
shifted the cac and cmc to higher values; the effects were more pronounced for the cmc and in acetate
Keywords:
buffer. The Lutrol F127 adsorption at the air/solution interface strongly depended on the equilibria in the
Triblock copolymers
Chitosan
bulk-phase, i.e. on the extent of the protonation of chitosan amino groups. The results were discussed in
Nonionic surfactants terms of electrostatic, hydrophobic and intra- and inter-polymer interactions.
Polyelectrolytes 2008 Elsevier B.V. All rights reserved.
Surface tension

1. Introduction groups. An outer shell of hydrated PEO groups surrounds the

PEO/PPO/PEO triblock copolymers are nonionic surface-active
agents with diverse applications (dispersing, antifoaming, emulsi-
fying and solubilizing agents) [1]. In novel drug and gene delivery
systems, PEO/PPO/PEO triblock copolymers are often considered
as functional excipients playing an important and essential role
in formulation [2,3]. They are usually referred to by their trade
names (Pluronics, Lutrol, Monolan, Synperonics and Supronic) or
generic name (poloxamers) [4]. Their aqueous solubility, aggre-
gation behaviour, and applications are strongly dependent on
compositions of the hydrophilic poly(ethylene oxide) (PEO) block
and hydrophobic poly(propylene oxide) (PPO) block, as well as
on the molecular weight of copolymers. Among others commer-
cially available triblock copolymers, Lutrol F127, denoted as F127
(poloxamer 407) has been chosen for this investigation because
of its aggregation behaviour. At low concentrations, F127 exists
in solutions as individual coils (unimers) and thermodynamically
stable micelles are formed with increasing copolymer concentra-
tion.
The micelle formation involving triblock copolymers appears
to be more complex than conventional low molecular weight sur-
factans [1]. The shape of F127 micelles is spherical. It is generally
accepted that the core of the micelles consists of dehydrated PPO
Corresponding author. Tel.: +385 1 46 12 608; fax: +385 1 46 12 691.
jelena.lipovic-grcic@fbf.htnet.hr
E-mail addresses: ipepic@pharma.hr (I. Pepic),
(J. Filipovic-Gr ijalsenjak@gmail.com (I. Jalsenjak).
cic),
hydrophobic core [5,6]. Wanka et al. [5] have studied phase dia-
grams of pluronic copolymer and found that F127 has a relatively
simple behaviour. In the temperature region up to about 37 C, the
copolymer solution is present as an isotropic phase up to approx-
imately 20% of copolymer, and beyond that concentration, the
cubic-phase is present. The solution with copolymer micelles are
expected at more than 10-fold lower concentration of F127. The
copolymer forms micelles when present above the critical micelle
concentration (cmc). Micelle formation is also critically depen-
dent upon temperature, and the temperature at which micelles
are formed is critical micelle temperature (cmt). In F127 solutions,
there is the strong decrease in the cmc with increasing temper-
ature, which can be more than four orders of magnitude with a
temperature increase from 15 to 40 C.
Besides concentration and/or temperature, an important factor
of copolymers aggregation to be considered is their sensitivity to
the presence of additives, since they constitute usual drug delivery
formulations (e.g. electrolytes, low molecular weight surfactant or
drugs). Pandit et al. [7] showed that addition of salting-out elec-
trolytes (Na3 PO4 , Na2 SO4 and NaCl) in F127 solutions decreases
cmt, whereas salting-in electrolyte (NaSCN) has the opposite effect.
The slight changes in the cmc of F127 in the presence of naproxen,
indomethacin [8] and pilocarpine HCl [9] were noticed, whereas
propofol induced micelle formation in aqueous F127 solutions at
lower cmc [10]. The interaction between F127 and nonionic sur-
factants (hexaethylene glycol mono-n-dodecyl ether) resulted in
spontaneous formation of mixed micelles indicating that nonionic
surfactants interacted strongly with both the unassociated and

0927-7757/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfa.2008.06.009
96 I. Pepic et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 327 (2008) 95102
associated forms of F127 [11]. Although several researches have
explored fundamental behaviour of F127 copolymers, as well as the
effect of some additives (electrolytes, drugs and surfactants), little is
known about F127 micellization in the presence of polyelectrolyte
additives.
The physicochemical aspects of F127/polyelectrolyte mixtures
could have fundamental and practical importance (e.g. drug
delivery systems). Since chitosan is a positively charged linear
polysaccharide having numerous pharmaceutical applications (e.g.
pharmaceutical forms and drug delivery systems) [12], it was cho-
sen as an example of a polyelectrolyte. Chitosan is a random
copolymer, containing (1 4) d-glucosamine and N-acetyl-d-
glucosamine units and it is water-soluble in dilute acid aqueous
solutions. The physicochemical properties of chitosan solutions can
be controlled by the manipulations of solution conditions (tem-
perature, pH, ionic strength, concentration, and solvent). Chitosan
carries the free amino and hydroxyl groups along its backbone.
The pKa value of free amino group is around 6.5. Because of
that chitosan exhibits pH dependent solubility; at pH > 6.5 chi-
tosan solutions exhibit phase separation, while for pH < 6.5 it
is soluble and carries a positive charge due to the presence of
protonated amino groups [13]. The different degree of deacety-
lations and/or different solution conditions have inuence on
charge density along the chains and, therefore, on the exibil-
ity of chitosan chains. Conformation of the chitosan polymer is
a function of its chain exibility and solution conditions [14].
Chain exibility facilitated inter- and/or intra-molecular interac-
tion between chitosan chains [15,16]. Within investigated pH range
(i.e. pH 15), chitosan molecules of the same deacetylation degree
have different inter- and/or intra-molecular electrostatic repul-
sive forces [15]. Chitosans with different degree of deacetylations,
in different solution conditions may be in a rod shape, a ran-
dom coil, or a compacted sphere [14]. These properties, together
with the hydrophobicity of the backbone, may play an important
role on its interactions with amphiphilic molecules [17]. Addi-
tionally, Amiji [18] has found the self-aggregation behaviour of
chitosan in aqueous solution caused by intermolecular hydropho-
bic interactions, with strong inuence of the ionic strength of the
medium.
To the best of our knowledge, there is no report related to the
aggregation behaviour and surface activity of systems containing
F127 and chitosan polyelectrolyte. The aim of this work is to pro-
vide more information on the interactions between the nonionic
surfactants, F127, and cationic polyelectrolyte, chitosan. The cho-
sen experimental conditions are of considerable importance for the
stability during the storage and during the possible application (e.g.
ocular surface) of the mixed systems.
2. Materials and methods
2.1. Materials
The triblock copolymer, Lutrol F127 (BASF) (generically polox-
amer 407) (molecular weight 12 600) was used as received. On
the basis of molecular weights, Lutrol F127 can be represented by
the formula (EO)100 (PO)65 (EO)100 . Low-viscous chitosan (molecu-
lar weight 150 000, deacetylation degree 84.5%, Fluka) was used as
received. According to the supplier, chitosan contained 0.35% insol-
uble matter and 0.60% ash as impurities. Analytical grade acetic
acid and sodium acetate (Sigma) were used for the preparations
of acetate buffer solutions pH 6.0 (100 mM acetic acid: sodium
acetate, ionic strength 0.1), and pH 6.5 (500 mM acetic acid: sodium
acetate, ionic strength 0.5). Double distilled water (specic conduc-
tivity <1 S cm1 ) was used for the preparation of all solutions. All
others chemicals were reagent grade.
2.2. Systems preparation
Stock solutions of the copolymer (10%, w/v) were prepared by
dissolving F127 copolymers in double distilled water or acetate
buffer for 48 h at 4 C. Stock solution of chitosan (1%, w/v) was pre-
pared in aqueous acetic acid (0.5%, v/v) by stirring for 24 h at room
temperature. The solution was ltered through medium porosity
lter in order to exclude the trace of undissolved chitosan. The
mixed F127/chitosan systems were prepared from F127 and chi-
tosan stock solutions with a constant chitosan concentration (0.005
or 0.01 or 0.015%, w/v) and variable F127 concentrations (1 105
to 5%, w/v) by diluting with water or acetate buffer (pH 6.0 or 6.5).
Additionally, the solutions of chitosan only or F127 only were
prepared by dilution of stock solutions with water or acetate buffer
(pH 6.0 or 6.5) in the concentration range 1 105 to 5 102 %
(w/v). All systems were kept in water circulating bath at 25 0.02 C
or 34 0.02 C to equilibrate for at least 24 h before surface tension
measurements. The pH measurements were made for all solutions
prepared with Seven Multi pH meter (Mettler Toledo). All sub-
sequent experiments were carried out in triplicate with freshly
prepared solutions.
2.3. Time effect on surface tension
The solutions were gently stirred after being placed in the cell to
enhance diffusion. In order to reach equilibrium value, conditioning
time was allowed between the placement of the solution in the
measurement cell and the actual reading [19,20]. Equilibrium of
30 min was found to be sufcient for F127, chitosan, and mixed
F127/chitosan systems, so this period was utilized for subsequent
measurements.
2.4. Surface tension measurement
The surface tension measurements were performed with K-
100C tensiometer (Krss) applying the density corrections needed.
Surface tension values (), as a function of concentration (c), were
determined employing the Du Noy ring method. The surface ten-
sion of the neat water and acetate buffer solutions pH 6.0 and 6.5
were always higher than 71.49 mN m1 at 25 C and 70.28 mN m1
at 34 C in each set of measurements. The measurements were
made at two different temperatures using Heating Immersion
Circulator MB (Julabo) attached to water bath with temperature
stability within 0.02 C. The readings were taken in triplicate and
were reproducible, with the standard deviation being less than 4%
of the mean value.
3. Result and discussion
3.1. Surface properties of chitosan solutions
The surface tension of chitosan in water and acetate buffer solu-
tions (pH 6.0 and 6.5) were determined for the concentration range
6.66 1010 to 3.34 106 mol dm3 at two temperatures (25 and
34 C). The surface tension data for chitosan in water are given in the
insert of Figs. 1 and 2 at 25 C (pH 5.06) and 34 C (pH 5.21), respec-
tively. The constant values of surface tension (71.45 mN m1 at
25 C and 70.26 mN m1 at 34 C) close to the surface tension
of pure water (71.99 mN m1 at 25 C and 70.52 mN m1 at 34 C)
[21] were observed. The constant values of surface tension indi-
cate that chitosan molecules are excluded from the air/solution
interface, indicating absence of surface activity. At a given pH, the
amino groups of chitosan molecules are protonated, which gave
the chitosan hydrophilic character in bulk solutions. Schultz et al.
[22] described analogous result explaining that chitosan molecule
 I. Pepic et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 327 (2008) 95102
Fig. 1. The isotherm of surface tension for F127 and mixed F127/chitosan systems in
water containing various F127 and constant chitosan concentrations at 25 C: F127
(); F127 with 0.005% (w/v) chitosan (); F127 with 0.01% (w/v) chitosan (); F127
with 0.015% (w/v) chitosan (). Insert: chitosan in water solution () without F127
at 25 C.
has no large hydrophobic groups that may be adsorbed on the
air/solution interface.
In contrast to the surface tension of chitosan in water, the slight
decrease of surface tensions for chitosan in acetate buffer solu-
tions was observed. In the inserts of Figs. 3 and 4 are the results
of surface tension of chitosan in acetate buffer at 25 C (pH 5.88)
and 34 C (pH 5.92), respectively. The surface tension changes were
 = 2.47 mN m1 at 25 C and  = 2.17 mN m1 at 34 C for chi-
tosan concentrations investigated. The surface tension changes for
chitosan in acetate buffer at 25 C (pH 6.38) and 34 C (pH 6.44)
(Figs. 5 and 6, in the inserts) were similar to changes at lower
pH of the acetate buffer solution, although the values were gen-
erally higher;  = 6.07 mN m1 at 25 C and  = 7.14 mN m1 at
34 C. Geng et al. [23] have studied chitosan/acetic acid system
and found surface tension change produced by an increasing acetic
acid concentration in water. The similar effect in chitosan/acetate
buffer solutions could not be expected, due to the almost con-
stant pH values in the systems. At higher pH of the solutions, i.e.
6.0 and 6.5, the free amino groups of chitosan molecules become
less protonated and hydrophobic character along chitosan chain
Fig. 2. The isotherm of surface tension for F127 and mixed F127/chitosan systems in
water containing various F127 and constant chitosan concentrations at 34 C: F127
(); F127 with 0.005% (w/v) chitosan (); F127 with 0.01% (w/v) chitosan (); F127
with 0.015% (w/v) chitosan (). Insert: chitosan in water solution () without F127
at 34 C.
97
Fig. 3. The isotherm of surface tension for F127 and mixed F127/chitosan systems
in acetate buffer (pH 6.0, ionic strength 0.1) containing various F127 and constant
chitosan concentrations at 25 C: F127 (); F127 with 0.005% (w/v) chitosan ();
F127 with 0.01% (w/v) chitosan (); F127 with 0.015% (w/v) chitosan (). Insert:
chitosan solution in acetate buffer () without F127 at 25 C.
become stronger. Therefore, the chitosan self-aggregates could be
formed in acetate buffer solutions by intra- and inter-molecular
hydrophobic interactions. Since the increasing ionic strength of
the medium enhances hydrophobic associations [18], the improved
self-aggregation might be expected for chitosan in acetate buffer
solutions with ionic strength 0.1 and 0.5 at pH 6.0 and 6.5, respec-
tively. The decrease of surface tension with increased chitosan
concentration in acetate buffer solutions may be explained by
adsorption of hydrophobic sequences of chitosan molecules, at least
partially, on the air/solution interface. Amiji [18] and Wang et al.
[24] have found self-aggregation properties of chitosan by means of
the uorescence measuring using dye solubilization methods. The
concentration at which aggregates started to form was around 0.1%
(w/v) chitosan in acetic acid solutions (0.1 mol dm3 ). Although, the
formation of chitosan self-aggregates in our study is not evident,
probably because of the different method used, there is certain
surface activity of chitosan itself in acetate buffers conditions. In
future, this interesting phenomenon should be further investi-
gated.
Fig. 4. The isotherm of surface tension for F127 and mixed F127/chitosan systems
in acetate buffer (pH 6.0, ionic strength 0.1) containing various F127 and constant
chitosan concentrations at 34 C: F127 (); F127 with 0.005% (w/v) chitosan ();
F127 with 0.01% (w/v) chitosan (); F127 with 0.015% (w/v) chitosan (). Insert:
chitosan solution in acetate buffer () without F127 at 34 C.
98 I. Pepic et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 327 (2008) 95102
Fig. 5. The isotherm of surface tension for F127 and mixed F127/chitosan systems
in acetate buffer (pH 6.5, ionic strength 0.5) containing various F127 and constant
chitosan concentrations at 25 C: F127 (); F127 with 0.005% (w/v) chitosan ();
F127 with 0.01% (w/v) chitosan (); F127 with 0.015% (w/v) chitosan (). Insert:
chitosan solution in acetate buffer () without F127 at 25 C.
3.2. Surface properties of mixed F127/chitosan systems
3.2.1. Adsorption behaviour before the rst break
Both F127 and mixed F127/chitosan systems were analyzed
simultaneously as a function of the pH, ionic strength, and temper-
ature in order to discriminate the aggregation behaviour of F127
in the presence of chitosan. Figs. 16 displays the surface ten-
sion data obtained. Few observations are immediately apparent.
A slope with two breaks in the surface tension/logarithm concen-
tration (/ln c) plot characterizes the general shape of adsorptions
isotherms for F127 and mixed F127/chitosan systems for all con-
ditions investigated. The results point out the existence of three
regions in which the adsorption at the air/solution interface is
clearly different. Other investigators observed three distinct sep-
arate regions for F127 solutions [19,25,26]. These ndings can be
summarized briey as follows. There is general agreement that the
second break in the curve is due to the formation of micelles, while
for the rst break at lower copolymer concentrations there is no
general consent. At low bulk concentration before the rst break
F127 adsorbs as individual molecules (unimers) and no aggregates
Fig. 6. The isotherm of surface tension for F127 and mixed F127/chitosan systems
in acetate buffer (pH 6.5, ionic strength 0.5) containing various F127 and constant
chitosan concentrations at 34 C: F127 (); F127 with 0.005% (w/v) chitosan ();
F127 with 0.01% (w/v) chitosan (); F127 with 0.015% (w/v) chitosan (). Insert:
chitosan solution in acetate buffer () without F127 at 34 C.
are thought to form. Polat and Chander [19] have suggested that
the rst break is a consequence of unimer aggregation into dimers
and other larger aggregates. Yet another explanation is by Alexan-
dridis and Hatton [1] who have proposed that the rst break is
due to a change in conguration of the copolymer molecules at the
air/solution interface.
In Figs. 1 and 2, the surface tension isotherms at two tempera-
tures for F127 and mixed F127/chitosan systems in water are shown.
It can be seen that at the same temperature all systems attained the
same and constant surface tension value beyond the cmc. However,
the addition of chitosan caused differences in the slopes of the sur-
face tension isotherm before the rst break. These differences were
independent on the chitosan concentration. Moreover, the surface
tension values before the rst break are always above that of the
F127 solutions by itself. Barreiro-Iglesias et al. [27] have studied
surface behaviour of F127 in the presence of 0.25% (w/w) carbopol
in water. The surface tension values for mixed F127/carbopol sys-
tem remained above the values obtained for F127 alone. It was
attributed, according to Maloney and Huber [28], to a disturbance
of the interfacial F127 layer by coadsorption of carbopol. According
to our results, chitosan molecules in water solution are excluded
from the air/solution interface. Therefore, one could not expect the
same behaviour for mixed F127/chitosan system in water. Mata et
al. [29] have ascribed any difference in the surface tension between
PEO/PPO/PEO-free and PEO/PPO/PEO-added systems to interac-
tions. Barreiro-Iglesias et al. [27], who have cited the results of other
investigators, observed that the interaction with nonionic surfac-
tant is usually weak except for the association of poly-(acrylic acid)
with ethoxylated surfactants.
From the results of surface tension, it can be speculated that an
interaction between the chitosan and F127 takes place in the water
solutions. It is possible that surface-active F127/chitosan complexes
are formed. These complexes are less surface-active and desorb
from the surfaces, which leads to a steeper slope of the isotherms
and higher surface tension values before the rst break in the chi-
tosan containing solutions. This might be due to the fact that the
pH of F127 solutions in water are 6.40 and 6.50 at 25 and 34 C,
respectively, and the pH of mixed F127/chitosan systems in water
are in range of 4.504.90 at both temperatures. At those pH values
of mixed F127/chitosan systems, the free amino groups of chitosan
molecules are protonated. The interaction between chitosan and
F127 may be governed by an electrostatic effect. More precisely,
electronegative oxygens of PEO units might bind to the electropos-
itive chitosan chains by electrostatic dipole-ion interaction. Since
the increased amount of chitosan in F127 water solutions retained
the surface behaviour before the rst break practically identical,
one might conclude that interactions between F127 and chitosan
are not depended on the chitosan concentration. They depend on
the chitosan charge and are sensitive to both pH and ionic strength.
On the other hand, the interactions are insensitive to temperature. It
is interesting to compare these results with those obtained by Bak-
shi et al. [30]. These authors, interpreting aggregation behaviour
of PEO/PPO/PEO copolymers with cationic surfactants in aqueous
solution, have found an electrostatic interaction of copolymers with
cationic surfactants due to the electronegative PEO unit.
A comparison of the surface tension values of F127 in acetate
buffer 6.0 (ionic strength 0.1) solutions with or without chitosan
added at 25 C is shown in Fig. 3. Both lower starting values and
subsequent values of the surface tension before the rst break for
mixed F127/chitosan systems could be explained by the surface
tension reduction caused with chitosan added. The slopes of the
isotherms before the rst break become steeper with increasing
concentration of chitosan in the systems. These results are related
with pH and ionic strength, since the chitosan molecules are less
protonated at higher pH and ionic strength. Therefore, due to the
 I. Pepic et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 327 (2008) 95102
lack of sufcient electropositive chitosan charges the electrostatic
interactions are weaker, compared to water systems. Under these
circumstances, chitosan could disturb the interfacial F127 layer by
coadsorbtion, consequently leading the surface tension of mixed
F127/chitosan systems to lower values.
In the case of these systems at 34 C (Fig. 4), as well as mixed
F127/chitosan systems at pH 6.5 (ionic strength 0.5) at both tem-
peratures (Figs. 5 and 6), the situation is rather complicated.
The evident similarity for mixed F127/chitosan systems in acetate
buffer 6.0 and 6.5 both at 34 C are present. The surface ten-
sion of F127 with 0.015% (w/v) chitosan began to decline from
64.24 and 62.29 mN m1 and become almost constant at 38.94 and
39.79 mN m1 , respectively, to pH 6.0 and 6.5 both at 34 C. The
reduction of the surface tension at 0.015% (w/v) chitosan resulted in
a steeper slope of both isotherms and lower surface tension values
before the rst break, compared to F127 acetate buffers alone.
Although, some changes of surface tension were observed when
comparing the systems in presence and absence of chitosan for
mixed F127/chitosan systems in acetate buffer 6.5 at 25 C (Fig. 5),
these changes as well as the slopes are not much different from the
F127 adsorption isotherm in the absence of chitosan.
Tentatively, it might be stated that multiple interaction process
are governed both by electrostatic and hydrophobic effect in the
dilute region investigated depending on pH, ionic strength, tem-
perature and chitosan concentrations, but the precise pattern is not
clear yet.
3.2.2. Critical aggregation and micelle concentration for F127
solutions
For all solutions cac (at the rst break) and cmc (at the second
break) values were obtained, from the intersections of two straight
lines. The cac and cmc values listed in Table 1 are discussed in this
section in terms of temperature, pH and ionic strength.
For F127 in water the cac values were 0.20 and
0.13 106 mol dm3 at 25 and 34 C, respectively. In rough
approximation, all calculated cac values occurred in the same
order of magnitude. This is in a good accordance with the literature
data, which have established that the cac is practically temperature
independent [26,31,32].
The cmc values for F127 in water are 4.70 and
0.19 104 mol dm3 at 25 and 34 C, respectively. These results
also correlates with previous ndings of other investigators [31].
The decrease of the cmc values observed with 9 C increase of
temperature was approximately 25 times in water, 10 times in
acetate buffer pH 6.0 (ionic strength 0.1) and 2 times in acetate
buffer pH 6.5 (ionic strength 0.5). The theory about the heat
induced micellization of the PEO/PPO/PEO aqueous solutions [31],
seems to be compromised in acetate buffers pH 6.5 for temperature
region investigated, since there are no signicant changes in the
cmc at 25 and 34 C.
The decrease of the cmc with pH and ionic strength increase is
observed at both temperatures, although it is more pronounced at
25 C.
The effect of pH and/or ionic strength may be ascribed
to the inuence of electrolyte contained in buffers on water
structure properties and, as a consequence, moderately hydra-
tion/dehydration of PPO, which shifts the F127 micellization at
higher/lower concentration.
3.2.3. Critical aggregation and micelle concentration for mixed
F127/chitosan systems
The cac values obtained for mixed F127/chitosan systems in
water and acetate buffers at both temperatures (25 and 34 C) are
listed in Table 1. The addition of chitosan to water solution of F127
caused the increase of the cac values. Vincekovic et al. [33] have
99
summarized that the charge density, conformation and exibility
of the polyelectrolyte chains are important factors affecting the cac.
Having in mind this observation and according to before discussed
possible electrostatic interactions between F127 and chitosan in
dilute region, certain repercussions on F127 onset of aggregation
could be accepted. However, the changes of cac values with increas-
ing chitosan concentration in the systems are more pronounced
at 25 C than at 34 C, comparing F127 solutions alone with chi-
tosan added solutions. Vincekovic et al. [33] have found a very
weak effect of polyelectrolyte concentration on the cac in the mix-
tures of anionic polyelectrolyte and cationic surfactant at 30 C. In
contrast to cac values, the addition of chitosan has not strongly
affected the cmc values of F127 in water. The effect was particu-
larly weak at 25 C, indicating only 5 to 25% increase of cmc values
for all chitosan added solutions. Therefore, certain cmc increases
(up to 64%) are observed at 34 C, depending of chitosan amount
added.
Comparison of cmc values for mixed F127/chitosan systems at
pH 6.0 reveals that the difference in cmc increased as the chitosan
concentration in systems increased. In fact, at 25 C we obtained
46.2, 129.2, and 421.5% increase in the cmc for 0.005, 0.01, and
0.015% (w/v) chitosan added, respectively. At higher temperature
we obtained 292.3, 407.7, and 600% increase in cmc for the same
chitosan amount added compared to F127 at pH 6.0 alone. In the
same way, one could compare the cmc data for mixed F127/chitosan
systems at pH 6.5 for both temperatures. The maximal increase in
the cmc values as a function of increased chitosan concentration
could be observed at 34 C, compared to cmc values for F127 alone
at the same temperature. In fact, the results show 9.6, 19.6, and
104.5 times increase in the cmc values compared to cmc value for
F127 alone. In the other words, the most pronounced effect of chi-
tosan on the cmc increase was shown in pH 6.5 at 34 C, compared
to all others investigated systems at the same temperature. At the
same time, at lower temperature (25 C) the differences between
cmc values for mixed F127/chitosan systems at 6.5 are less pro-
nounced, i.e. about 1.2, 1.9, and 3.8 times increase in the cmc for
0.005, 0.01, and 0.015% (w/v) chitosan concentrations, respectively.
The discussed cac and cmc results can be summarized briey as
follows. In F127 water solutions, the presence of chitosan generally
increased cac values at both temperatures, while the changes of
cmc values are less pronounced at both temperatures. The increase
of chitosan concentration in water systems has comparatively little
effect on the cac and cmc values at both temperatures. Evidently,
chitosan affected the onset of F127 aggregation in water (cac), but it
did not inuenced micelles formation (cmc). The cac and cmc values
found to be dependent on chitosan concentration in acetate buffer
solutions at both temperatures. In both acetate buffer solutions, the
cac values increased as the chitosan concentrations in the systems
increased, although the orders of magnitude of increased cac val-
ues are generally lower comparing to the systems in water at both
temperatures. It could be concluded that chitosan in both acetate
buffer solutions generally less affect the onset of F127 aggregation
comparing to the systems in water at both temperatures, which
depends on the chitosan concentration in systems. Contrary to the
water systems, the effect of chitosan on the cmc increase is much
more pronounced in acetate buffer systems at both temperatures.
In fact, the cmc values for mixed F127/chitosan systems increase as
a concentration of chitosan increases at both temperatures, indi-
cating that systems with higher amount of chitosan form micelles
at higher F127 concentration. It appears that chitosan signicantly
affects micelles formation at higher pH/ionic strength of the system.
Particularly extreme increase (about 104.5 times) of cmc values
are observed for F127/chitosan 0.015% (w/v) composition at high-
est pH/ionic strength and temperature investigated. Additionally,
it should be emphasized that established enthalpy of F127 micel-
100 I. Pepic et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 327 (2008) 95102

Table 1
The critical aggregation concentration (cac), the critical micelle concentration (cmc) and area per surfactant molecule at the air/solution interface (a) for F127 and mixed
F127/chitosan systems in water and acetate buffers at 25 and 34 C

cac 106 (mol dm3 ) cmc 104 (mol dm3 ) a (nm2 molecule1 )

25 C 34 C 25 C 34 C 25 C 34 C

Water system
F127 0.20 0.13 4.70 0.19 3.43 4.94
F127/0.005% (w/v) chitosan 3.35 0.54 4.91 0.22 2.99 3.54
F127/0.01% (w/v) chitosan 4.35 0.61 5.63 0.28 2.90 3.31
F127/0.015% (w/v) chitosan 5.10 0.70 5.90 0.31 2.61 3.26

Acetate buffer system pH 6.0 (ionic strength 0.1)

F127 0.72 0.45 1.30 0.13 2.32 2.38
F127/0.005% (w/v) chitosan 0.82 0.77 1.90 0.51 2.60 3.17
F127/0.01% (w/v) chitosan 1.02 0.88 2.98 0.66 3.07 3.82
F127/0.015% (w/v) chitosan 1.75 1.31 6.78 0.91 3.44 4.98

Acetate buffer system pH 6.5 (ionic strength 0.5)

F127 0.47 0.39 0.24 0.11 1.98 2.24
F127/0.005% (w/v) chitosan 0.61 1.08 0.29 1.06 2.08 3.76
F127/0.01% (w/v) chitosan 1.01 1.39 0.45 2.16 2.51 4.24
F127/0.015% (w/v) chitosan 1.08 1.41 0.92 11.5 4.26 8.68

lization is compromised in the case of all mixed F127/chitosan
systems at 34 C and pH 6.5, since the cmc values at 34 C are
higher then at 25 C. This observation suggests antagonistic mix-
ing behaviour between F127 and chitosan. Having in mind both,
thermodynamic stability of the investigated systems as well as the
dilution of the systems in biological uids upon administration,
the mixed F127/chitosan systems in acetate buffer pH 6.5 might be
associated with several problems.
Taking into account previously published literature data there
are several possible explanations for observed cac and cmc
behaviour. According to Bakshi et al. [30], an electrostatic ion-dipole
interaction might be occurring in a dilute region below the cac,
particularly in water system. The anticipated electrostatic inter-
actions could shift cac at higher values because electronegative
EO units might be bounded to the electropositive protonated chi-
tosan chains. In that sense, the self-aggregation starts at higher
F127 concentration, i.e. at higher cac values. As pH increases up
to 6.5, the chitosan become less protonated and the possibility
of electrostatic interaction is reduced. Consequently, less shifting
of the cac values is observed in the acetate buffers. As regard
to mixed F127/chitosan systems in water, cmc behaviour in the
presence of chitosan is evidence of poor interaction. Because the
changes in the cmc values are less pronounced for water solutions,
one might speculate that pure F127 micelles are formed indepen-
dently of chitosan chains. Since micelles formation are found to be
antagonistic in nature for mixed F127/chitosan systems in acetate
buffers at both temperatures, it might be attributed to the differ-
ent patterns of self-aggregation. Namely, as the pH is increased
the formation of hydrophobic sequences of chitosan molecules
are found, and consequently, the interaction between hydropho-
bic sequences of chitosan and PPO sequences of F127 could be
governed by hydrophobic effect. Zeghal and Auvray [34] have pro-
vided an evidence of interaction between PEO/PPO/PEO copolymer
and polyacid due to the formation of complex that preserves the
corecorona structure of micelles. Moreover, the non-dissociated
polyacid sequences are bound to the PPO sequences of copoly-
mer forming the hydrophobic core of the complex, whereas the
ionized polyacid sequences form a stabilizing hydrated corona
with the PEO. It appears that similar reasoning can be accepted
for shifting cmc values at higher concentration in acetate buffers
at both temperatures. Yet Varade et al. [35] provided another
explanation. The authors have explained the cmc increase in
the presence of hydrotropes by enhancing the solubility of PPO
sequences making Pluronic L62 behave like a more hydrophilic
block copolymer (salting-in nature) that would micellize at higher
concentration.
3.2.4. Surface excess and area per surfactant molecules at the
air/solution interface
Apart of cac and cmc determinations, surface tension experi-
ments provide a second important characteristic parameter, i.e. the
slope d/d ln c before cmc. The slope before cmc can be used in
connection with the Gibbs adsorption isotherm for the calcula-
tion of the area per molecule (a) which a copolymer occupies at
air/solution interface:
1 1
 d 
a= , and  =
Nav RT d ln c
where  is the surface excess concentration of copolymer
molecules which are adsorbed at monolayer coverage at the inter-
face and c is the bulk concentration. The area calculated gives
information on the degree of packing and the orientation of the
adsorbed molecule [36]. The calculated values for a are given in
Table 1 for all investigated systems at both temperatures. The area
of a single PO group is 0.11 nm2 [19]. As F127 consist of 65 PO
groups it follows that the total packing area is 7.15 nm2 for extended
molecule. All the results obtained for water systems show lower
values for a, indicating that the molecules are not adsorbed with
their extended conguration, and only a fraction of PPO sequences
reside at the surface. Furthermore, the presence of chitosan in F127
water systems cause additionally decreases a values and there-
fore results with less extended conguration regarding to F127 in
water alone. However, dissipated results for mixed F127/chitosan
systems in water are difcult to explain. At the beginning, a gross
error/artefact was thought, but a number of repeated experiments
produced the same results. For the time being, it is not possible to
explain each of any patterns.
By considering the data in Table 1, the results obtained for
the acetate buffers are consistent. The higher pH values and ionic
strength of F127 solutions without chitosan produced smaller a val-
ues and furthermore the temperature increase in all cases increased
them. The results of other investigators are only partly correspond-
ing with ours. For instance, Alexandridis et al. [31] have observed
a decrease in the a with an increase in temperature, which were
attributed to either less PEO hydration water at higher temperature,
or an increase in hydrophobicity of the PPO sequence, or both. Lo
Nostro and coworker [37] have found that increasing temperature
results in higher a due to the higher exibility of the hydrophobic
 I. Pepic et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 327 (2008) 95102
part of the molecule and, consequently, expansion of monolay-
ers spread at air/solution interface. Discrepancies might be related
with difference in surface tension values due to the rather broad
distribution of copolymer species present in a particular sample
and the presence of impurities.
For mixed F127/chitosan systems in acetate buffers at both tem-
peratures could be generally observed the increase in the a values
with increasing chitosan concentrations, comparing to F127 acetate
buffer solutions alone under same conditions (Table 1).
Evidently, the presence of chitosan polyelectrolyte in F127 sys-
tems at higher pH and ionic strength than water solutions produces
in different excess surface concentration and packing at air/solution
interface. Since the a value for the acetate buffer solution (pH 6.5,
ionic strength 0.5) with the highest amount of chitosan used at 34 C
is 8.68 nm2 molecule1 , the extended conguration of the molecule
might be speculated.
Future experiments are aimed at an incorporation of selected
drugs into systems studied here in order to establish whether a
certain F127/chitosan complex with its physicochemical properties
favours inclusion of different drugs (hydrophilic or hydrophobic).
Also the stability and behaviour in biological surroundings in situ,
as well as general applicability as drug formulations should be elu-
cidated.
4. Conclusions
The investigations of mixtures of nonionic surfactant, Lutrol
F127, and cationic polyelectrolyte, chitosan, revealed that the
solution conditions (pH, ionic strength, chitosan concentration)
inuence the self-aggregation in bulk solution and adsorption at
the air/solution interface. In water solutions, chitosan shifted the
onset of self-aggregation in mixed systems to higher concentra-
tions possible by electrostatic interactions, while the changes of
cmc values are less pronounced but dependent of chitosan concen-
tration, indicating evidence of poor interactions near cmc. On the
other hand, at pH 6.0 (ionic strength 0.1) and 6.5 (ionic strength 0.5)
the chitosan generally less affected the onset of self-aggregation in
mixed F127/chitosan systems, although it was dependent of chi-
tosan concentration. At the same time, the cmc values for mixed
F127/chitosan systems at pH 6.0 and 6.5 increased with increasing
chitosan concentration at both temperatures, indicating that chi-
tosan antagonistically affected micelle formation at higher pH and
ionic strength of the medium. The antagonistic mixing behaviours
were observed for mixed F127/chitosan systems at pH 6.5, since
the enthalpy of micellization were compromised. The observed
effect can be driven by combination of different factors among
which: (i) the hydrophobic interactions between PPO sequence of
F127 and hydrophobic sequence of chitosan, (ii) solubilization of
chitosan hydrophobic sequence and (iii) quite different, enhanced
solubility of PPO sequence resulting that F127 micellized at higher
concentrations. The systems studied are very complex from a
physicochemical standpoint. Even small changes in the system pro-
duced quite a different outcome. Therefore, careful consideration
is needed when/if drug delivery systems are envisioned, because
an incorporation of new drug species into micelle systems might
completely change the pattern previously known.
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