Investigation of Micellization and Vesiculation of Conjugated Linoleic Acid by Means of Self-Assembling and Self-Crosslinking

Article abstract of DOI:10.1007/s11743-014-1591-4

Bioactive and biocompatible conjugated linoleic acid (CLA) has been only considered as a food or medicine ingredient due to its rare natural occurrence. In this work, the surface activities and pH-induced self-assembling behaviors of the semi-synthetic CLA molecules into micelles or vesicles were systematically investigated. First, the self-assembling of CLA was studied in detail, and it was found that aside from temperature and ionic strength, pH is the prominent factor affecting the self-assembling of CLA. Moreover, stable CLA ufasomes (unsaturated fatty acid liposomes) in uniform size were obtained by self-crosslinking of the CLA ufasomes, and the morphologies of the crosslinked CLA assemblies were recorded by transmission electron microscopy, which made known the pH-induced formation of the CLA ufasomes or the CLA micelles. The crosslinked CLA assemblies presented improved properties such as a higher calcium stability, a lower lime soap dispersing requirement and a better solubilization ability than that of the CLA molecules themselves or the pre-crosslinked linoleic acids. These investigations could be helpful for comprehensively understanding effects of environment factors on self-assembling behaviors of conjugated fatty acids and responsive polymerization of polymerizable surfactants.

Full text of DOI:10.1007/s11743-014-1591-4

J Surfact Deterg
DOI 10.1007/s11743-014-1591-4
ORIGINAL ARTICLE
Investigation of Micellization and Vesiculation of Conjugated
Linoleic Acid by Means of Self-Assembling and Self-Crosslinking
•
Ye Fan Yun Fang Lin Ma Hang Jiang
•
•
Received: 2 December 2013 / Accepted: 21 April 2014
Ó AOCS 2014
Abstract Bioactive and biocompatible conjugated lino-
leic acid (CLA) has been only considered as a food or
medicine ingredient due to its rare natural occurrence. In
this work, the surface activities and pH-induced self-
assembling behaviors of the semi-synthetic CLA molecules
into micelles or vesicles were systematically investigated.
First, the self-assembling of CLA was studied in detail, and
it was found that aside from temperature and ionic strength,
pH is the prominent factor affecting the self-assembling of
CLA. Moreover, stable CLA ufasomes (unsaturated fatty
acid liposomes) in uniform size were obtained by self-
crosslinking of the CLA ufasomes, and the morphologies
of the crosslinked CLA assemblies were recorded by
transmission electron microscopy, which made known the
pH-induced formation of the CLA ufasomes or the CLA
micelles. The crosslinked CLA assemblies presented
improved properties such as a higher calcium stability, a
lower lime soap dispersing requirement and a better solu-
bilization ability than that of the CLA molecules them-
selves or the pre-crosslinked linoleic acids. These
investigations could be helpful for comprehensively
understanding effects of environment factors on self-
assembling behaviors of conjugated fatty acids and
responsive polymerization of polymerizable surfactants.
Keywords Conjugated linoleic acid ꢀ Micelle ꢀ Ufasome ꢀ
Self-assembling ꢀ Self-crosslinking ꢀ Surface activity ꢀ
Polymerizable surfactant
Introduction
Over the past decades, polymerizable surfactants have
attracted more attention and been used in a variety of
application areas [1]. Among these polymerizable surfac-
tants, polymerizable lipids, especially unsaturated fatty
acids, are topics of great interest. Conjugated linoleic acid
(CLA), a rare natural lipid present in animals and plants,
has attracted considerable attention because of its excellent
anticarcinogenic [2], antiatherogenic [3] and anti-inflam-
matory [4] functions. However, the rare natural occurrence
of CLA is still the limitation for application. Recently, the
chemical synthesis of CLA has been reported, which has
now made it possible to use CLA extensively for nutra-
ceuticals. From what has been reported, to date, CLA has
mainly been synthesized through dehydration of ricinoleic
acid [5], isomerization of linoleic acid (LA) catalyzed by
metal [6], enzyme and cultured cells [7], and alkali isom-
erization of LA [8]. Nevertheless, these chemical synthesis
routes have several obvious limitations: (1) the traditional
dehydration of ricinoleic acid generally provided low CLA
yield and complicates the chemical compositions of pro-
ducts [9]; (2) the metal-catalyzed isomerization may result
in some toxic and environmental problems that would
undoubtedly impede CLA from being used in food, drugs
and cosmetics [10]; (3) though the chemoenzymatic syn-
thesis can obviously enhance the product selectivity, low
CLA yield still restricts its availability and further appli-
cation [11]. Therefore, a semi-synthesis method, the alkali
isomerization of LA (Scheme 1), certainly becomes the
Electronic supplementary material The online version of this
article (doi:10.1007/s11743-014-1591-4) contains supplementary
material, which is available to authorized users.
Y. Fan ꢀ Y. Fang (&) ꢀ L. Ma ꢀ H. Jiang
The Key Laboratory of Food Colloids and Biotechnology,
Ministry of Education, School of Chemical and Material
Engineering, Jiangnan University, 1800 Lihu Avenue,
Wuxi 214122, Jiangsu, People’s Republic of China
e-mail: yunfang@126.com
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J Surfact Deterg
Scheme 1 Semi-synthesis of
CLA from LA by alkali
isomerization
H
H
C
C
C
H
H
C
HOOC(CH2)7
H
H
(CH2)5CH3
C
C
cis-9,trans-11 CLA
CH2
HOOC(CH2)7
(CH2)4CH3
H
alkali isomerization
C
C
H
HOOC(CH2)8
H
C
(CH2)4CH3
H
LA
C
C
H
C
H
trans-10,cis-12
CLA
most promising synthesis strategy, with many advantages
such as low-cost, high safety, high product yield and
selectivity, which is favorable for eliminating the worry
about the availability of CLA [12].
spectroscopy and transmission electron microscopy (TEM).
Subsequently, the CS and the encapsulation of 5-fluoro-
uracil (5-FU) using the self-crosslinked CLA assemblies
were tested. These studies could be helpful for compre-
hensively understanding the effects of environment factors
on the self-assembling behaviors of conjugated fatty acids
and responsive polymerization of polymerizable surfac-
tants. The investigation of microenvironment driven-
self-assembling of the CLA molecules and the resultant
self-crosslinked ufasomes would be of benefit to fabricate
biocompatible carriers for medicine capsules.
Fatty acid vesicles, especially ufasomes (‘‘unsaturated
fatty acid liposomes’’) assembled from unsaturated fatty
acids, have been considered as very attractive carriers for
food, drug and cosmetics capsules. Interestingly, many of
their assembling processes can be adjusted by pH, tem-
perature, salinity and water hardness [13, 14]; for instance,
decanoic acid in water being self-assembled into vesicles at
pH 6.8–7.8, and evolved into micelles at pH 11.0 [15–17],
as well as oleic acid [18, 19]. Much of the attention
regarding CLA has only been focused on its nutrition and
bioactivity [20, 21] rather than its surface activities, self-
assembling behaviors and further applications as biocom-
patible and polymerizable surfactants for its rare natural
occurrence. However, the colloid properties of CLA and
the reactive conjugated double bonds in the CLA molecule
could make it a potential capsule wall material through
self-assembly followed by self-crosslinking of CLA mol-
ecules. Actually, a very interesting phenomenon was pre-
viously observed when testing the lime soap dispersing
requirement (LSDR) of CLA in our laboratory. The aque-
ous solution of CLA in hard water gradually turned from
cloudy to clear after a long standing time or moderate
heating and the aged CLA solution became more resistant
to hard water and less sensitive to lowing of the pH than the
parent solution. Giving the fact that CLA was easily
polymerized by moderate heating [22], we speculated that
part of the CLA molecules in the aged solution may self-
crosslink to form polycarboxylates that display better cal-
cium stability (CS), and the self-crosslinked CLA, if it
exists, could be a new microcapsule material with more
stable vesicle structure and a wider environmental toler-
ance window.
Experimental Section
Materials
Safflower oil was purchased from Cofco Co., Ltd. Urea,
ethylene glycol, potassium hydroxide, ammonium persul-
phate (APS), calcium acetate, calcium chloride, magne-
sium sulfate, toluene and 5-FU were all analytical reagent
grades and purchased from Sinopharm Chemical Reagent
Co. Ltd. Sodium dodecyl sulfate (SDS, 99 wt%) was pur-
chased from Acros Organics Co. Ltd. All materials were
used without further purification. Ultrapure Millipore water
(18.2 MX cm) was used throughout.
Methods
Preparation of Linoleic Acid
LA was prepared from saponification of safflower oil and
enriched by urea inclusion. Typically, 400 mL sodium
hydroxide solution (4 wt% in ethanol) was added dropwise
in 4 h into a flask containing 100 g safflower oil. The
reaction mixture was stirred at 60 °C for another 2 h, and
then cooled to room temperature. The produced sodium
soap of fatty acid mixture was obtained by filtration, while
the unreacted components were discarded with the filtra-
tion solution. The filtrate was acidified with 10 wt% HCl to
pH 2, and washed alternatively with distilled water and
Thus, in this paper, the self-assembling behavior of the
CLA molecules induced by changes of pH and ionic
strength was firstly investigated by the tensiometry method.
Then, thermo-induced self-crosslinking of the CLA
assemblies was further studied by UV spectroscopy, FT-IR
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saturated saline. The resultant fatty acid mixture was dried
over Na2SO4. Then, 30 g fatty acid mixture was added
dropwise in 2 h into the mixture of 75 g urea in 240 mL
ethanol (95 v%) at 80 °C under N2 atmosphere, and
refluxed for another 30 min. The reaction mixture was
placed at -18 °C for 12 h; the produced crystal was
obtained after filtration and acidified again with 10 wt%
HCl to pH 2. The acidified mixture was extracted with
anhydrous diethyl ether, and the ether phase was then
washed with distilled water and saturated salt water alter-
natively and repeatedly, and finally dried over Na2SO4 to
obtain purified LA (97.6 wt%) after remove the ether.
(20 mmol/L) and adjusting the solution to pH 8.6 with a
little borate solution (0.2 mol/L). Then the flask stood at
room temperature overnight to let CLA molecules self-
assemble into CLA ufasomes.
Self-Crosslinking of CLA Assemblies
After adding initiator APS (5 wt% of CLA) into the above
CLA micelle solution and bubbling N2 for 30 min, the
mixture was heated to 70 °C for 24 h under N2 atmosphere,
initiating the self-crosslinking of the CLA micelles.
Similarly, APS (5 wt% of CLA) was added into the
above CLA ufasomes solution, and N2 was bubbled
through the mixture for 30 min; it was heated to 70 °C for
24 h under N2 atmosphere, initiating the self-crosslinking
of the CLA ufasomes.
Semi-Synthesis of Conjugated Linoleic Acid
CLA was semi-synthesized by alkali isomerization of LA
with KOH and ethylene glycol. 10 g LA was dropped in
30 min into a flask containing 5 g KOH and 15 mL eth-
ylene glycol at 170 °C under N2 atmosphere, and reacted
for another 4 h. The reaction mixture was acidified with
10 wt% HCl to pH 3, and extracted with anhydrous diethyl
ether. The ether phase was then washed with distilled water
and saturated salt water alternatively, and finally dried over
Na2SO4. A light yellow liquid of CLA was obtained after
removal of the ether. The obtained CLA was characterized
with a FTLA2000-104 infrared spectrometer (ABB Bo-
mem Co. Ltd., Canada), a Persee T6 UV–Vis spectroscopy
(Beijing Pgeneral Co. Ltd.), a 400 MHz Avance III NMR
spectrometer (Bruker, Germany) and a FULI 9790 gas
chromatograph (Fuli Analytical Instrument Co. Ltd.)
equipped with PEG 20000 capillary column and FID
The absorbance of the reaction solution at 234 nm was
used to monitor the reaction progress. The morphologies of
self-crosslinked CLA assemblies were imaged with TEM
(JEM-2100, 200 kV, JEOL Ltd., Japan).
Measurement of Surface Tension
The surface tensions (c) of the aqueous CLA solution at
different pHs and temperatures were measured using the
pendant drop method with an optical contact angle tensi-
ometer (OCA 40 micro, DataPhysics Instruments GmbH,
Germany). The pH values of all the tested solutions were
adjusted with NaOH–borate or boric acid–borate buffer,
and ionic strength was adjusted with NaCl. The critical
micelle concentration (CMC) and the critical vesicular
concentration (CVC) were determined according to the
turning points of c–log c curves.
detector, respectively. IR: 3,015, 1,709, and 1,654 cm-1
=CH, C=O and C=C, respectively; 946 and 982 cm-1
,
,
conjugated double bond of trans-, cis-CLA and cis-, trans-
CLA, respectively [23]. The characteristic absorption peak
of conjugated double bond at 234 nm [24] was observed in
Calcium Stability
1
UV spectrum. H NMR (CDCl3, ppm) d: 5.68 (m, 4H),
2.34 (t, 2H), 2.12 (q, 4H), 1.63 (m, 2H), 1.32 (m, 16H),
Calcium stability is defined as the maximum concentration
of calcium ion with which the sample solution could
remain clear. CS was determined using a modified Hart’s
method [25]. Concretely, 40 mL of the tested sample
solution (3 mmol/L) was titrated with a 1 wt% calcium
acetate solution in a 100-mL beaker at pH 10.5. The degree
of developed turbidity, which just obscured the typewriter
print on paper fastened to the other side of the breaker as
viewed horizontally through the solution, was taken as the
end point of the titrations. CS was expressed as ppm of
CaCO3 and calculated as Eq. (1):
0.89 (t, 3H).
Self-Assembling of CLA
One hundred milliliters of the aqueous CLA solution
(3 mmol/L) was prepared in a 250-mL flask by dissolving
CLA in a NaOH–borate buffer (20 mmol/L) and adjusting
the solution to pH 10.5 with a little NaOH solution
(0.1 mol/L). The flask then stood at room temperature
overnight to let CLA molecules self-assemble into CLA
micelle.
100
158
0:01V ꢁ
6
Calcium tolerance ðppmÞ ¼
ꢁ 10
ð1Þ
V þ 40
The procedure of preparation of CLA ufasome is similar
to the above preparation process of CLA micelle. 100 mL
of the aqueous CLA solution (3 mmol/L) was prepared in a
250-mL flask by dissolving CLA in a boric–borate buffer
where V is the consumed calcium acetate solution (mL);
158 and 100 the molecular weight of calcium acetate and
calcium carbonate, respectively; 0.01 the concentration of
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calcium acetate solution (1 wt%); and 40 is the volume of
tested sample solution (mL).
shaken in a thermostat oscillator at 80 rpm for 24 h, fol-
lowed by dialysis using a membrane with 3,500 molecular
weight cut-off against PBS buffer (0.1 mol/L, pH 7.4) for
1 h. The dialysis tube was then relocated in 200 mL fresh
PBS buffer, and incubated at 37 0.5 °C and 80 rpm in a
thermostat oscillator for the following sustained releasing
test. The UV absorbance (266 nm) of the solution was
recorded every 15 min. Fresh PBS was recruited to make
up the solution volume. The cumulative release was cal-
culated based on the calibration of standard 5-FU solution.
The entrapment efficiency (EE) of 5-FU from the cross-
linked CLA assemblies was also investigated according to
the above dialysis method and calculated as the following
Eq. (4):
Lime Soap Dispersing Requirement
Lime soap dispersing requirement is a numerical measure of
the effectiveness of lime soap dispersants. It is the minimum
amount of dispersant causing dispersion of the lime soap
deposit. In this paper, LSDR was tested with a modified
LSDP assay [26] in which SDS was used as the given dis-
persant, and the tested soap samples were used instead of
sodium oleate. LSDR was similarly defined as the minimum
amount of SDS required to completely disperse the resultant
lime soap deposit formed by 100 g of the tested soap
samples in hard water (1,000 ppm) at pH 10.5 and 30 °C,
which was calculated as Eq. (2). Thus, a soap sample with a
low LSDR is better than one with a high LSDR.
m1 ꢂ m2
EE % ¼
ꢁ 100
ð4Þ
m1
where m1 is the total amount of 5-FU in the crosslinked
CLA assemblies solution (g); and m2 is the amount of free
5-FU unencapsulated in the crosslinked CLA assemblies
(g). m2 was calculated based on the calibration of standard
5-FU solution.
V1 ꢁ 2:5
LSDR ð%Þ ¼
ꢁ 100
ð2Þ
V2 ꢁ 5:0
where V1 and V2 are the volume (mL) of SDS solution and
sample solution, respectively; and 2.5 and 5.0 are the
concentration (g/L) of SDS and the tested sample,
respectively.
Results and Discussion
Solubilization Capacity
Microenvironment Manipulated Self-Assembly
at Surface Layer and in Aqueous Solution of CLA
Briefly, 5 mL of the tested soap samples (3 mmol/L) was
mixed with 0–0.12 mL toluene in nine volumetric flasks
(10 mL), respectively. After shaking (80 r/min) in a ther-
mostat oscillator at 30 °C for 1 h, the absorbance of the
sample solution was recorded at 560 nm with a Persee
T6 UV–Vis spectroscopy. According to the absorbance–
toluene volume curve, the maximum amount of toluene
solubilized in the CLA assemblies was determined based
on the fact that the absorbance increases suddenly upon
reaching the solubilization limitation. The solubilization
capacity (AM) was calculated as the following Eq. (3) [27].
Although CLA is insoluble in water; sodium conjugated
linoleate (SCL) is an amphiphilic surfactant that can sol-
ubilize and self-assemble in water, and thus the mixture of
CLA and SCL is expected to assemble together and to
cause the variation of surface activity parameters of CLA
solution. As reported [19, 28, 29], most assemblies of
unsaturated fatty acid can undergo a transformation from
micelles to ufasomes responsive to pH variation, and the
transformation point mostly relies on pKa of the relevant
fatty acids. Since the disassociation constant (pKa) of CLA
was 8.5 obtained by acid-base titration, at pH 8.6 where the
amount of C17H31COO- in solution is comparable to that
of C17H31COOH, it is possible to induce a unique self-
assembling of an equal amount of C17H31COO- and
C17H31COOH. When the pH is increased to 10.5, a popular
pH value usual for soaps in applications, the amount of
C17H31COO- in the solution would be 100 times greater
than that of C17H31COOH. Upon further increasing the pH
to 13, the amount of C17H31COO- in the solution would be
10,000 times greater than that of C17H31COOH, which
means C17H31COO- is almost the exclusive CLA species
in the solution. Thereby, an investigation of the pH-
induced self-assembling of CLA was carried out at these
aforementioned three typical pH values along with various
A
AM
¼
ð3Þ
V ꢁ C
where AM is solubilization capacity (L/mol); A the maxi-
mum amount of solubilized toluene corresponding to
turning point (mL) in the curve of absorbance versus tol-
uene volume; V the volume of the tested soap sample
solution (mL), and C is the concentration of the testing
soap sample solution (mol/L).
Microencapsulation and Sustained Release of 5-FU Using
Crosslinked CLA Ufasome
0.0153 g 5-FU was mixed with 10 mL of crosslinked CLA
assemblies solution (3 mmol/L, pH 8.6) at 37 0.5 °C,
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Fig. 2 c–log c curves of CLA at pH 13 (a), pH 10.5 (b) and pH 8.6
(c) at different temperatures
hꢀ
ꢁ
i
dc ¼ ꢂRT CðB1ꢂÞ þ Cð1Þ dðln aÞ
ð6Þ
HB
Fig. 1 Microenvironmental influence on the self-assembly of CLA
In a dilute solution, dðln aÞ ꢃ dðln xÞ ꢃ dðln cÞ, c is
concentration of the surfactant in mol/L. Assuming
Cm ¼ CBð1ꢂÞ þ CðH1BÞ , then, the maximum adsorbed quantity
ionic strengths and temperatures, and the c–log c curves of
CLA solutions are indicated in Figs. 1 and 2.
As SCL is a typical anionic surfactant composed of an
active anion (B-) and a counter cation (A?), the Gibbs
equation is:
(Cm, mol/cm2), and minimum area per molecule (amin, A ),
2
˚
can be calculated as following:
1
dc
Cm ¼ ꢂ
ð7Þ
ð8Þ
2:303RT dðlog cÞ
1020
h
ꢀ
ꢁ
dc ¼ ꢂRT CAð1þÞdðln aAþ Þ þ CBð1ꢂÞ þ Cð1Þ dðln aB
Þ
ꢂ
HB
ꢀ
In the presence of excess electrolyte that provides same
ꢁ
i
ð5Þ
amin
¼
þ C þ CðB1ꢂÞ ꢂ CðA1þÞ dðln aH
Þ
ð1Þ
NACm
þ
HB
where R is the gas constant, 8.314 J/(mol K); T the abso-
lute temperature in K, and NA is the Avogadro constant.
A decreased tendency of both CMC (or CVC) and cCMC
of CLA with decreasing pH is observed in Fig. 1a. As
þ
counterion and buffering effect, aA and pH will be con-
stants, and CðA1þÞ ¼ CBð1ꢂÞ ¼ Cð1Þ; aB ¼ a, therefore,
ꢂ
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Table 1 Parameters of the surface self-assembly/the micellization (or the vesiculation) of the aqueous CLA solution at different pH
Parameter
pH 13
pH 10.5
pH 8.6
T (K)
303.15
0.13
308.15
0.13
313.15
0.13
303.15
0.13
308.15
0.13
313.15
0.13
298.15
0.41
303.15
0.41
308.15
0.41
I (mol/kg)
cCMC (mN/m)
Cm (10-6 mol/m2)
30.77
3.42
30.39
3.51
29.72
3.58
29.15
3.62
28.75
3.74
28.02
3.80
26.30
6.83
25.85
6.90
25.52
6.98
2
amin (A )
˚
48.6
47.3
46.4
45.9
44.4
43.7
24.3
24.1
23.8
CMC (mmol/L)
d(log CMC)/dT
DGHm (kJ/mol)
DHmH (kJ/mol)
DSHm [kJ/(mol K)]
0.42
0.38
0.34
0.41
0.37
0.35
0.20a
0.18a
0.17a
0.0091
0.0066
0.0061
-62.14b
20.76b
0.2781b
-59.43
-60.95
-62.46
-59.53
-60.97
-62.25
-63.70b
21.47b
-65.04b
22.18b
32.02
33.09
34.17
23.23
24.00
24.78
0.3017
0.3052
0.3086
0.2730
0.2757
0.2779
0.2810b
0.2830b
a
CVC
b
Thermodynamic parameters at CVC
shown in Fig. 1b and c, both higher surface activities
[lower CMC (or CVC) and cCMC] and more compacter
aggregation (higher Cm and lower amin) result from
increasing temperature and increasing ionic strength. All
the surface activity parameters of the aqueous CLA solu-
tion at different combinations of pH, temperature and ionic
strength are listed in Table 1. Nevertheless, pH is the
prominent factor affecting the self-assembling of CLA, and
is superior to temperature and ionic strength. At pH 13, the
CLA molecules mainly present in C17H31COO- species
(SCL), and the strong electrostatic repulsion among the
SCL ions favors forming loose molecular assembly struc-
tures, thus leading to a large amin. This signifies small
curvature radius of the self-assembly structures and bene-
fits to form spherical micelle. At pH 10.5, the values of Cm
and amin are similar with those at pH 13, which indicates a
similar surface molecular arrangement and a similar self-
assembly, and may be reasonably explained based on the
occurrence of the pH-dependent species. Whereas, at pH
8.6, the values of amin corresponding to the CLA species is
much closer to those of fatty acids at the insoluble mono-
In addition to the surface activity parameters, the ther-
modynamic parameters of micellization or vesiculation
of CLA at different pHs are calculated according to
log CMC–T curves of CLA (Fig. 3) based on the pseudo-
phase separation model [31]. Under constant temperature
and pressure t, the standard free energy change of micel-
lization DGHm, standard enthalpy change of micellization
DHmH, and standard entropy change of micellization DSHm,
are calculated, respectively, as following:
CMC
DGHm ¼ 4:606RT log
DHmH ¼ ꢂ4:606RT2
ð9Þ
x
dðlog CMCÞ
dT
ð10Þ
ð11Þ
ꢂTDSHm ¼ DGHm ꢂ DHmH
where x is the concentration of H2O in mol/L. The ther-
modynamic parameters of micellization or vesiculation of
CLA at different pHs are also listed in Table 1.
As one can see, DGHm is negative and decreases with
increasing temperature in all the experimental temperature
ranges, which is beneficial to spontaneous micellization
and vesiculation. The corresponding DSHm and DHmH are all
positive and TDSHm is obviously greater than DHmH, indi-
cating that the common driving forces of the micellization
and vesiculation of the CLA solution are entropy increases.
2
˚
layer film (20.5 A ) [30]. This means that the co-assembly
of an equal proportion of C17H31COOH molecules and
C17H31COO- ions may cause a sharp decrease of electro-
static repulsion between the ions and molecules, and then
the space between the C17H31COOH molecules and the
C17H31COO- ions drops down dramatically, which is
favorable for the formation of a large assembly, such as of
rod-like micelles, lamellar micelles or vesicles. As shown
in Fig. 2 and Table 1, the values of Cm increase and the
values of both CMC (or CVC) and cCMC decrease slightly
with increasing temperature. This is because the molecular
motion of CLA is enhanced and electrostatic repulsion
between hydrophilic head groups of the CLA molecules is
screened with increasing temperature, resulting in a tight
arrangement of hydrophobic chains.
Thermo-Induced Self-Crosslinking of CLA Assemblies
After confirmation of the proposed microenvironment
manipulated self-assembling of CLA, the self-crosslinking
of the CLA assemblies is then reasonably expected. The
CLA assemblies were heated in aqueous phase separately
at 50 and 70 °C, and were referred to as CLA-50 and CLA-
70, respectively. The thermo-induced self-crosslinking of
the CLA assemblies was verified by UV and IR spectra
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Fig. 4 UV (a) and IR spectra (b) of CLA before and after
crosslinking at pH 10.5
images of the thermo-induced self-crosslinked CLA
assemblies, one can clearly find the stable spherical
micelles at pH 10.5 (Fig. 5a), and the stable ufasomes with
diameters of *48 nm at pH 8.6 (Fig. 5b). The fact shows
that the CLA species self-assemble into micelles and ufa-
somes corresponding to different pH values, and then
structural fixation of the CLA assemblies is completed by
the self-crosslinking among the conjugated double bond in
the CLA molecules. Interestingly, the micelles obtained at
pH 10.5 are quite larger than classical micelles. It may be
that the thermo-inducement procedure disturbs the normal
micelle formation and results in formation of the large
particles, and thus a less disturbing method is being studied
in our laboratory to obtain the fixed normal micelle.
Moreover, the bilayer thickness of the crosslinked CLA
ufasome is greater than the length of an extended CLA
molecule but much shorter than twice the length of
CLA chain, suggesting that the molecular arrangement
within the bilayer membrane of the CLA vesicle is not the
normal ‘‘tail-to-tail’’ model. This is possibly because there
is intercalation of fatty acid chains from the outer and the
inner bilayer leaflets, or the fatty acid chains in the bilayer
membrane are considerably distorted due to the conjugated
Fig. 3 log CMC–T curves of CLA at pH 13 (a), pH 10.5 (b) and pH
8.6 (c)
(Fig. 4). With increasing temperature, the characteristic
absorption peak of the conjugated double bond at 234 nm
is shrunk, which is usually interpreted as the disappearance
of the conjugated double bond in the CLA molecules.
Additionally, when the crosslinking degree of double
bonds is approximately completed, the peaks correspond-
ing to the conjugated double bonds, i.e., 3,015, 1,654, 982
and 946 cm-1, are no longer presented in the FT-IR
spectrum.
Furthermore, based on the aforementioned self-assem-
bling behavior of CLA, the CLA assemblies were further
attempted to be chemically tethered through self-cross-
linking at pH 10.5 and pH 8.6, respectively. From TEM
123

J Surfact Deterg
Fig. 5 TEM images of the self-
crosslinked spherical micelles at
pH 10.5 (a) and the ufasomes at
pH 8.6 (b) prepared by self-
crosslinking at 70 °C, and the
illustration of the pH-induced
formation of the micelle or the
ufasome of CLA (c)
double bonds situated in the middle of the hydrophobic
chain. The size distribution and the molecular arrangement
of CLA molecule within the bilayer membrane of the
crosslinked CLA ufasome are similar to those of the
docosahexaenoic acid (DHA) vesicle [14]. Thus, a tenta-
tive mechanism is proposed to account for the formation of
the spherical micelles and the ufasomes induced by pH
changes as shown in Fig. 5c. At high pH, the C17H31COO-
ions are the main species and result in the formation of
spherical micelle in solution; with decreasing pH,
C17H31COO- ions are protonated to form C17H31COOH
molecules that are expected to weaken the interaction
among C17H31COO- ions and cause the formation of the
ufasomes of CLA. Moreover, it should be emphasized that
the transition between micelles and ufasomes occurs only
before self-crosslinking.
the same time, CLA, LA and pre-crosslinked LA (named as
LA-50 and LA-70, obtained by heating LA solution at 50
and 70 °C with the same reaction conditions as in
‘‘Methods’’, respectively) were taken as the controls. Their
dispersing abilities and solubilization abilities were inves-
tigated over lime soap and toluene, respectively. From the
results shown in Fig. 6, it can be found that CS (Fig. 6a),
LSDR (Fig. 6b) and solubilization ability (Fig. 6c) of the
crosslinked CLA assemblies are superior to those of all
LA species, and the higher crosslinking degree of CLA
assemblies is, the better the above properties are. More
importantly, the above three properties of CLA-50 and
CLA-70 are better than that of CLA itself, which is con-
sistent with the aforementioned phenomenon that the aged
CLA solution became less sensitive to pH and was
accompanied by an increase of resistance to hard water.
Additionally, the microencapsulation and the sustained
release of 5-FU were assayed using the crosslinked CLA
ufasome fabricated at pH 8.6 and 70 °C. The cumulative
release of 5-FU from the crosslinked CLA ufasome is
51.4 % in 1 h, compared to the 95.7 % of cumulative
release of 5-FU in the unencapsulated control. Nevertheless,
Other Miscellaneous Properties
The other miscellaneous properties, such as CS, dispersing
ability, and solubilization ability of CLA-50 and CLA-70,
were examined and the results are exhibited in Fig. 6. At
123

J Surfact Deterg
capacity (EC, mol/mol) of vesicles is usually represented as
a mass ratio or a mole ratio of solute and lipid, and it was
expressed as the mole ratio of solute and lipid in this paper.
The EC was calculated based on the encapsulation results
and using the following equation:
moles of entrapped 5 ꢂ FU
EC ¼
moles of CLA
gross weight of 5 ꢂ FU ꢁ EE =M5ꢂFU
ð12Þ
¼
moles of CLA
where M5-FU is the molar mass of 5-FU (g/mol). From the
microencapsulation and the sustained release results of
5-FU, the EE was determined and calculated according to
Eq. (4) as 39.7 %. Then the EC was calculated as 1.6,
suggesting that the mole of solute is higher than that of
lipid, which supports the above deduction that the cross-
linked CLA ufasome is vesicle-like.
Conclusions
In present experiments, a semi-synthetic lipid CLA was
obtained by alkali isomerization of LA, and then its self-
assembling behaviors at surface layer and in solution were
investigated by surface tension strategy. It was found that
pH, temperature and ionic strength obviously influenced
the surface self-assembling behaviors and micellization (or
vesiculation) of CLA, but pH mainly manipulated the self-
assembling of the CLA molecules. Furthermore, the TEM
images revealed that the stable, homogeneous and cross-
linked CLA assemblies were obtained from pH dependent
ufasomes by self-crosslinking of the CLA molecules. The
crosslinked CLA assemblies are micelles at pH 10.5 and
ufasomes at pH 8.6, respectively, because at pH 10.5 the
C17H31COO- ions are the main species of CLA that trend
to assemble into micelles, whereas pH 8.6 is slightly higher
than the pKa of CLA and one-half of CLA species in the
solution are the CLA molecules that are prone to form
ufasomes. The experimental results showed that the
crosslinked CLA assemblies possessed superior CS, LSDR
and solubilization ability to CLA itself and all the LA
species, and the higher crosslinking degree induced better
miscellaneous properties. These findings will help to enrich
colloid chemistry properties of unsaturated lipids and
reveal the interesting potential to generate new stable
ordered microcapsules from ufasomes through combining
pH-induced self-assembling and self-crosslinking.
Fig. 6 Calcium stability (a), lime soap dispersing requirement
(b) and solubilization ability (c) of various CLA species
the non-crosslinked CLA ufasome collapsed in PBS buffer
at pH 7.4, and the cumulative release of 5-FU was unmea-
sured. Moreover, the ratio of entrapped volume is one of the
most significant properties of vesicles. In this paper, the ratio
of entrapped volume of the crosslinked CLA ufasome can be
abstractly calculated according to the size and the bilayer
thickness of the crosslinked CLA ufasome obtained from
TEM results and expressed as V2/(V1 - V2). The volumes of
the crosslinked CLA ufasome and the internal aqueous
compartment are named as V1 and V2, respectively. Then the
ratio of entrapped volume was calculated as 2.3, indicating
that the volume of the internal aqueous compartment is
greater than that of the external compartment. So large a
ratio indicates that the crosslinked CLA ufasome looks like
a vesicle rather than a micelle. In addition, the entrapment
Acknowledgments The authors gratefully acknowledge support
from the National Natural Science Foundation of China (No.
21276113), Jiangsu Provincial Post Graduate Innovation Plan (No.
CXZZ11_0467) and Doctor Candidate Foundation of Jiangnan Uni-
versity (No. JUDCF10006). Mr Yuhang Liu is also acknowledged for
his contribution in the LSDR experiment.
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J Surfact Deterg
transformation of micelles to vesicles in an aqueous oleic acid/
oleate system. Langmuir 17:4223–4231
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Ye Fan received her B.Sc. (2007) and M.Sc. (2009) in applied
chemistry from Jiangnan University, Jiangsu, People’s Republic of
China. Currently she is a Ph.D. candidate of the School of Chemical
and Material Engineering at Jiangnan University. Her research
interests focus on colloid and interface chemistry.
Yun Fang is a professor of the Key Laboratory of Food Colloids and
Biotechnology, Ministry of Education; and the School of Chemical
and Material Engineering at Jiangnan University, Jiangsu, People’s
Republic of China. He received his Ph.D. degree in 2011 from Fudan
University. His main research interests are the fields of colloid and
interface chemistry.
Lin Ma is a graduate student of the School of Chemical and Material
Engineering at Jiangnan University.
Hang Jiang is an undergraduate student of the School of Chemical
and Material Engineering at Jiangnan University.
19. Fukuda H, Goto A, Yoshioka H, Goto R, Morigaki K, Walde P
(2001) Electron spin resonance study of the pH-induced
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