Surface modification of vesicles with methylol urea

Article abstract of DOI:10.1007/s11746-002-0633-0

Surface-modified vesicles were prepared using N-[3-(dimethylamino)propyl]-octadecanamide and stearic acid as bilayer-forming lipids, and N-methylol urea-dodecylamine conjugates (MU-DOA) as a surface modifier. The conjugation of MU to DOA was confirmed by FTIR spectra. MU-DOA was incorporated into the vesicles by co-homogenization of the lipids and MU-DOA, and the incorporated MU-DOA was then reacted with MU in aqueous bulk phase through a self-condensation reaction between the methylols under an acidic condition at 70°C. On a scanning electron microscope, the vesicles were spherical and multilamellar, and they exhibited thin polymer films on their surfaces. The incorporation of MU-DOA into the bilayer and the surface coating of the vesicles did not significantly influence the transition temperature of the vesicles. The absolute values of zeta potentials of the surface-modified vesicles were smaller than those of the unmodified vesicles, and the point of zero charge was shifted from ca. pH 9.5 to ca. pH 6.5 by the surface modification.

Full text of DOI:10.1007/s11746-002-0633-0

Surface Modification of Vesicles with Methylol Urea
Jin-Chul Kim* and Jong-Duk Kim
Department of Chemical Engineering, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea
ABSTRACT: Surface-modified vesicles were prepared using
(MU–DOA) was used as a surface modifier of vesicles.
N-[3-(dimethylamino)propyl]-octadecanamide and stearic acid MU–DOA was incorporated into the vesicles by co-homoge-
as bilayer-forming lipids, and N-methylol urea–dodecylamine
conjugates (MU–DOA) as a surface modifier. The conjugation
of MU to DOA was confirmed by FTIR spectra. MU–DOA was
incorporated into the vesicles by co-homogenization of the
lipids and MU–DOA, and the incorporated MU–DOA was then
reacted with MU in aqueous bulk phase through a self-conden-
sation reaction between the methylols under an acidic condi-
tion at 70°C. On a scanning electron microscope, the vesicles
were spherical and multilamellar, and they exhibited thin poly-
nization with bilayer-forming lipids, N-[3-(dimethylamino)-
propyl]-octadecanamide (DMAPODA), and stearic acid
(
SA). DMAPODA and FA are reported to form a building
block for vesicle formation by creating a salt bridge between
+
−
NH3 of DMAPODA and COO of FA (13,14). The incorpo-
rated MU–DOA was reacted with MU in the aqueous phase
through a self-condensation reaction between the methylols
under acidic conditions at 70°C. The complete process is rep-
mer films on their surfaces. The incorporation of MU–DOA into resented in Scheme 1. Observation of the physical properties
the bilayer and the surface coating of the vesicles did not signif- of the surface-modified vesicles using thermal phase transi-
icantly influence the transition temperature of the vesicles. The tion and microelectrophoresis is included in this report.
absolute values of zeta potentials of the surface-modified vesi-
cles were smaller than those of the unmodified vesicles, and the
point of zero charge was shifted from ca. pH 9.5 to ca. pH 6.5
by the surface modification.
MATERIALS AND METHODS
Materials. DMAPODA (M.W. 369) was a gift from Inolex
Chemical Co. (Philadelphia, PA). SA (99%), urea (99.5%),
formaldehyde (37% solution), and dodecylamine were pur-
chased from Sigma (St. Louis, MO). DMAPODA and SA
were used together as bilayer-forming materials for vesicle
formation. Urea, formaldehyde, and dodecylamine were used
Paper no. J10284 in JAOCS 79, 1235–1239 (December 2002).
KEY WORDS: N-Methylol urea, self-condensation, surface-
modification, vesicles.
Encapsulation of food ingredients, pharmaceuticals, cosmet- for modification of the vesicular surface.
ics, and agrochemicals has been of much interest among sci-
Preparation of the MU precondensate. Urea (80 g, 1.33
entists involved in the area of vesicles, microcapsules, and mi- mol) was dissolved in 400 g of an aqueous solution of
crospheres. Microencapsulation techniques are used for pro- formaldehyde (30 wt%, 3.99 mol). After pH was adjusted to
tecting unstable substances from oxidation and atmospheric 9.0 with 1 N NaOH, the solution was heated to 70°C and
moisture, converting volatile materials into a nonvolatile form, stirred at constant temperature. After a 1-h reaction, the re-
releasing the active ingredients on demand, and targeting phar- acted solution was cooled to room temperature and kept for
maceuticals to the site of action (1,2). Various methods, such further use. This step is shown in step 1 in Scheme 1.
as emulsification–solvent evaporation (3,4), spray-drying
5,6), coacervation (7,8), interfacial polycondensation (9), in 53.9 mmol) was added to 120 mL of distilled water contained in
situ polymerization (10,11), and complexation (12) have been a 250-mL round-bottomed flask, and the mixture was heated to
used to prepare microcapsules and microspheres.
80°C. After dissolving 0.73 g of borax in the mixture, 23.3 g of
Preparation of the MU–DOA conjugate. Dodecylamine (10 g,
(
Our method of preparing microcapsules by forming sur- MU solution (64.7 mmol of urea), prepared as in the previous sec-
face-modified vesicles involves a self-condensation reaction, tion, was added and the reaction mixture was stirred at 80°C for
a key technology of in situ polymerization where N-methylol 1.5 h. This step is shown in step 2 in Scheme 1. Upon standing,
urea or N-methylol melamine is used as a precondensate for the reaction was separated into the upper layer of oil (MU–DOA)
the wall material of microcapsules; they are polymerized in and the lower layer of aqueous phase. The upper layer was iso-
the continuous phase of oil-in-water (O/W) emulsions lated from the lower layer using a separation funnel.
through the self-condensation of the N-methylol groups.
IR spectroscopy. FTIR spectra were recorded on a Nicolet
In this study, N-methylol urea–dodecylamine conjugate 560 spectrometer (Madison, WI) purged with N . Aliquots of
2
samples (MU precondensate, DOA, and MU–DOA) were ap-
*
To whom correspondence should be addressed at Department of Chemical plied onto a discoid (32 mm × 3 mm) internal reflection element
Engineering, Korea Advanced Institute of Science and Technology, 373-1
Kusung-Dong, Yousong-Gu, Taejon 305-701, Korea.
E-mail: jinkim@lgcare.co.kr
(
KRS-5; Nicolet) for data collection. Bulk water in the MU pre-
condensate was removed using a dry N stream.
2
Preparations of vesicles. DMAPODA (2.82 g, 7.64 mmol)
and 2.18 g of SA (7.66 mmol) were added to a 250-mL
Present address of first author: LG Household and Health Care, #84,
Jang-dong, Yusong-gu, Taejon 305-343, South Korea.
Copyright © 2002 by AOCS Press
1235
JAOCS, Vol. 79, no. 12 (2002)

1236
J.-C. KIM AND J.-D. KIM
SCHEME 1
beaker. The mixture was then heated in a water bath at 70°C scanned on a differential scanning calorimeter (TA Instru-
until it melted and became clear. Distilled water, preheated to ments DSC 2010). For each sample, 10–20 mg was weighed
7
(
0°C, was added to the melt so that the total mass was 200 g into an aluminum DSC pan and scanned from 10–70°C at a
2.5 wt% lipids). The dispersion was stirred at 500 rpm until heating rate of 3°C/min.
it had cooled to room temperature. The procedure for Microelectrophoresis. Zeta potentials of vesicle suspen-
the preparation of DMAPODA/SA vesicles incorporating sions of bare vesicles, vesicles incorporating MU–DOA, and
MU–DOA was the same as that for preparation of the surface-modified vesicles were measured using ZetaPlus
DMAPODA/SA vesicles except that 0.54 g of MU–DOA was (Brookhaven Instrument Co., Holtsville, NY). The concen-
added to the mixture of DMAPODA and SA in the melting trations of the vesicles in the suspensions were adjusted to ap-
step. This step is shown in step 3 in Scheme 1. The proximately 0.06%. The ionic strengths of the suspensions
DMAPODA/SA vesicles coated with polymerized MU were were fixed to 1 mM with NaCl. The pH of the suspensions
prepared as follows. MU solution (1.26 g, 3.5 mmol of urea) were varied from 3.0 to 11.0 with 1 mM NaOH and 1 mM
was added to a 100-g suspension (2.5 wt% lipids, 0.27 wt% HCl solutions.
MU–DOA) of the vesicles incorporating MU–DOA. After the
pH was adjusted to 4.8 with acetic acid (10 wt%), it was
stirred at approximately 70°C for 1 h. This step (i.e., surface
RESULTS AND DISCUSSION
modification by the self-condensation reaction between Conjugation of MU and DOA. Figure 1 shows FTIR spectra
MU–DOA on vesicle bilayers and MU in the bulk aqueous of the MU precondensate, DOA, and MU–DOA. A peak
−1
phase) is shown in step 4 in Scheme 1. Finally, the pH was around 3320 cm of spectrum A, characteristic of the OH al-
adjusted to 8.0 and then the suspension was cooled to room cohol stretching, indicates the presence of N-methylol groups
temperature.
in the MU precondensate (step 1 in Scheme 1). The peak
−
1
Scanning electron microscopy (SEM). To verify whether around 3350 cm of spectrum B, the vibration characteristic
the surfaces of vesicles were modified with polymerized MU, of amine groups of DOA, disappeared and a small and broad
−1
a structural analysis was carried out using a cold-stage scan- band of 3250–3550 cm , corresponding to the OH stretching
ning electron microscope (JEOL JSM-840A). An aliquot of of N-methylol groups of MU–DOA, was observed following
the vesicle suspensions was placed on a metal stub and then the reaction between DOA and the MU precondensate (step 2
immersed in liquid nitrogen. The specimen was transferred to in Scheme 1), indicating that DOA was successfully conju-
a prechamber (Bio-Rad E7450) located outside the scanning gated to the MU precondensate (see spectrum C). The rela-
electron microscope and then etched at −60°C, 10− mbar, for tively weak OH signal of MU–DOA, compared with the sig-
5
2
h. The specimen was then sputtered with gold at −130°C to nal of the MU precondensate, was caused by a portion of the
a thickness of 100–200 Å and viewed in the scanning elec- OH groups of the MU precondensate reacting with the amine
tron microscope using an accelerating voltage of 10 kV at a groups of DOA.
stage temperature of −180°C.
Structures of vesicles. Figure 2 shows electron micro-
DSC. Suspensions of bare vesicles, vesicles incorporating graphs of the surface-modified vesicles. The particles were
MU–DOA, and surface-modified vesicles were thermally spherical and less than 20 µm in size. Wrinkled coats show-
JAOCS, Vol. 79, no. 12 (2002)

SURFACE MODIFICATION OF VESICLES
1237
FIG. 1. FTIR spectra of N-methylol urea (MU) precondensates (A), dodecylamine (DOA) (B),
and MU–DOA conjugates (C). The spectrum in the solid box was obtained by intensifying the
spectrum in the dotted box.
ing a polymeric membrane of MUwere observed on the sur- cles incorporating MU–DOA, and the surface-modified vesi-
face. In the process of microcapsule preparation using in situ cles were 52.7, 51.5, and 52.7°C, respectively (there was no
polymerization, MU in the aqueous phase of the O/W emul- significant difference in the transition temperatures). The ther-
sion was deposited onto the surface of oil droplets by a self- mal transition of MU–DOA disappeared when it was in the
condensation reaction, leading to the formation of microcap- vesicle suspension. This indicates that hydrocarbon chains of
sules (10,11). It was postulated that the MU precondensate MU–DOA were incorporated into the lipid bilayer of vesicles.
became hydrophobic during the condensation reaction and The incorporation of MU–DOA did not significantly influence
that the hydrophobicity could be a driving force in its deposi- the transition temperatures of vesicles but led to broader peaks.
tion. In our systems, MU–DOA intercalated into vesicle bi-
Microelectrophoretic behavior. Figure 4 shows the pH-
layers would play a major role in depositing the MU precon- dependent zeta potentials of bare vesicles, vesicles incorpo-
densate onto the surfaces of vesicles because the vesicular rating MU–DOA, and the surface-modified vesicles. The zeta
MU–DOA could react with the MU precondensate in the bulk potentials of bare vesicles decreased with increasing pH, and
aqueous phase through their reactive methylol groups. On the the isoelectric point was observed around 9.5. FFA carboxyl
other hand, the cross-section micrographs revealed that the groups have a pK of approximately 5, so they will be depro-
vesicles had a mutilayered structure, indicating that the sur- tonated above a pH of 5, increasing the negative charge on
face modification did not affect the integrity of the vesicles. the surface of the vesicles. Similarly, the number of proton-
DMAPODA and FA are reported to form a building block for ated tertiary amine groups of DMAPODA will decrease with
+
3
vesicle formation by creating a salt bridge between the NH
of DMAPODA and the COO of FA (13,14).
increasing pH, decreasing the positive charge on the surface.
Therefore, the deprotonations of the carboxyl groups and the
−
Thermal phase transition. Figure 3 shows thermograms of protonated tertiary amine groups would be responsible for the
DOA, MU–DOA, and vesicles. The transition temperature of reduction of the zeta potential with increasing pH. The incor-
MU–DOA (21.1°C) was lower than that of DOA (30.3°C). poration of MU–DOA into the vesicles did not significantly
This is probably because the intermolecular interaction of affect the zeta potentials. The surface-modified vesicles, how-
MU–DOA would be hindered by its bulky MU group. On the ever, exhibited quite a different curve. The absolute values of
other hand, the transition temperatures of bare vesicles, vesi- zeta potentials were smaller than those of the others and had
JAOCS, Vol. 79, no. 12 (2002)

1238
J.-C. KIM AND J.-D. KIM
FIG. 3. Thermograms of DOA (a), MU–DOA (b), bare vesicles (c), vesi-
cles incorporating MU–DOA (d), and surface-modified vesicles (e). For
abbreviations see Figure 1.
be responsible for the decrease in zeta potential with increas-
ing pH.
In conclusion, the surfaces of vesicles were coated by self-
condensation reactions between MU–DOA in the vesicle bi-
layers and MU in the bulk aqueous phase. The coating
changed the surface property of the vesicle, as shown by the
FIG. 2. Scanning electron micrographs of the surfaces (A,B) and a cross-
section of surface-modified vesicles (C).
the point of zero charge around pH 6.5. The microelec-
trophoretic behavior of the surface-modified vesicles was
dominated not by the surface of DMAPODA/SA but by the
polymer coats formed on the surface of the vesicles. The de-
FIG. 4. pH-Dependent zeta potentials of bare vesicles (●), vesicles in-
corporating MU–DOA (●), and surface-modified vesicles (■). For ab-
protonations of the MU moieties in the polymer coats would breviation see Figure 1.
JAOCS, Vol. 79, no. 12 (2002)

SURFACE MODIFICATION OF VESICLES
1239
zeta potentials, but it did not influence the internal structure
or the transition temperature of the vesicle. Therefore, the
method presented in this study covers the surfaces of vesicles
with thin polymer films without deteriorating their integrity.
The polymer film is expected to act as a protective layer
against the destabilization of the vesicles, e.g., their fusion,
size reduction, and even solubilization.
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9
ACKNOWLEDGMENTS
We thank Joo-Ok Kim for technical assistance in the experiment
with DSC.
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[Received March 28, 2002; accepted July 20, 2002]
JAOCS, Vol. 79, no. 12 (2002)