32 Biomacromolecules 2009, 10, 32–40

Micelles and Polymersomes Obtained by Self-Assembly of

Dextran and Polystyrene Based Block Copolymers
Clément Houga,† Joanna Giermanska,‡ Sébastien Lecommandoux,† Redouane Borsali,§
Daniel Taton,† Yves Gnanou,*,† and Jean-François Le Meins*,†
Université de Bordeaux, Laboratoire de Chimie des Polymères Organiques UMR5629, ENSCPB-CNRS,
16 avenue Pey Berland, 33607, Pessac cedex, France, Université de Bordeaux, Centre de Recherche
Paul Pascal CNRS-UPR 8641, Avenue Albert Schweitzer, 33600 Pessac, France, and Centre de
Recherche sur les Macromolécules Végétales CERMAV and Joseph Fourier University,
BP53, 38041, Grenoble Cedex 9, France
Received March 25, 2008; Revised Manuscript Received November 10, 2008

The self-assembly of dextran-block-polystyrene (dex-b-PS) block copolymers was investigated in solution. The
hydrophobic PS weight fraction in these block copolymers ranges from 7 to 92% w/w, whereas the average
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number molar mass of dextran was kept constant at 6600 gmol-1. Self-assembly by direct dissolution in water
could be performed only for block copolymers with a low hydrophobic content (7% w/w), whereas mixtures of
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tetrahydrofuran and dimethylsulfoxide were required for higher PS content, before transferring the structures into
water. Core-shell micelles, ovoı̈ds, and vesicles could be identified upon characterization by light and neutrons
scattering, atomic force microscopy, and transmission electron microscopy. Most of the morphologies observed
were not expected considering the chemical composition of the block copolymers. Finally, the size and shape of
these nanoparticles were fixed upon cross-linking the dextran block through reaction of the hydroxyl groups with
divinylsulfone. The role of the dextran conformation on the self-assembly process is discussed.

Introduction
The self-assembly of block copolymers in a selective solvent
provides a great variety of morphologies such as spherical and
wormlike micelles, vesicles, and numerous other microstruc-
tures. Such a process is the result of two driving forces that
interplay oppositely: on the one hand, long-range repulsive
interactions between incompatible blocks, and on the other hand,
short-range attractive interactions due to the chemical bond
linking the two blocks, which leads to microphase rather than
macrophase separation. The morphologies observed at thermo-
dynamic equilibrium result from the minimization of the free
energy of the self-assembled structures and are controlled by
molecular parameters such as the chemical nature of the blocks,
the volume fraction of each block, the molar mass and the
overall architecture of the copolymer.1 Solution parameters such
as polymer concentration, temperature, solvent quality, pH, and
ionic strength2 also play a crucial role on the self-assembly.
Finally, the dissolution process is another key parameter that
impacts morphological aspects of the nanoparticles formed.3-6
The self-assembly phenomenon in solution is therefore ex-
tremely complex; numerous theoretical works have been devoted
to the subject7-10 as well as experimental studies, which have
helped to clarify the micellization process and the way to
generate stable morphologies.3,5,11
Discher and Eisenberg11 proposed a general empirical law
which stipulates that block copolymers possessing a hydrophilic
weight fraction (f) >45% are expected to form spherical
* To whom correspondence should be addressed. Tel.: 00 33 5 57 00 36
96. Fax: 00 33 5 57 40 00 84 87. E-mail: lemeins@enscpb.fr (J.F.L.M.);
gnanou@enscpb.fr (Y.G.).
†
Laboratoire de Chimie des Polymères Organiques UMR5629.
‡
Centre de Recherche Paul Pascal CNRS-UPR 8641.
§
Centre de Recherche sur les Macromolécules Végétales CERMAV and
Joseph Fourier University.
10.1021/bm800778n CCC: $40.75
Published on Web 12/11/2008
micelles, while those with f < 25% self-assemble into inverted
nanostructures. Block copolymers with hydrophilic weight
fraction around 35 ( 10% are predicted to form polymeric
vesicles also referred to as polymersomes.
Nanostructures formed by self-assembly in solution of block
copolymers make them attractive materials in various applica-
tions, in particular, as nanocarriers for the encapsulation and
controlled release of drugs. For example, polyester-based block
copolymers are the subject of numerous studies12-17 aiming at
the development of biomaterials or drug delivery systems.
In addition to providing abundant sources of raw materials,
naturally occurring oligosaccharides and polysaccharides exhibit
attractive properties such as biodegradability, biocompatibility,
texturing, or gelifying properties. Harnessing these properties
in nanostructures obtained from the self-assembly of block
copolymers could be of great interest for numerous applications
from cosmetics to medicine. Pioneer work in this field has been
performed in the early 1980s by Ziegast and Pfannemüller who
coupled an oligosaccharide functionalized with an aldonolactone
at its reducing end to an R,ω-diamino poly(ethylene oxide)
(PEO).18 In recent years, other routes, though scarce, have been
proposed to develop new “hybrid” polysaccharide-based block
copolymers. A first strategy is based on the coupling of an
amino-terminated synthetic polymer to the terminal aldehyde
of an oligosaccharide,19-23 possibly followed by the enzymatic
growth of the oligosaccharide block.21,24,25 A second route starts
from the chemical modification of the reducing ends to
subsequently grow the synthetic block by means of a controlled/
living polymerization. In this method, sugar derivatives or
cyclodextrine are derivatized into macroinitiators of controlled
radical polymerization process26,27 to create “end sugar” func-
tionalized synthetic polymers. Recently, our group has reported
a methodology to synthesize dextran-block-polystyrene (dex-
b-PS) diblock copolymer by atom transfer radical polymerization
 2009 American Chemical Society
Micelles and Polymersomes
Table 1. Molecular Characteristics of the Block Copolymers Used
in this Study
sample Mn SECa DPn NMRb PDIa ΦPS %w/w
dex40-b-PS5 17500 5 1.4 7
dex40-b-PS270 82200 270 1.7 81
dex40-b-PS775 160000 775 1.9 92
a
Determined by SEC in THF (calibration with PS standards). b Overall
composition determined by 1H spectroscopy, knowing the molar weight
of commercial dextran (Mn ) 6600 g · mol-1).
(ATRP).28 From a synthetic viewpoint, most of reported works
take advantage of the terminal aldehyde of the oligosaccharide
that is in equilibrium with a hemiacetal function, except in a
few references.21,24 However, the number of saccharide based
block copolymers systems that have been thoroughly investi-
gated is limited and among existing studies only a few contain
informations about their solution behavior. For instance, Liu
and Zhang20 have shown by transmission electron microscopy
(TEM) and dynamic light scattering (DLS) that dextran-b-poly-
(ε-caprolactone) self-assembles in water into polydisperse
micellar structures with an average diameter of 100 nm. pH-
responsive carboxymethyl dextran-b-poly(ethylene oxide) were
also shown to self-assemble in aggregates with an average size
of 100 nm in acidic media by intra- and intermolecular hydrogen
bonding.22 The self-assembly of PEO-b-amylose in chloroform
results in the formation of reverse micelles in which the amylose
block is believed to adopt a helical structure.29 The behavior of
sugar end-functionalized PS (from glucose to maltohexaose) has
also been studied in toluene.30 Reverse micelles were obtained
with an aggregation number varying with the overall composi-
tion of the block copolymer. In a recent addition, the self-
assembly of amylose-b-PS in THF revealed that nonequilibrium
aggregates with different sizes were obtained,31 whereas crew
cut micelles with a diameter ranging from 10 to 30 nm were
observed in water. In the latter case, the amylose corona was
found to exhibit a rod-like structure. It is important to note that
depending on their chemical nature, polysaccharide chains can
present a rod conformation (Xanthan, amylose,...) or a coil
conformation like, for instance, pullulan or dextran, although
the latter one behaves as a rod for molar mass below 2000
g/mol.32 This coil or rod character can have a marked influence
on the self-assembly process, as it will be discussed later in the
paper.
From these findings, it appears difficult to establish general
principles with respect to the self-assembly of polysaccharide
based block copolymers. For instance, the influence of the helical
structure of a polysaccharide such as amylose on the self-
assembly process is not yet clearly understood. In the present
report, we describe the self-assembly properties of a series of
dex-b-PS block copolymers in organic solvent but also in water.
The degree of polymerization (DP) of the polysaccharide was
kept constant (∼40) but that of PS blocks was varied from 5 to
770. The synthesis of these hybrid block copolymers has been
already described in a previous report.28 The influence of the
block copolymer composition on the micellar structures formed
has been thoroughly investigated by means of dynamic and static
light scattering, atomic force microscopy and freeze fracture
transmission electronic microscopy. We discuss at the end of
this work the possible influence of the dextran block on the
self-assembly process.
Experimental Section
Materials. Molecular features of dex-b-PS diblock copolymers
previously synthesized28 are reported in Table 1. All organic solvents
Biomacromolecules, Vol. 10, No. 1, 2009 33
used were of technical grade. Divinyl sulfone (Aldrich) was used
without purification. The following nomenclature was used for the
different block copolymers: dexa-b-PSb, where a and b stand for the
degree of polymerization of dextran and polystyrene, respectively.
Preparation of the Self-Assembled Nanoparticles. The direct
dissolution of dex40-b-PS5 in water was achieved after stirring for two
days at 90 °C, followed by sonication. The direct dissolution in water
for systems with a larger PS content was just not possible. As no
common solvent for both PS and dextran could be found, a mixture of
THF and DMSO was used to dissolve these diblock copolymers, DMSO
being selective for the dextran block and THF for the PS one. This
method is slightly different from the procedure generally used that
consists in the dissolution of the block copolymer in a common solvent
for both blocks, the self-assembly being induced by adding a selective
solvent for one of the blocks.2,6,33-35 All dex-b-PS block copolymer
samples could be dissolved in DMSO/THF mixtures in a composition
range from 50 to 75% in DMSO for dex40-b-PS270 and from 30 to 45%
in DMSO for dex40-b-PS775 (dotted lines in Figure 3). In these
compositions, no clear scattered signal was detected, indicating that
the block copolymers behave as unimers. Then, the solvent composition
was changed by addition of either DMSO or THF to induce self-
assembly and engineer different morphologies.
Cross-Linking Procedure of the Nanoparticles. Divinyl sulfone
(DVS) was added under nitrogen to dex40-b-PS270 dissolved in a DMSO/
THF mixture with a composition of 40/60 to cross-link dextran in the
core of the nanoparticles or in a DMSO/THF mixture with a composi-
tion of 90/10 to cross-link dextran in the shell of the nanoparticles. A
large excess (>10 equiv) of DVS was used relative to the hydroxyl
groups present in the dextran chains. The reaction was performed at
room temperature during 24 h.
Atomic Force Microscopy (AFM). Samples for AFM analysis were
prepared by evaporation at room temperature on substrates starting from
water solutions. Practically, for aqueous media, 20 µL of a dilute
solution (∼5 mg/L) was dropped on a 1 × 1 cm2 freshly cleaved mica
substrate. Samples were analyzed after complete evaporation of water
at room temperature. For solution in THF, 20 µL of a dilute solution
(∼0.5 mg/L) was spin-coated on freshly cleaved HOPG. For solution
in DMSO, 20 µL (∼5 mg/mL) were dropped on HOPG surfaces and
the evaporation of the solvent was performed under dynamic vacuum.
AFM images were recorded in air with a dimension microscope (Digital
Instruments, Santa Barbara, CA) operated in tapping mode. The probes
were commercially available silicon tips with a spring constant of 40
N/m, a resonance frequency lying in the 270-320 kHz range and a
radius of curvature in the 10-15 nm range. In this work, both the
topography and the phase signal images were recorded with the highest
sampling resolution available, that is, 512 × 512 data points.
Dynamic and Static Light Scattering. Dynamic and static light
scattering measurements were performed using an ALV laser goniom-
eter, which consists of a 22 mW HeNe linear polarized laser operating
at a wavelength of 632.8 nm and an ALV-5000/EPP multiple τ digital
correlator with 125 ns initial sampling time. The copolymer solutions
were maintained at a constant temperature of 25.0 ( 0.1 °C in all
experiments. All the scattering measurements were carried out from
40 to 120° by steps of 10°. Data were collected using ALV Correlator
Control software, and the counting time was fixed at 300 s for each
angle. Dynamic light scattering measurements were evaluated by fitting
of the measured normalized time autocorrelation function of the
scattered light intensity. The data were fitted with the help of the
constrained regularization algorithm (CONTIN), which provides the
distribution of relaxation times τ, A(τ), as the inverse Laplace transform
of g(1)(t) function
∫
∞
g(1)(t) ) A(τ) exp(-t ⁄ τ)dτ (1)
0
Apparent diffusion coefficients D were obtained by plotting the
relaxation frequency, Γ (Γ ) τ-1) versus q2, where q is the wavevector
defined as
34 Biomacromolecules, Vol. 10, No. 1, 2009 Houga et al.

4πn θ
q)
λ
sin ()
2
(2)

and λ is the wavelength of the incident laser beam (632.8 nm), θ is the
scattering angle, and n is the refractive index of the media. Single
nanoparticle diffusion coefficient were determined by extrapolation to
zero concentration and hydrodynamic radius (RH) was calculated from
the Stokes-Einstein relation

kBT 2 kBT
RH ) q ) (3)
6πηΓ 6πηDreal
where kB is the Boltzmann constant, Γ is the relaxation frequency, T is
the temperature, and η is the viscosity of the medium.
The size dispersity of the particles was then calculated by the 〈∆Γ2〉/
Γ2 ratio, in which 〈∆Γ2〉 was determined by analysis of the first-order
correlation function by cumulant analysis.

1 1 Figure 1. Autocorrelation function at 90 ° and time distribution function

g1(t)H ) exp[-〈Γ〉t + 〈∆Γ2 〉t2 - 〈∆Γ3 〉t3 + ...] (4)
2 6
The reduced elastic scattering I(q)/kC, with K ) 4π2n0(dn/dc)2(I090°/
R90°)/λ04NA, was measured in steps from 40 to 120° scattering angle,
where n0 is the refractive index of the standard (toluene), I090° and R90°
are the intensity and the Rayleigh ratio of the standard at θ ) 90°,
respectively, dn/dc is the increment of the refractive index, C is the
concentration, and I(q) is the intensity scattered by the sample. Elastic
(static) intensity was calculated according to standard procedures using
toluene as the standard with known absolute scattering intensity. A
curvature of the angular dependence in a Zimm plot is often observed
in our self-assembled nanoparticles which present hydrodynamic radii
larger than about 100 nm. This curvature becomes largely linearized
by the Berry representation.36 The radius of gyration (Rg) and the second
virial coefficient (A2) were obtained using the equation
by CONTIN analysis for dex40-b-PS5 at 0.05 mg/mL in water. Inset:
Relaxation frequency vs q2.
sulfuric acid and water). The detached replicas were then rinsed with
water and cleaned from copolymer with DMSO/THF mixture, before
being collected on the 200 mesh copper grid. Observations by trans-
mission electron microscopy were made with a FEI Tecnai 12
Microscope working at 120 keV.
Some nanoparticules were directly observed by TEM after evapora-
tion of the solvent. For that purpose, one drop of solution (∼20 µL) at
0.1 mg/mL was deposited on copper EM grid coated with Formvar
film. The solvent was dried at atmospheric pressure at room temperature.
Results and Discussion

  (
Kc 1 1
R(q, c)
)
Mw 6 )
1 + Rg2q2 (1+A2Mwc) (5)
(Kc/∆R(θ))1/2 was plotted against (sin 2(θ/2) + kC), with k being an
adjustable constant. Extrapolation of the experimental data to zero
concentration and zero angle gave the micellar parameters of MW, Rg,
and A2. We only considered the calculated radius of gyration in our
work.
Small Angle Neutrons Scattering (SANS). SANS experiments were
performed at the Léon Brillouin Laboratory (Orphée reactor, Saclay)
on the PACE spectrometer. Two spectrometer configurations have been
used to cover a q range from 5 × 10-3 Å-1 to 0.15 Å-1. The main
parameters used to calculate the corresponding resolution functions37,38
are listed in Supporting Information (SI; Table S1).
The sample (a solution of dex40-b-PS270 at 10 mg/mL in a mixture
DMSO-d/THF-d (40/60)) was introduced into a 5 mm thick rectangular
quartz cell. The blank sample was pure DMSO-d/THF-d (40/60) v/v.
Data treatment was done with the PAsidur software (LLB). Absolute
values of the scattering intensity (I(q) in cm-1) were obtained from
the direct determination of the number of neutrons in the incident beam
and the detector cell solid angle.39 Contribution due to incoherent
scattering of the dex40-b-PS270 solute was determined by plotting q4I(q)
versus q4 of the sample signal. At large q values, this plot is linear and
its slope gives the incoherent contribution of the solute. The magnitude
of this slope was around 0.0065 cm-1 and was subtracted from the
data.
Freeze-Fracture Transmission Electron Microscopy (FFTEM)
Technique. A drop of the water solution of dex-b-PS (0.1 mg/mL)
was placed between two copper planchettes of a sandwich holder and
freezed by plunging into liquid propane. Sample was then fractured at
-150 °C and pressure of the order of 10-6 mBar in a BAF 300 Balzers
apparatus. The fractured surfaces were replicated with platinum
evaporated at a 45° angle, followed by carbon deposition normal to
the fracture surface to increase mechanical strength. The copper
planchettes were dissolved in chromerge (a mixture of chromic acid,
I. Self-Assembly of a 7% w/w PS Fraction dex-b-PS
Copolymer. The solution properties of dex40-b-PS5 with a PS
weight fraction of 7% were first investigated. Dilute solutions
were analyzed by dynamic light scattering (DLS) at different
angles at 25 °C. Figure 1 illustrates the autocorrelation function
obtained at a scattering angle of 90° with the corresponding
CONTIN analysis. Three main time distributions were observed:
the main population exhibits a hydrodynamic radius RH ) 28
nm that was calculated from the diffusion coefficient obtained
by plotting the characteristic relaxation frequency versus q2
(inset in Figure 1). The other populations were not observed at
all the scattering angles and were thus considered as physically
meaningless. Moreover, the few characteristic times that could
be extracted were not representative of a diffusive mode, as
illustrated in Figure S1, SI. The hydrodynamic radius (28 nm)
of the main population is characteristic of micelles formed by
self-assembly. These micelles were found to be stable for weeks.
Given the block copolymer composition (∼7% w/w PS),
core-shell micellar structure with a dextran-based corona
oriented toward the continuous aqueous phase is the expected
morphology and should be at thermodynamic equilibrium.
Indeed, the size and dispersity of these micelles remained
unchanged even after prolonged heating time. The formation
of spherical objects was confirmed by AFM experiments carried
out in a tapping mode (Figure 2): the measured diameter (∼40
nm) being in reasonable agreement with DLS findings.
A comparison of the size of the micelles (RH ∼ 28 nm in
solution) with the characteristic length of the block copolymer
analyzed has been made to check the compatibility with a
core-shell structure. Assuming a fully extended conformation
and average monomer length of 0.25 nm, the contour length
for the PS block would be 1.2 nm. A solvated dextran of the
same molar mass than in our block copolymers, presents a
Micelles and Polymersomes
Figure 2. AFM image obtained from a drop of a solution in water at
5 mg/L of dex40-b-PS5 evaporated on a mica surface.
Figure 3. Evolution of hydrodynamic radius vs DMSO volumic fraction
for different block-copolymers: (O) dex40-b-PS270, (4) dex40-b-PS775.
Lines are guides for the eyes. Dotted curve indicates the area where
no scattered signal could be detected. Schematic morphologies are
inserted in the graph. Red color illustrates dextran and blue color
illustrates polystyrene.
hydrodynamic radius in the range of 2 nm. Therefore, it is
expected that the hydrophilic chains in core-shell micelles adopt
an extended rather than a coil conformation in water.39 Assum-
ing a fully extended or “rod-like” conformation of dextran and
considering a characteristic size of 0.5 nm for each sugar unit,
a value of 20 nm (for DP ) 40) for its contour length is
obtained. This hypothesis is in good agreement with our
experimental observations, confirming the formation of micellar
core shell structure, with dextran chains in their extended con-
formation.
II. Self-Assembly of Larger PS Fraction dex-b-PS (>81%)
Block Copolymers. In the first instance, each solution was
analyzed by DLS at different angles, and the resulting RH were
plotted as a function of the solvent composition (Figure 3). As
the procedure is based on the addition of THF or DMSO to
change the solvent composition, this of course induces dilution
of the solution. We have first checked that dilution had no
influence on the morphology of the nanostructures. This was
realized by diluting up to four times a given solution in which
self-assembled structures were obtained, without changing its
solvent composition and by checking the invariance of hydro-
dynamic radius. We can thus conclude that variations observed
in Figure 3 are exclusively due to the modification of solvent
Biomacromolecules, Vol. 10, No. 1, 2009 35
Figure 4. Autocorrelation function at 90° and time distribution function
after CONTIN analysis for a dex40-b-PS270 in THF region (92% THF).
Frequency relaxation vs q2 are represented in inset.
mixture. Insets in Figure 3 show a schematic representation of
the obtained morphologies in the following three distinct
domains: DMSO region, THF region, and water, as discussed
in the following sections.
In all cases, self-assembly occurred irrespective of the solvent
composition, giving self-assembled structures with hydrody-
namic radii ranging from 70 to 145 nm. For dex40-b-PS270, RH
values of about 140 nm were measured at low DMSO volume
fraction. Upon increasing the DMSO content, no scattered signal
(dotted curve in Figure 3) could be detected from ∼50 to ∼75%
of DMSO. Further addition of DMSO (>80%) brought about
the formation of smaller nanoparticles (RH ) 120 nm). For the
dex40-b-PS775 sample the behavior was similar with hydrody-
namic radii ranging from ∼115 to ∼65 nm, when increasing
the DMSO content. The domain with no scattering signal was
estimated from ∼30 to 45% DMSO. It has to be noted that the
same hydrodynamic radii are obtained, whatever the evolution
of this solvent composition (from DMSO to the THF region or
from THF to the DMSO region), attesting to the reversibility
of the self-assembly process.
Given the sizes of the nanoparticles obtained for dex40-b-
PS775 and dex40-b-PS270 samples and the fact that the RH of dex40-
b-PS775 is smaller than that of dex40-b-PS270, the formation of
spherical core-shell micelles is very unlikely. Thus, further
investigations were carried out to identify the type of nano-
structures formed in THF and DMSO regions.
II-1. BehaVior in THF Region. In a first approach, both DLS
and SLS experiments served to assess the Rg/RH value. This
ratio is well-known to be indicative of the morphology
obtained,36,40 (Rg/RH ∼ 0.77 for a hard sphere, Rg/RH ) 1 for a
vesicle, Rg/RH ) 1.7 for a coil), provided the nanoparticles
present a relatively narrow size distribution. The different
structures were also characterized by AFM and FFTEM. A
relatively narrow distribution of relaxation time was detected
by DLS in all cases. It is worth mentioning that the slow mode
observed in Figure 4 (∼2000 nm) was not reproducible and
not representative of a diffusive mode. This can be attributed
to the existence of few and nonreproducible aggregates and was
therefore considered as physically meaningless. Autocorrelation
function and relaxation frequency versus q2 are illustrated in
Figure 4 for dex40-b-PS270. Similar results are available as ESI
for dex40-b-PS775 (Figure S2, SI).
Values of the hydrodynamic radii extracted from these
experiments are summarized in Table 2. Static light scattering
36 Biomacromolecules, Vol. 10, No. 1, 2009
Table 2. Radius or Gyration, Hydrodynamic Radii, and Polydispersity Indexa
solvent system Rg (nm)
THF region dex40-b-PS270 (92% THF) 200
dex40-b-PS775 (95%THF) 115
DMSO region dex40-b-PS270 (95%DMSO) 250
dex40-b-PS775 (90%DMSO) 77
water dex40-b-PS270 60
dex40-b-PS775 70
a
Given at 90° (cumulant analysis) obtained by dynamic light scattering.
Figure 5. (Top) AFM height micrographs obtained in the THF region
(92%) for (a) dex40-b-PS270 and (b) dex40-b-PS775 (95% THF). (Bottom)
Section profile of (b).
experiments were also performed and a Berry plot was used to
extract the Rg values (SI, Figures S4,5). Size dispersity of the
objects formed, estimated by the cumulant analysis, is also
indicated in Table 2. Considering the light scattering results,
dex40-b-PS775 would afford vesicular morphology (Rg/RH ∼ 1),
the case of dex40-b-PS270 being less clear. Indeed, the ratio Rg/
RH of 1.4 in that case does not correspond to any known
morphology in literature. This can be explained by the relatively
high polydispersity (0.30) of the structures obtained.
To get more insight into the morphologies obtained, AFM
experiments were performed on dex40-b-PS270 and dex40-b-PS775.
As illustrated in Figure 5, for both block copolymers, a donut-
type morphology is observed. In the case of dex40-b-PS270
(Figure 5a, top), it appears that large number of objects are in
the range of ∼100-400 nm in reasonable agreement with the
distribution of RH observed in Figure 4. However, it is difficult
to evaluate the polydispersity of these objects because of their
insufficient number on the picture. In the case of dex40-b-PS775
(Figure 5b, top), less objects are observed, with a characteristic
size between 300 nm and 1 µm, in relative good agreement
with distribution of RH obtained by DLS (Figure S2, SI). Again,
this technique confirms the formation of vesicles (polymer-
somes) that appear as being adsorbed on the surface. The force
induced by the AFM probe causes deformation of the vesicles
as verified by analysis of cross section profiles (Figure 5b,
bottom). Such a profile was drawn using Monte Carlo simula-
tions on adsorbed lipid vesicles.41 This deformation is presum-
ably due to the presence of residual DMSO, which interacts
Houga et al.
RH (nm) Rg/RH expected morphology dispersity index 〈∆Γ2〉/Γ2
145 1.38 polydisperse vesicles 0.30
110 1.04 vesicle 0.20
115 2.17 elongated nanoparticle 0.11
77 1 vesicle 0.10
64 0.94 vesicle 0.074
77 0.91 vesicle 0.10
Figure 6. SANS intensity as a function of q for dex40-b-PS270 in
DMSO-d6/THF-d5 (40/60) mixture (10 mg/mL).
with the inner membrane of dextran playing the role of a
plasticizer and giving the vesicles the aspect of circular plates
on the mica surface. Very similar AFM results have been
obtained on vesicles resulting from the self-assembly of discostic
liquid crystalline molecules42 and diblock codendrimers based
on poly(benzylether) and poly(methallyl dichloride), above the
Tg of both blocks.43 In both cases, results were also interpreted
in terms of deformation of the spherical vesicle following
adsorption on the surface, this deformation being possibly
enhanced by the force of the AFM probe. Based on these
elements, it can be concluded that dex40-b-PS775 and dex40-b-
PS270 self-assemble into vesicles when dissolved as unimers in
a mixture of DMSO and THF and then further diluted with THF
(92% for dex40-b-PS270 and 95% for dex40-b-PS775). It has to be
noted that a few very small objects appearing as non hollow
structures on AFM images could be due to the existence of
spherical micelles which could result from a “residual” kinetic
influence on the micellar structure formation. Due to their size
and their low number compared to the vesicular morphologies,
these objects are not detected in light scattering experiments.
The solvent composition (DMSO/THF 40/60) was also
investigated in the case of the dex40-b-PS270 sample, to gain
more information on the morphology generated before dilution
to a high THF content (>92%). The Rg/RH ratio was found
roughly the same as in the case of the THF-rich solutions (∼1.4),
which is not relevant to draw a clear conclusion about the type
of self-assembled nanostructures. SANS experiments were thus
performed on a solution that was prepared in this case with a
deuterated mixture of solvents (DMSO-d6/THF-d5 40/60 v/v).
The corresponding scattering curve is shown in Figure 6. Due
to the large diameter and high polydispersity of the particles,
the scattered intensity did not present any characteristic oscil-
lation from the form factor that could easily help the data
interpretation. However, this curve presented a clear q-2 slope
of the scattered intensity as a function of q, attesting to the
Micelles and Polymersomes
Figure 7. Autocorrelation function at 90° and time distribution function
after CONTIN analysis for dex40-b-PS270 in DMSO region (95%
DMSO). Frequency relaxation vs q2 is presented in inset.
existence of a flat interface, which is characteristic of vesicle
membranes. We thus conclude that the dex40-b-PS270 copolymer
self-assemble in the DMSO/THF 40/60 solution into vesicles
with its PS chains oriented toward the solvent. The size and
morphology of these vesicles remained unaffected upon addition
of THF.
II-2. BehaVior in the DMSO Region. Each block copolymer
solutions exhibited a transition region where no scattering signal
was detected “entering” either the THF- or the DMSO-rich
regions. This means that morphologies obtained in THF dis-
assemble into free copolymer chains (unimers) when the solvent
mixture solvates efficiently both blocks. These free chains
subsequently self-assemble into reverse nanoparticles when
DMSO content increases. In this case, the nanoparticles consist
of dextran blocks oriented toward the solvent, whereas PS chains
are shielded from the continuous DMSO medium in the inner
part of the nanoparticles. Each solution of the block copolymers
has been studied in the DMSO region by DLS and SLS.
Autocorrelation functions and CONTIN analysis are shown in
Figure 7 for dex40-b-PS270 as an example, the other results being
provided in SI (Figure S3).
All the block copolymers exhibit a single relaxation time with
a relatively narrow size distribution, as indicated by cumulant
analysis (Table 2). The Berry plot are available in SI (Figures
S6, S7). One can note that the characteristic sizes of the
nanoparticles are lower compared to the ones measured in the
THF region. When diluting the THF-rich solution with DMSO,
the morphology of the dex40-b-PS270 is modified as the Rg/RH
ratio stands above 2, a value that is characteristic of an
anisotropic structure. It has to be noted that no rotational
diffusion mode in depolarized DLS experiments was detected,44
meaning that the anisotropy of the nanoparticles formed is
relatively weak. In the case of dex40-b-PS775, a vesicular structure
is expected in DMSO according to its Rg/RH ratio. Unfortunately,
no reliable results could be obtained by an imaging technique
in the DMSO-rich region to confirm these results. Indeed,
evaporating DMSO under vacuum caused the disruption of the
nanoparticles formed. In order to further elucidate the exact
structure, we then tried to transfer the nanoparticles from DMSO
to water.
II-3. BehaVior in Water. Starting from solution containing
95% of DMSO and 5% THF, which means that the dextran
blocks are oriented toward the solvent and PS chains are shielded
Biomacromolecules, Vol. 10, No. 1, 2009 37
Figure 8. TEM image obtained for the dex40-b-PS270 in water (0.1
mg/mL).
Figure 9. Freeze-fractured TEM image of dex40-b-PS775 (down) and
dex40-b-PS270 (up; from water solution, 0.1 mg/mL). Inset: schematic
representation of the fracture and platinum shadowing (arrows).
within the core of the nanoparticles, water was added dropwise
until a total volume fraction of 50% was reached. The
hydrodynamic radii measured under these conditions remained
unchanged upon addition of water onto dex40-b-PS775. In
contrast, for dex40-b-PS270, the RH value decreased from 115 to
64 nm (Figure 7 and Figure S9, SI). This could be ascribed to
a morphology change of the self-assemblies and will be
discussed further. Dialysis was performed against distilled water
to remove all traces of DMSO and THF. Results of SLS and
DLS experiments are given in Table 2, and those drawn from
imaging techniques (TEM and FFTEM) are shown in Figures
8 and 9. After dialysis, no significant change of the hydrody-
namic radius of the nanoparticles could be noted for both block
copolymers.
In water, the Rg/RH ratio is close to one, irrespective of the
block copolymer composition, suggesting a vesicular morphol-
ogy. This confirms the occurrence of a morphological change
from an anisotropic structure to spherical vesicles for dex40-b-
PS270 from its initial DMSO-rich solution, where the Rg/RH was
higher than 2. Hollow spheres with low dispersity are clearly
visible on TEM image (Figure 8), their characteristic sizes being
in good agreement with those obtained by DLS. The replica of
freeze fractured samples of dex40-b-PS775 and dex40-b-PS270 were
further analyzed by TEM (Figure 9). In the case of dex40-b-
PS775, the holes inside the spheres are clearly evidenced by the
presence of a shadow. The fracture seems to occur as schema-
tized in Figure 9. The shadowing by platinum images the
vesicular structure of the sample. For the dex40-b-PS270 nano-
38 Biomacromolecules, Vol. 10, No. 1, 2009
Figure 10. AFM image obtained for cross-linked dex40-b-PS270 nanoparticles from solution DMSO/THF 95/5.
particles, one can notice a black core surrounded by a thick
structured ring. We believe that the particles were just cut off
in the plane as shown in the inset, which leads the inside of the
vesicle to appear black. The images obtained are compatible
with the formation of nanoparticles, exhibiting a vesicular
structure, and thus confirm the results obtained from scattering
experiments.
III. Cross-Linking of the Nanoparticles. To better elucidate
the morphologies obtained in the DMSO-rich region, we
attempted to cross-link them directly from the organic solution.
Indeed, cross-linking is an appropriate method to fix the self-
assembled morphologies and modify their properties such as
their permeability or strength. This can be realized by different
approaches that have been recently reviewed,45 with radical
cross-linking polymerization and chemical reaction between two
functional groups being mainly used. Cross-linking can be
performed within the core 46-50 or in the shell of the micellar
structures.51-60 If the cross-linking of core shell micelles has
been widely investigated, vesicles made from the self-assembly
of block copolymer have also been the subject of such treatment.
61-64
In this work, DVS was used to cross-link the dextran
chains of the self-assembled nanostructures in DMSO/THF
solution. This cross-linker has been successfully used in
polyacrylate derivatives54,65 or to cross-link vesicles made from
the self-assembly of amphiphilic comb-like PEG.66 In DMSO/
THF medium, nanoparticles with a core constituted of dextran
or PS can be obtained reversibly, depending on the composition
of the solvent mixture. The cross-linking of dextran was
successfully accomplished irrespective of the location of dextran
in the nanoparticle (in the inner core or as the shell). To this
end, a solution of dex40-b-PS270 in DMSO/THF (40/60) was
prepared to obtain a vesicle with dextran oriented in the inner
membrane before adding DVS. The evolution of the nanopar-
ticles was then followed by DLS. A slight increase of RH (from
∼140 to ∼155 nm) was observed, which could be explained
by an increase of the rigidity of the dextran membrane, resulting
in a slight decrease of its curvature. Dilution with THF of this
solution left RH almost unchanged, confirming the stability of
the nanoparticles with their PS chains located at the surface
of the vesicle. In contrast, a progressive addition of DMSO
induced the irreversible precipitation of the cross-linked nano-
particles. Indeed, the structure cannot disassemble and form
stable reverse vesicles with dextran oriented toward the continu-
ous medium because of intermolecular cross-linking of dextran.
The same approach was used for nanoparticles presenting
dextran at the surface. A solution of dex40-b-PS270 was prepared
in 90/10 DMSO/THF, thereafter, DVS was added. A marked
increase of the hydrodynamic radius from 115 to 140 nm is
again observed, and the subsequent dilution of this solution with
THF brought about the particles precipitation, attesting to the
efficiency of the cross-linking process.
Houga et al.
Figure 11. AFM image obtained for cross-linked dex40-b-PS270
nanoparticles after dialysis in water.
In the case of dex40-b-PS270, we anticipated the formation of
an anisotropic (elongated) self-assembled structure in DMSO.
This hypothesis could not be confirmed by imaging techniques.
Indeed, as indicated above, the evaporation of DMSO under
vacuum led to a disruption of the nanoparticles. After cross-
linking, this could be done without destroying the nanoparticles.
AFM imaging was then performed as shown in Figure 10. Ovoïd
nanoparticles are clearly observed with characteristic dimensions
of ∼250 nm along the main axis and ∼100 nm perpendicular
to it, confirming the anisotropy and the size measured by
scattering techniques. Detailed information about these aniso-
tropic objects, whether they are filled or hollow structures, have
not yet been obtained. However, as vesicles were formed from
noncross-linked samples upon addition of water and dialysis
(see Figure 9 and Table 2), the formation of hollow anisotropic
objects can be contemplated here. Upon addition of water (up
to 50% v/v) and dialysis, these cross-linked anisotropic nano-
particles became spherical as shown in Figure 11. The charac-
teristic size is in reasonable agreement with the one obtained
by dynamic and static light scattering on the cross-linked system
in water (RH ) 145 nm; Rg ) 210 nm; Rg/RH ) 1.44, see Figure
S11). The Rg/RH ratio is different from what is expected for a
vesicular structure. At this point, it is difficult to conclude on
the morphology obtained in water from cross-linked samples.
We think that vesicular structure with relatively pronounced size
dispersity could be possible. Finally, these results suggest that
in spite of cross-linking, the mobility of some dextran chains
increases when DMSO is replaced by water, the constraints and
the stresses existing in the anisotropic structure being likely
relaxed by a morphology change. For noncross-linked mor-
phologies, the phenomenon is supposed to be the same but with
Micelles and Polymersomes
greater amplitude, as a decrease of hydrodynamic radius from
115 to 65 nm is observed when water is added to a solution of
dex40-b-PS270 in DMSO. Obviously, it is difficult to describe
precisely what really happens at the molecular level during the
transfer from DMSO to water as the morphology in DMSO is
not yet fully elucidated (full or hollow ovoïds).
Conclusions
The self-assembly process of dex-b-PS diblock copolymers
in organic and aqueous media is strongly influenced by their
overall composition. The reversibility of the self-assembly
process (from DMSO to THF and vice versa) demonstrates that
some thermodynamic equilibrium can be reached. Core-shell
micelles are generated in water for a content of dextran equal
to 93% (w/w). In all other conditions, block copolymers self-
assemble into vesicular structures. These morphologies were
not expected and do not correspond to the prediction and
classification proposed by Discher and Eisenberg11 (e.g., for
hydrophilic fraction of 19 and 8%) for coil-coil type block
copolymers, because crew-cut micelles would have been
expected. Although dextran is considered to exhibit a coil
conformation, it can adopt a rod-like conformation for molar
masses below 2000 g/mol.32 Because the average molar mass
of the dextran in this study is equal to 6600 g/mol and its
polydispersity index is around 1.6, it is obvious that some of
the dextran chains are below the 2000 g/mol range and
accordingly adopt a rod conformation. It is well-known in the
self-assembly of rod-coil block copolymer that their stiffness
asymmetry promotes self-assembly into flat membranes and in
diluted state, into vesicles.67-70 Therefore, the slight stiffness
asymmetry induced by the dextran chains that adopt a rod
conformation could play a crucial role in the formation of a
planar bilayer membrane, resulting in the formation of vesicles
either in water, THF, or DMSO. The study of block copolymers
with longer dextran chains and narrow size distribution could
bring interesting information about this aspect.
This study revealed some specificities of dextran-based block
copolymers (unexpected vesicles, ovoïds,...) and that further
studies are needed to get a better insight, especially on the
influence of the dextran conformation to the self-assembly
behavior. Indeed, if vesicular structure could be explained by a
rod conformation of short dextran chains, the origin of the
formation of the ovoı¨d structures is not yet understood. In
addition, the exact role of the dextran chains in the morphologi-
cal transition observed during transfer from DMSO to water
has to be further elucidated.
Morphologies obtained in DMSO could be transferred in
water paving the way of potential applications as carriers or
vehicles. The vesicles (or polymersomes) obtained are particu-
larly interesting because of their ability to encapsulate both
hydrophobic and hydrophilic molecules. The morphologies can
be efficiently cross-linked via the hydroxyl groups of the
dextran, which can be a way to tune the membrane permeability
of these vesicles. Moreover, low concentration regime can be
reached without disruption of the nanoparticles, which could
be an advantage in many applications.
Acknowledgment. The authors would like to thank Prof.
Olivier Mondain Monval (CRPP, Bordeaux) for helpful com-
ments on freeze-fractured TEM images, Michel Schappacher
for AFM measurements, Annie Brûlet (LLB, CEA Saclay) for
SANS measurements, and referees for their helpful comments.
Supporting Information Available. Experimental details
about light scattering measurements, experimental details about
Biomacromolecules, Vol. 10, No. 1, 2009 39
the transfer of the morphologies from organic solvent to water,
as well as dynamic and static light scattering results. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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