Sweet block copolymer nanoparticles: Preparation and self-assembly of fully oligosaccharide-based amphiphile

Article abstract of DOI:10.1021/bm3000138

The preparation of biocompatible nanocarriers that have potential applications in the cosmetic and health industries is highly desired. The self-assembly of amphiphilic block copolymers displaying biosourced polysaccharides at the surface is one of the most promising approaches. In the continuity of our works related to the preparation of "hybrid" amphiphilic oligosaccharide-based block copolymers, we present here the design of a new generation of self-assembled nanoparticles composed entirely of oligosaccharide- based amphiphilic block co-oligomers (BCO). These systems are defined by a covalent linkage of the two saccharidic blocks through their reducing end units, resulting in a sweet "head-tohead" connection. As an example, we have prepared and studied a BCO in which the hydrophilic part is composed of a free maltoheptaosyl derivative clicked to a hydrophobic part composed of a peracetylated maltoheptaosyl derivative. This amphiphilic BCO self-assembles to form spherical micelles in water with an average diameter of 30 nm. The efficient enzymatic hydrolysis of the maltoheptaose that constitutes the shell of the micelles was followed by light scattering and colorimetric methods.

Full text of DOI:10.1021/bm3000138

Article
pubs.acs.org/Biomac
Sweet Block Copolymer Nanoparticles: Preparation and Self-
Assembly of Fully Oligosaccharide-Based Amphiphile
Samuel de Medeiros Modolon, Issei Otsuka, Sebastien Fort, Edson Minatti,† Redouane Borsali,*
́
and Sami Halila*
Centre de Recherches sur les Macromolec
́ ́ ́ ́
ules Vegetales, CERMAV-CNRS (Affiliated with Universite Joseph Fourier, and member of
the Institut de Chimie Moleculaire de Grenoble), BP 53, 38041 Grenoble cedex 9, France
́
ABSTRACT: The preparation of biocompatible nanocarriers that
have potential applications in the cosmetic and health industries is
highly desired. The self-assembly of amphiphilic block copolymers
displaying biosourced polysaccharides at the surface is one of the
most promising approaches. In the continuity of our works related
to the preparation of “hybrid” amphiphilic oligosaccharide-based
block copolymers, we present here the design of a new generation
of self-assembled nanoparticles composed entirely of oligosacchar-
ide-based amphiphilic block co-oligomers (BCO). These systems
are defined by a covalent linkage of the two saccharidic blocks
through their reducing end units, resulting in a sweet “head-to-
head” connection. As an example, we have prepared and studied a BCO in which the hydrophilic part is composed of a free
maltoheptaosyl derivative clicked to a hydrophobic part composed of a peracetylated maltoheptaosyl derivative. This amphiphilic
BCO self-assembles to form spherical micelles in water with an average diameter of 30 nm. The efficient enzymatic hydrolysis of
the maltoheptaose that constitutes the shell of the micelles was followed by light scattering and colorimetric methods.
coupled to a functional synthetic polymer block. For instance,
we have reported the thermo-responsive PNIPAM-b-malto-
heptaose block copolymers that self- assembled into narrow
vesicular morphologies in water.12 It was noteworthy that the
strong interfacial incompatibility represented by the χN
(Flory−Huggins constant) between the natural block and the
synthetic block enables such self-organization even for low
molecular weight oligosaccharide.
This result prompted us to design a new class of linear
amphiphilic block co-oligomers (BCO) exclusively constituted
of biocompatible and biodegradable saccharidic blocks. As an
example, we describe herein the preparation and physicochem-
ical studies of a BCO consisting of a bare maltoheptaose
derivative as the hydrophilic block “clicked” to its peracetylated
maltoheptaose form as the hydrophobic block. Maltoheptaose 1
is a linear α-(1,4) glucan of seven glucosyl units obtained from
the ring-opening of β-cyclodextrin and can be hydrolyzed by
starch-degrading enzymes such as glucoamylase or α-amylase.
The hydrophobic block was generated by “masking” the
hydroxyl groups thanks to ester linkage-mediated hydrophobic
protecting groups. Then, the BCO was synthesized by
regioselective modification of each block and then linked
through Cu(I)-catalyzed azide−alkyne cycloaddition
(CuAAC).13 The self-assembly behavior in aqueous solution
INTRODUCTION
■
The sustained interest in amphiphilic block copolymers (BCPs)
arises from their ability to self-assemble into a wide variety of
morphologies in solution and in thin films, leading to numerous
applications such as drug delivery systems,1,2 nanoreactors,3,4
and nanotemplates.5,6 Nanoparticles formed from BCPs are
one of the most promising nanocarriers for delivery of
hydrophobic or hydrophilic active molecules.7 In such
applications, some important parameters have to be considered,
such as (i) the use of biocompatible and safe biodegradable
polymers and (ii) the narrow size distributions of nanoparticles
in the size range of 10−100 nm that are ideal for intravenous
injection because they are much smaller than the inner
diameter (≥4 μm) of blood capillaries.8,9 Thus, many
polymeric materials have been reported including poly(L-lactic
acid), poly(ε-caprolactone), and polypeptides that constitute
the hydrophobic inner core, while poly(ethylene oxide),
polypeptides, and, more rarely, polysaccharides constitute the
hydrophilic outer shell. Most of the combinations between
these hydrophobic and hydrophilic polymers have been
recently reviewed.10 Among them, polysaccharides are the
most interesting biopolymers because they are abundant,
derived from renewable resources, and display various natural
biological activities. Typically they are involved in inflamma-
tion, cell interactions, pathogen host adhesion, signal trans-
duction development, and a myriad of other processes.11
Very recently, we have carried out work on the preparation
and the study of the self-assembly of different “hybrid” block
copolymers, based on a well-defined sized oligosaccharide block
Received: January 4, 2012
Revised: March 2, 2012
Published: March 7, 2012
© 2012 American Chemical Society
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Scheme 1. Synthetic Route to Mal7-“click”-AcMal7 (4) Block Co-oligomer
at 90°. The aqueous solutions of Mal7-“click”-AcMal7 (4) were filtered
directly into the glass cells through a 0.45 μm Millipore Millex PES
hydrophilic filter. Data were collected using digital the ALV Correlator
Control software. The relaxation time distributions A(t) were obtained
using Contin analysis of the acquired autocorrelation functions C(q,t).
Morphological examination of the micelles was conducted using a
Kodak SO163 film using a CM200 Philips transmission electron
microscope operating at 18 kV. One drop of micelle suspension was
placed on a copper mesh covered with nitrocellulose membrane and
dried in air before being stained with phosphotungstic sodium solution
(1% w/v). An atomic force microscope (AFM PicoPlus; Molecular
Imaging) was used to study the surface morphology of the
nanoparticles. A 1 drop aliquot of properly diluted micelles was
placed on the surface of a clean silicon wafer (ABC, Munich,
Germany) and dried under nitrogen flow at room temperature. The
AFM observation was performed in tapping mode, with a silicon
cantilever of resonant frequency 145−230 kHz.
Synthesis of AcMal7alkyne (3). The synthesis of N-
(2I−VII,3I−VII,4VII,6I−VII-docosa-O-acetyl-β-maltoheptaosyl)-3-acetami-
do-1-propyne (3, AcMal7alkyne) was prepared by a method modified
from a previously published procedure.12 Briefly, a suspension of
maltoheptaose (10.0 g, 8.67 mmol) in neat propargylamine (11.9 mL,
174 mmol) was stirred vigorously at room temperature for 72 h. The
reacting mixture was dissolved in MeOH and then precipitated in
CH2Cl2. The solid was filtrated and washed with a mixture of MeOH/
CH2Cl2 (1:3, v/v). The acetylation was carried out by mixing the solid
in pyridine with acetic anhydride. The reaction mixture was evaporated
was investigated and its sensitivity to in vitro enzymatic
hydrolysis was carried out.
EXPERIMENTAL SECTION
■
Materials. β-Cyclodextrin, sodium ascorbate, copper sulfate, and
amyloglucosidase (10115, 67.4 U/mg) were purchased from Sigma-
Aldrich and used as received. Water was purified by a Milli-Q water
purification system (Billerica, MA, U.S.A.). Maltoheptaose and β-
maltoheptaosyl azide (2, Mal7N3) were prepared according to the
literature.14,15
Instrumentation. Reactions were followed by thin-layer chroma-
tography on silica gel 60F254 (E. Merck) by charring with diluted
H2SO4. Flash-column chromatography was performed on silica gel 60
(230−400 mesh, E. Merck) or size-exclusion chromatography on a
1
HW40 Toyopearl. H NMR spectra were recorded using 400 MHz
Bruker Avance DRX400. Infrared (IR) spectra were recorded using a
Perkin-Elmer Spectrum RXI FTIR Spectrometer. MALDI-TOF
measurements were performed on a Bruker Daltonics Autoflex
apparatus. Fluorescence spectra were recorded on a LS-50B Perkin-
Elmer spectrofluorimeter, equipped with a thermostatted cell holder.
Characterization. Mean diameter and the light scattering intensity
of the micelles suspension were determined, both by static and
dynamic light scattering (SLS and DLS) methods. Measurements were
performed using an ALV laser goniometer, which consists of 35 mW
HeNe linear polarized laser operating at a wavelength of 632.8 nm, an
ALV-5004 multiple τ digital correlator with 125 ns initial sampling
time, and a temperature controller. The measurements were recorded
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under reduced pressure and the residue was purified by flash
chromatography on silica gel (eluent EtOAc−petroleum ether, 70:30
→ 90:10) to afford 3 as a white solid, 9.4 g (50%). Rf = 0.4 (7:3
EtOAc−petroleum ether); IR (KBr): ν = 3100−2700 (C−H, sugars),
of β-cyclodextrin using Ac2O/FeCl3 then deacetylation under
Zemplen conditions (catalytic amount of sodium methoxide in
́
methanol) as reported by Liptak et al.14 Initially the free
reducing oligosaccharide Mal7 (1) was chemoselectively
functionalized with a propargylamine linker12,16,17 and then
peracetylated to give AcMal7alkyne (3; Scheme 1). In the
second step of our strategic pathway, the maltoheptaosyl azide,
Mal7N3 (2), was prepared according to Shoda’s methodology.15
The 2-chloro-1,3-dimethylimidazolinium chloride (DMC)
reagent was demonstrated to be an excellent dehydrating
agent for direct activation of the anomeric hydroxyl group of
unprotected sugars in aqueous media. The subsequent
substitution by azide ions led to the direct synthesis of β-
glycosyl azides on various sugars with very good yields. A major
concern with this approach is the use of huge quantities of
reagent salts and bases, which makes the purification difficult,
especially on a large scale. Recently, Vinson et al.18 have
proposed a purification procedure (precipitation and acidic ion-
exchange column) that has been ineffective in our hands. Our
purification was achieved conveniently by successive steps of
acetylation and deacetylation leading to 2 with 77% overall
yield.
1
1644 (CO, esters, amide) cm−1; H NMR (400 MHz, CDCl3): δ=
5.93 and 5.40 (d, J1−2= 9.35 Hz, 1H, rotamers; H-1GlcI,Ac), 5.40 (m,
GlcI‑VII,Ac
6H; H-1GlcII‑VII,Ac), 5.30−4.80 (m, 44H; H-2, 3, 4, 5, 6
and
NCH2), 2.32−1.96 ppm (m, 70H; CH3 (OAc and NAc) and C
CH); MALDI-TOF: M + Na+ m/z: 2178.46.
Synthesis of Mal7-“click”-AcMal7 (4). The synthesis of 4-
[maltoheptaosyl]-1-[N-acetyl-N-(2I−VII,3I−VII,4VII,6I−VII-docosa-O-ace-
tyl-β-maltoheptaosyl)]-5H-[1,2,3]-triazole (4, Mal7-“click”-AcMal7)
was performed by mixing 2 (270 mg, 0.24 mmol) and 3 (500 mg,
0.24 mmol) in water/t-BuOH (1:1 v/v) and by adding sodium
ascorbate (95 mg, 0.48 mmol) and 0.1 m copper(II) sulfate (2.5 mL,
0.25 mmol). The reaction mixture was stirred at 50 °C for 24 h. The
reaction mixture was then evaporated in the presence of silica and the
crude solid purified by flash column chromatography (eluent:
CH3CN−H2O, 90:10 → 80:20) to yield the “clicked” product 4 as
a white solid, 500 mg (63%). Rf = 0.42 (7:3 CH3CN-H2O); IR (KBr):
ν 3600−3100 (O−H, sugars), 3100−2700 (C−H, sugars and alkyl),
1
1644 (CO, esters, amide) cm−1; H NMR (400 MHz, THF-d8/
D2O): δppm 8.29 and 8.17 (2x s, 1H rotamers; H-5triazole), 5.85 (m, 1H;
H-1GlcI,Ac), 5.60 (m, 6H; H-1GlcII‑VII,Ac), 5.00 (m, 6H; H-1GlcII‑VII), 4.60
(m, 1H; H-1GlcI), 5.40−3.50 (m, 86H; H-2, 3, 4, 5, 6 GlcI‑VII, and GlcI‑VII,Ac
and NCH2), 2.50−1.80 (m, 69H; CH3 (OAc and NAc)); HRMS (ESI,
m/z): [M + Na]+ Calcd for C133H192N4O93Na, 3356.0315; Found,
3356.0270.
In the final step of the synthesis, the Mal7N3 (2) was
“clicked” with AcMal7alkyne (3) to produce the block co-
oligomer Mal7-“click”-AcMal7 (4) with a good yield (64%). The
later was purified by liquid chromatography on silica gel and
then freeze-dried. We are aware that the original form of the
CuAAC reaction requires a copper catalyst that is toxic at high
micromolar concentrations to cells and organisms. As a result,
we have taken care to remove most of the copper salts
especially using flash chromatography purification. But we note
that one can use other catalytic systems,19,20 even Cu-free
CuAAC21 that could solve the potential problem of Cu toxicity.
The FT-IR spectra showed that the azide group within,
Mal7N3 (2) at 2105 cm−1 (Figure 1b), completely disappeared
Enzymatic Activity. A total of 3 mg/mL of Mal7-“click”-AcMal7
(4) was incubated with 6 μL of glucoamylase from Aspergillus niger
(0.15 mg/mL) in 0.1 M of lithium chloride at 40 °C.
For the bicinchoninic acid (BCA) assay, the reaction tubes were
incubated at 40 °C in a reciprocal shaker where 36 μL of sample was
regularly taken and diluted up to 500 μL then immediately mixed with
BCA reagent (500 μL). The assay is based on the reduction of Cu(II)
to Cu(I). The subsequent complexation of Cu(I) and BCA at alkaline
pH and elevated temperatures produces an intense purple color. In all
experiments, samples of 500 μL were incubated with 500 μL of reagent
and heated for 35 min to 75 °C in an oven. The eppendorf tubes were
cooled on ice after this and the absorbance was measured at 550 nm
using a Bio-Rad 680 microplate reader.
For the follow in SLS and DLS, the biodegradation was conducted
at 40 °C inside the cuvette to measure in situ the effective diameter
and light scattering intensity with time.
RESULTS AND DISCUSSION
■
Synthesis and Structural Characterization of Mal7-
“click”-AcMal7 (4). One of the major problems during the
synthesis of amphiphilic molecules is the solubility arising from
the different nature of the two blocks. By using proper solvent
combinations such as N,N-dimethylformamide or hydro-
organic medium (acetone, THF, alcohol), the solubility of
both blocks can be achieved. The CuACC reaction is perfectly
adapted for such solvent systems and since it is highly
chemoselective and compatible with many functional groups
such as the hydroxyl groups of polysaccharides, CuAAC is
particularly relevant. To address the CuAAC reaction, we
introduced the azide and alkyne function at the reducing end of
maltoheptaose leading to Mal7N3 (2) and AcMal7alkyne (3),
respectively. This method results in a head-to-head structure,
thereby forming the so-called rod−rod block co-oligomers. It is
clearly advantageous to directly modify native unprotected
sugar because this opens up an avenue for future incorporation
of natural polysaccharides without extensive synthetic manip-
ulations.
Figure 1. IR spectra of (A) Mal7N3 (2) and (B) Mal7-“click”-AcMal7
(4).
in Mal7-“click”-AcMal7 (4; Figure 1). Moreover, the spectra
demonstrated the presence of the acetyl group, as indicated by
CO stretching at 1752 cm−1 and the broad band at 3400
cm−1 confirmed hydroxyl groups of the free maltoheptaosyl
block.
Figure 2a shows the 1H NMR spectrum of BCO in THF-d8/
D2O obtained using the “click” reaction. In such a good solvent
for both maltoheptaosyl blocks, the proton signals can be
clearly seen and the dynamic light scattering (DLS) showed
that Mal7-“click”-AcMal7 (4) essentially exists as unimers whose
Maltoheptaose (1) denoted by Mal7 was the key saccharidic
block of our synthetic pathway. It was obtained by ring-opening
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0.1 mg/mL. The CMC value is within the range reported for
low molecular weight nonionic surfactants such as n-
dodecylmaltoside (0.097 mg/mL) or pentaethyleneglycol-
monododecylether (0.027 mg/mL).22 In the field of drug
delivery vehicles, this small value is promising and allows us to
anticipate the efficient in vivo performance and integrity of the
micelles even after dilution in bloodstream.
The self-assembly properties of Mal7-“click”-AcMal7 (4) were
investigated in order to highlight their micellar behavior.
Nanoparticles were prepared by direct dissolution in deionized
water to reach a 0.3 wt % concentration. The size and
morphology of Mal7-“click”-AcMal7 (4) were probed by DLS
measurements (Figure 4a) through the angular dependence
(q2-behavior) of the decay rates of the autocorrelation functions
(Figure 4b). The results showed that the BCO self-aggregates
in water to form micelles with a hydrodynamic diameter of
about 56 nm. The small deviation of the data from the linear
fitting in Figure 4b is caused by the very small polydispersity in
size of the micelles.
1
Figure 2. H NMR spectra of Mal7-“click”-AcMal7 (4) in THF-d8/
D2O (1:1 v/v) (above) and D2O (below).
The TEM images of Mal7-“click”-AcMal7 (4) micelles
(Figure 5a) indicate that the micelles had approximately
spherical morphology and a smooth surface with minimal
aggregation, and the average sizes of the micelles were about 27
4 nm. The investigation by the AFM height image of the
micelles was also carried out (Figure 5b). Separate and
randomly spherical particles are deposited on the mica surface
and appear to be comparatively uniform in size. The height
profile was determined along the horizontal line crossing
several micelles, as seen in the AFM image (white line). Cross-
sectional analysis indicated a micelle diameter of 30−35 nm, in
good agreement with those obtained using TEM.
The difference in the diameter of the spherical micelles
measured using TEM or AFM (∼30 nm) comparatively to DLS
(∼56 nm) can be accounted for considering the conditions of
observations since in DLS the corona of the micelles is fully
solvated while in TEM and AFM the nanoparticles are in dry
state. Both the morphologies and the sizes of the micelles did
not change within several weeks.
Degradation Behavior of Glucoamylase-Catalyzed
Mal7-“click”-AcMal7 Micelles. The presence of free malto-
heptaose block at the shell of the micelles offers one possibility
of enzymatic-mediated drug release. Glucoamylase or amylo-
glucosidase (EC 3.2.1.3) catalyzes the release of glucose from
the nonreducing ends of starch and related maltodextrins23
such as maltoheptaose (Mal7). Accordingly, the studies of
glucoamylase-catalyzed degradation of Mal7-“click”-AcMal7 (4)
size are a few nm (results not shown). The BCOs exhibited
characteristic peaks of free and acetylated maltoheptaose. The
N-acetyl group around 2.4 ppm displayed restricted rotations
(rotamers), giving rise to a multiplicity of signals in the proton
NMR spectra. Moreover, the 1H NMR spectra showed
resonances at δ ≈8.2−8.3 ppm, corresponding to the olefinic
proton associated with the 1,4-disubstituted 1,2,3-triazole
isomer moiety indicating good agreement with assigned
structure.
Self-Assembly of Mal7-“click”-AcMal7 Co-oligomer in
Water. The micelle formation of the diblock co-oligomers in
an aqueous condition was first confirmed by comparing the 1H
NMR spectra in THF-d8/D2O (1:1, v/v) and D2O (Figure 2b).
The spectrum recorded in D2O shows clear signals for the
hydrophilic Mal7 shell while those for the hydrophobic core
AcMal7 and the triazole group are less intense and even
disappeared. This indicates that the aggregated AcMal7 blocks
are distributed preferably inside a confined core of the micelles
resulting in restricted molecular motion compared to Mal7
blocks that are located at the outer shell of the micelles in an
aqueous phase.
The CMC value for Mal7-“click”-AcMal7 (4) was determined
by the pyrene fluorescent probe method. The measurements of
the pyrene fluorescence intensity ratios at I1/I3 were measured
(Figure 3). This ratio was plotted as a function of BCO
concentration to determine the CMC value and the results gave
Figure 3. Pyrene fluorescence emission spectra and the corresponding variation in the I1/I3 ratio as a function of Mal7-“click”-AcMal7 (4)
concentration ([pyrene] = 6.10−7 mol/L).
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Figure 4. (A) Dynamic light scattering autocorrelation function (circle) and relaxation-time distribution (solid line) of Mal7-“click”-AcMal7 (4) in
water (conditions: Mal7-“click”-AcMal7 (4) = 3 g·L−1; scattering angle, θ = 90°; temperature, 25 °C). (B) Variation of the relaxation frequency (Γ) vs
the square of the wave vector (q2).
Figure 5. (A) Transmission electron micrograph (bar 200 nm) with size distribution and (B) tapping mode AFM phase images with sectional
analysis of Mal7-“click”-AcMal7 micelles (4; 3 mg/mL).
were done by static and dynamic light scattering (SLS and
DLS) measurements and by reducing sugar assays. The change
in light scattering intensity and hydrodynamic size of Mal7-
“click”-AcMal7 micelles related to the action of glucoamylase
were used to probe the enzymatic hydrolysis. Figure 6a shows
an increase of the relative scattering intensity that levels off after
2 h. At this stage, one visually observes a precipitation in the
reaction mixture. In Figure 6b, the time-dependent average
hydrodynamic radius of the micelles indirectly shows that there
is a change in their molar mass during the biodegradation. It is
noteworthy that this behavior is very different from a
degradation of the hydrophobic block where the intensity
decreases and the micelle size remains unchanged.24 During the
enzymatic degradation, the shell, consisting of maltoheptaose
(DP 7), is hydrolyzed to smaller maltooligosaccharides (DP <
7), resulting in a decrease of important factors of stability such
as the steric repulsion of the shell and the interfacial free energy
between the water and AcMal7 core. These degraded micelles
with higher free energy are unstable in the aqueous solution
and must reassemble to form larger aggregates to minimize
their free energy that ultimately precipitates.
Alternatively, the degradation in the same conditions of Mal7-
“click”-AcMal7 micelles by glucoamylase was followed by BCA
(bicinchonic acid) method that is colorimetric assay, which
measures the concentration of reducing end-groups as shown in
Figure 7. The BCA assay has been shown to give a good
accuracy with low concentration of glucose and relatively
uniform values when applied to oligosaccharides derived from
starch, polygalacturonic acid, and chitin, independently of the
degree of polymerization.25 Initially, the hydrolysis rate was
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Figure 6. (A) Enzymatic hydrolysis time dependence of relative raw scattering intensity at 90° and (B) of the hydrodynamic radius with 3 mg/mL
Mal7-“click”-AcMal7 micelles (4). The error bars correspond to the width of the CONTIN size distribution peaks. After 1 h, the correlation function
was too bad to obtain a CONTIN fit.
future studies, we envisage a more detailed unraveling of the
mechanism of the micellization process and biodegradation.
Work on biological properties such as cytotoxicity, encapsula-
tion, vectorization, and so on will also be carried out.
AUTHOR INFORMATION
■
Corresponding Author
*Tel.: +33 4 76 03 76 66 (S.H.); +33 4 76 03 76 40 (R.B.). Fax:
+33 4 76 54 72 03 (S.H.); +33 4 76 03 76 29 (R.B.). E-mail:
sami.halila@cermav.cnrs.fr (S.H.); borsali@cermav.cnrs.fr
(R.B.).
Present Address
†Laboratory of Polymer and Surfactant Solutions, Federal
University of Santa Catarina, Brazil.
Figure 7. Variation of the absorbance over time when 3 mg/mL of
Mal7-“click”-AcMal7 micelles (4) were hydrolyzed by glucoamylase.
The experiment was carried out in duplicate and the values represent
the average.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
linear with time but decreased gradually after 30 min. After 75
min, the hydrolysis rate increased again linearly with time and
then decreased once more after 120 min. We suggest that in the
first instance the Mal7-containing shell is well-hydrolyzed by the
glucoamylase until it becomes less and less accessible. In the
second instance, the reassembly might allow a new cycle of
hydrolysis up to complete degradation or inaccessible
substrates.
■
Dedicated to Dr. Hugues Driguez on the occasion of his 70th
birthday. We are grateful for the help of I. Pignot-Paintrand and
A. Durand-Terrasson in the technical assistance with TEM and
C. Travelet for dynamic light diffusion experiments. We thank
S. Boullanger for MALDI-TOF MS analysis and D. Lesur for
ESI-HRMS. We acknowledge the financial support from the
CNRS and CAPES-COFECUB (Project 620/08).
CONCLUSION
■
REFERENCES
■
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dx.doi.org/10.1021/bm3000138 | Biomacromolecules 2012, 13, 1129−1135

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dx.doi.org/10.1021/bm3000138 | Biomacromolecules 2012, 13, 1129−1135