Polymer vesicles containing small vesicles within interior aqueous compartments and pH-responsive transmembrane channels

Article abstract of DOI:10.1002/anie.200704078

(Figure Presented) Multivesicle assemblies with pH-responsive transmembrane channels in the vesicle walls (see picture) were made by two-step double emulsion of copolymers comprising acrylic acid and acrylate of 1,2-distearoyl-rac-glycerol. These assemblies mimic eukaryotic cells, which contain functional organelles within the cell walls.

Full text of DOI:10.1002/anie.200704078

Angewandte
Chemie
DOI: 10.1002/anie.200704078
Polymer Vesicles
Polymer Vesicles Containing Small Vesicles within Interior Aqueous
Compartments and pH-Responsive Transmembrane Channels**
Hsin-Cheng Chiu,* Yue-Wen Lin, Yi-Fong Huang, Chih-Kai Chuang, and Chorng-Shyan Chern
Intermolecular packing of amphiphilic block copolymers into
vesicles is of particular interest, owing to the fundamental
importance of such systems as a new class of polymer
assemblies with well-controlled structures and potential
obtained from partial transesterification of poly(N-acryloxy-
succinimide) (poly(NAS)) with distearin and then thorough
hydrolysis of the unreacted NASto AAc units. Polymer
vesicles were prepared by a double emulsion technique in a
water/oil/water (w /o/w ) system, in which the copolymer was
[
1–4]
biomedical applications.
Similar to conventional lipo-
1
2
somes, polymer vesicles usually form a continuous bilayer
structure primarily consisting of the hydrophobic blocks of
copolymers, but they exhibit markedly enhanced stability and
feasibility of incorporating functional groups in response to
dissolved in the organic phase prior to emulsification. The
experimental methods are described in detail in the
Supporting Information. THF/CH Cl solutions of varying
3
ratios, depending on the target vesicle size, were employed as
the organic phase. Either water or buffers in the pH range of
4.0–5.5 were used as both the inner (w ) and outer (w )
[5]
external stimuli. However, the major limitation of polymer
vesicles as biofunctional containers arises from the lack of
permeation pathway for hydrophilic cargoes owing to the
1
2
aqueous phases. The vesicles formed upon the evaporation of
organic solvents in w /o/w emulsions. However, the copoly-
[
6,7]
requirement to maintain the architectural integrity.
The
1
2
vesicles obtained from block co-polypeptides are imparted
responsive channels upon the pH-induced conformational
mers assembled into micelles above pH 5.5 and large
precipitates below pH 4.0. The vesicles were obtained
mainly from copolymer with an average molecular weight
[7]
change of a polypeptide block. Redox control of the
permeability of multilayer microcapsules containing poly-
5
À1
of 2.97 ” 10 gmol and a composition of 9.1 mol% DSA,
unless stated otherwise. Figure 1a confirms that the resultant
assemblies are unilamellar vesicles. The laser scanning con-
focal microscopy (LSCM) image of polymer vesicles in
aqueous suspensions was revealed by the fluorescence of
Nile red associated with the vesicle membranes. The lyophi-
lized vesicles can be observed by scanning electron micro-
scopy (see the Supporting Information). The fact that such
polymer colloids maintain their structural integrity when
subjected to transition from the aqueous to dried state reflects
their robust stability. Transmission electron microscopy
(TEM) examination of the sectioned specimens (ca. 60–90-
nm thickness) of polymer vesicles indicates that the wall
thickness was approximately 25 nm (Figure 1b). The vesicle
[8]
(
ferrocenylsiliane) was reported. Incorporating channel-
forming proteins into the vesicle membranes while fully
retaining the protein functions represents an important
paradigm of equipping polymer vesicles with transmembrane
[
9,10]
channels.
Thus, the transport mechanism, being either
size-selective or substrate-specific, can be tailored by the pore
proteins selected. It is also desirable to have versatile
vesicular assemblies that contain small vesicles within the
interior aqueous compartments in a manner similar to
discrete organelles within eukaryotic cells, which perform
diverse functions and are one of the feature differences from
prokaryotic counterparts. Unfortunately, such assembly struc-
tural control has not yet been achieved. Herein, we show the
first example of polymeric multivesicle assemblies similar to
the architectural arrangement of eukaryotic cells, in which
both the vesicle membranes are equipped with pH-responsive
channels permeable for hydrophilic solutes (Scheme 1).
Copolymers comprising acrylic acid (AAc) and acrylate of
size can be controlled by adjusting either the THF/CH Cl
3
ratio used during emulsification or the DSA content of
copolymers to give vesicles with diameters ranging from 1 to
15 mm. For example, changing the DSA content of copoly-
mers from 9.1 to 13.1 mol% increases the vesicle diameter by
3–4 mm on the average. In contrast, increasing the THF
1
,2-distearoyl-rac-glycerol (distearin acrylate, DSA) were
content of the THF/CH Cl solution from 2 to 20% (v/v)
3
reduces the vesicle size significantly (Figure 2) because of the
increased miscibility with water and the resulting decreased
interfacial tension of the polymer-containing oil droplets in
the aqueous phase.
[
*] Prof. H.-C. Chiu, Y.-W. Lin, Y.-F. Huang, C.-K. Chuang
Department of Chemical Engineering
National Chung Hsing University
Taichung 402 (Taiwan)
Fax: (+886)4-2285-2636
When the ionization of AAc residues increases to some
extent with increasing pH value, the vesicles become equip-
ped with transmembrane channels that are permeable for
hydrophilic solutes. Figure 3 shows that, while transport of
calcein (a water-soluble fluorescence probe) across the
membrane was prohibited at pH 5.0, the probe molecules
freely diffused into the vesicular aqueous compartment when
the external pH value was increased to 8.0. Calcein was then
confined within the compartment simply by adjusting the
E-mail: hcchiu@dragon.nchu.edu.tw
Prof. C.-S. Chern
Department of Chemical Engineering
National Taiwan University of Science and Technology
Taipei 106 (Taiwan)
[
**] This work is supported in part by the National Science Council and
the Ministry of Education of Taiwan under the ATU plan.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2008, 47, 1875 –1878
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1875

Communications
responsive vesicles presented herein
offer an alternative pathway to encap-
sulate chemicals (e.g. globular proteins)
that are incompatible with organic sol-
vents used in vesicle preparation.
A molecular packing model is illus-
trated in Scheme 1. The distearin grafts
of copolymers are packed into discrete
bilayer regions by hydrophobic associa-
tion. The discrete bilayer islets act not
only as nucleation loci for vesicle-wall
formation but also as anchors for effec-
tively stabilizing the membrane struc-
ture. These bilayer islets are surrounded
by un-ionized AAc-rich regions. The
separation of the two distinct regions is
ascribed to the hydrophobicity differ-
ence. With the thickness of the DSA
bilayer islets estimated to be at most
5
nm and that of the vesicle membranes
approximately 25 nm, the AAc-rich
layers residing above and below the
bilayer islets are approximately 10 nm in
thickness on the average. The un-ion-
ized AAc-rich regions contribute mark-
edly to the stabilization of vesicles by
polymer-chain entanglements and cova-
lent connections of polymer backbones
to DSA residues in the bilayer islets in
addition to the extensive hydrogen
bonding and hydrophobic association.
The outermost charged AAc surface
layers prevent vesicles from aggregating.
The un-ionized AAc-rich regions on the
sides of the bilayer islets (parallel to the
aligned lipid chains) are responsible for
forming pH-responsive channels. It is
recognized that the AAc (co)polymers
embedded within sufficiently hydropho-
bic and compact domains are more
Scheme 1. Illustration of multivesicle assemblies equipped with pH-responsive transmembrane
channels from two-stage double emulsion of poly(AAc-co-DSA). The AAc-rich regions and the
bilayer islets within the vesicle membrane are not drawn to scale.
Figure 1. Images of a) polymer vesicles by LSCM and b) sectioned
specimens of vesicles by TEM. The scale bar in the inset of the TEM
image represents 500 nm.
pH value back to 5.0. This pH-responsive on/off process is
reversible, and it relies on change in pH value in a narrow
range between 6.4 and 6.5. These channels are also accessible
to large cargos such as hemoglobin (see the Supporting
Information). Polymer vesicles enclosing myoglobin or
Figure 2. Dependence of the number-average vesicle size on the THF
[
6,11]
hemoglobin as oxygen carriers were reported.
The pH-
content of organic THF/CH Cl solutions in preparing polymer vesicles.
3
1
876
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Angewandte
Chemie
were prepared by postmodification of polymeric precursor, it
is reasonable to postulate that distribution of DSA within the
copolymers occurred in a multiblock manner with pertinent
main-chain spacing from the AAc units (for decoupling the
[
14]
motion of the polymer main chain and the lipid grafts),
thereby promoting the intermolecular association
[
15]
and
formation of lipid bilayers and AAc-rich channels within
vesicle membranes. In contrast, randomly distributed DSA
units along the copolymer backbone may result in quite
different self-assembly behavior. The effects of the structure
of polymeric amphiphiles, especially the hydrophilic–lip-
ophilic balance values and geometric factors, on their
assembly through intermediate micelle structure into varying
[
16]
types of lyotropic mesophases were reported. Apart from
the copolymer of 9.1 mol% DSA, vesicles can also be
obtained from the copolymer of 13.1 mol% DSA in the
same buffer pH-value range, in contrast to micelle-like
assemblies from the copolymer of 4.3 mol% DSA, which
does not form vesicles owing to the insufficient hydrophobic
lipid grafts.
Owing to the vesiclesꢀ excellent stability and controllable
size over a wide range, the multivesicle assemblies shown in
Scheme 1 could be obtained by suspending small vesicles
(
prepared in the first stage) in the w phase of the second-
1
stage double emulsion process that was used to produce large
vesicles. Figure 4a shows the LSCM image of multivesicle
Figure 3. LSCM images of Nile-red-stained vesicle suspensions a) with
the addition of calcein at pH 5.0 (calcein could not enter the vesicle),
b) after pH adjustment to 8.0 (calcein diffused into the vesicle), and
c) after replacement with fresh buffer of pH 5.0 (calcein was confined
within the vesicle). Each multistained image (left) was derived from
combining the two images to its right, in which the upper image was
obtained at l =543 nm and l =560–700 nm (red color arising from
Figure 4. Images of a) multivesicle assemblies (LSCM) and b) a sec-
tioned specimen (ca. 80-nm thickness) of the multivesicle assembly
(TEM).
ex
em
Nile red) and the lower at l =488 nm and l =505–550 nm (green
ex
em
color arising from the combination of calcein and Nile red). The
intense colors of the membranes (yellow on the left photographs or
green on the lower right) were caused by the fluorescence of Nile red
associated with the membranes (see the Supporting Information).
assemblies. The TEM photograph also confirms the architec-
tural arrangement of multivesicle assemblies, as shown by
enclosing two small vesicles within a larger one in Figure 4b.
More LSCM images of multivesicles are illustrated in the
Supporting Information. While the multivesicles can be
readily obtained by the present approach, further control of
the number of small vesicles within a large one is needed.
Such assemblies represent a supramolecular organization
similar to biological eukaryotic cells and their subcellular
organelles. Furthermore, these synthetic vesicles, delicately
implemented with pH-responsive channels, show great poten-
tial as biofunctional encapsulants by providing individual
compartmentalization of varying chemicals within a vesicle
and facile control of their encapsulation and release through
the responses of the vesicle membrane to environmental
stimuli (pH in this case).
[12]
difficult to deprotonate than those in an extended state.
Hence, with increasing pH value of vesicle suspensions, AAc
ionization occurs mainly in the AAc-rich regions (those that
lack DSA), including the transmembrane channels. When the
pH value is increased to 6.5, the channels become permeable
upon abrupt disruption of hydrogen bonds and hydrophobic
association of un-ionized AAc units by the presence of a
critical amount of ionized AAc units within the channels. The
tendency toward forming vesicles is largely influenced by the
distribution of DSA grafts within the copolymers, the hydro-
philic/hydrophobic balance, and the stiffness of copoly-
[
2,13]
mers,
which, in turn, is closely related to the conjugation
approach and the extent of DSA and ionization degree of
AAc units along the polymer backbones. As the copolymers
Although many structural issues remain to be studied,
such functional multivesicular architectures represent a sig-
Angew. Chem. Int. Ed. 2008, 47, 1875 –1878
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
1877

Communications
nificant advance in mimicking eukaryotic cells and extend
Kamei, T. J. Deming, Nat.Mater. 2007, 6, 52; c) L. You, H.
Schlaad, J.Am.Chem.Soc. 2006, 128, 13336.
6] A. Kishimura, A. Koide, K. Osada, Y. Yamasaki, K. Kataoka,
Angew.Chem. 2007, 119, 6197; Angew.Chem.Int.Ed. 2007, 46,
potential applications of polymer vesicles. Further studies
concerning the effects of molecular parameters on perfor-
mance properties of vesicle assemblies and precise control of
the multivesicle morphology, such as the number of small
vesicles in large ones and channel size, are in progress.
[
6085.
[
7] E. G. Bellomo, M. D. Wyrsta, L. Pakstis, D. J. Pochan, T. J.
Deming, Nat.Mater. 2004, 3, 244.
[
8] Y. Ma, W.-F. Dong, M. A. Hempenius, H. Möhwald, G. J.
Vancso, Nat.Mater. 2006, 5, 724.
Received: September 4, 2007
Revised: November 8, 2007
Published online: January 4, 2008
[9] a) P. Broz, S. Driamov, J. Ziegler, N. Ben-Haim, S. Marsch, W.
Meier, P. Hunziker, Nano Lett. 2006, 6, 2349; b) A. Ranquin, W.
Versees, W. Meier, J. Steyaert, P. V. Gelder, Nano Lett. 2005, 5,
2
220; c) A. Graff, M. Sauer, P. V. Gelder, W. Meier, Proc.Natl.
Keywords: biomimetic systems · multivesicles · self-assembly ·
transmembrane channels · vesicles
.
Acad.Sci.USA 2002, 99, 5064; d) M. Sauer, T. Haefele, A. Graff,
C. Nardin, W. Meier, Chem.Commun. 2001, 2452.
[
10] a) M. Winterhalter, C. Hilty, S. M. Bezrukov, C. Nardin, W.
Meier, D. Fournier, Talanta 2001, 55, 965; b) R. Stoenescu, A.
Graff, W. Meier, Macromol.Biosci. 2004, 4, 930; c) C. Nardin, J.
Widmer, M. Winterhalter, W. Meier, Eur.Phys.J.E 2001, 4, 403;
d) A. Mecke, C. Dittrich, W. Meier, Soft Matter 2006, 2, 751.
11] D. R. Arifin, A. F. Palmer, Biomacromolecules 2005, 6, 2172.
12] O. E. Philippova, D. Hourdet, R. Audebert, A. R. Khokhlov,
Macromolecules 1997, 30, 8278.
[
[
[
1] a) D. E. Discher, A. Eisenberg, Science 2002, 297, 967; b) J.-M.
Lehn, Science 2002, 295, 2400.
2] a) M. Antonietti, S. Förster, Adv.Mater. 2003, 15, 1323; b) G.
Battaglia, A. J. Ryan, J.Phys.Chem.B 2006, 110, 10272.
3] a) R. Stoenescu, W. Meier, Chem.Commun. 2002, 3016; b) O.
Uzun, H. Xu, E. Jeoung, R. J. Thibault, V. M. Rotello, Chem.
Eur.J. 2005, 11, 6916; c) Y. Li, B. S. Lokitz, C. L. McCormick,
Angew.Chem. 2006, 118, 5924; Angew.Chem.Int.Ed. 2006, 45,
[
[
[
13] H. J. Lee, S. R. Yang, E. J. An, J.-D. Kim, Macromolecules 2006,
3
9, 4938.
5792; d) H.-J. Choi, C. D. Montemagno, Nano Lett. 2005, 5, 2538;
[
14] L. Laschewsky, H. Ringsdorf, G. Schmidt, J. Schneider, J.Am.
Chem.Soc. 1987, 109, 788.
15] Y. Chang, C. L. McCormick, Macromolecules 1993, 26, 6121.
16] a) H. Ringsdorf, B. Schlarb, J. Venzmer, Angew.Chem. 1988,
e) E. Donath, G. B. Sukhorukov, F. Caruso, S. A. Davis, H.
Möhwald, Angew.Chem. 1998, 110, 2323; Angew.Chem.Int.Ed.
[
[
1998, 37, 2201.
[
4] a) C. Dufes, A. G. Schätzlein, L. Tetley, A. I. Gray, D. G. Watson,
1
00, 117; Angew.Chem.Int.Ed.Engl. 1988, 27, 113; b) C. Viney,
D. Y. Yoon, B. Reck, H. Ringsdorf, Macromolecules 1989, 22,
088.
J.-C. Olivier, W. Couet, I. F. Uchegbu, Pharm.Res. 2000, 17,
1250; b) F. Ahmed, D. E. Discher, J.Controlled Release 2004, 96,
4
37.
[
5] a) A. Napoli, M. Valentini, N. Tirelli, M. Müller, J. A. Hubbell,
Nat.Mater. 2004, 3, 183; b) E. P. Holowka, V. Z. Sun, D. T.
1
878
www.angewandte.org
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