Angewandte
Chemie
The cellular environment can be regarded as a highly complex
synthetic medium, in which numerous multistep reactions
take place simultaneously with unsurpassed efficiency and
specificity. Nature employs several approaches to ensure the
integrity of these, mostly enzyme-catalyzed, synthetic path-
ways, one of the most important ones being compartmental-
ization. This approach, which isolates the catalytic cycle,
prevents interference by other compounds and enables
regulation of the flux of molecules in and out of the
microenvironment. Furthermore, to be efficient the biomo-
lecular catalysts need not only to be separated but also
positioned at specific sites within the cell.
because of the larger dimensions of the amphiphilic block
copolymers and their lower critical aggregation concentra-
[
10]
tion. The diffusion of water through their membranes is
also slower as a result of the larger thickness of these
[
10]
membranes.
To resolve this problem, researchers have
[
16]
incorporated channel proteins
polymersome membranes.
and proton pumps in
[
17]
In recent reports we have described the synthesis and
properties of the diblock copolymer polystyrene -b-poly(l-
4
0
isocyanoalanine(2-thiophen-3-yl-ethyl)amide)50 (PS–PIAT),
which consists of a rigid polyisocyanide block and a flexible
polystyrene tail, thus making it a rod–coil type of diblock
[
18,19]
Many studies have been reported in the literature with the
objective of mimicking this natural concept of enzyme
copolymer.
shown to form stable polymersomes, which could be used to
This well-defined block copolymer was
[
1–4]
[18,19]
assembly and encapsulation,
being based on phospholipid liposomes.
with the earliest examples
encapsulate a variety of guests.
A unique feature of these
[
5–8]
A major problem
polymersomes is that they are sufficiently porous by them-
selves to allow diffusion of small molecules across their
membranes, while large molecules, such as enzymes, remain
that accompanies the use of liposomes is their relative
thermodynamic and mechanical instability. To overcome
this limitation, other methods of enzyme compartmentaliza-
[
18]
trapped inside. These polymersomes, therefore, are ideal to
be used for compartmentalization, as they give protection to
the enzymes inside, whereas low-molecular-weight substrates
and products can diffuse in and out of the polymersomes.
Until now, in all cases enzyme encapsulation has been
obtained without a high level of control over positional
assembly. To mimic nature more closely it would be desirable
not only to encapsulate enzymes, but also to position different
types of enzymes in separate domains within the polymer-
some, for example, in the water pool and in the polymersome
[
1,2]
tion based on sol–gel chemistry
sition have been developed.
and layer-by-layer depo-
[
9]
Another method more closely related to the liposome
approach is one that makes use of block-copolymer amphi-
philes to construct a closed environment, a so-called polymer-
[
10]
some.
These block copolymers have the same basic
architecture as lipids, in that they possess a hydrophilic head
group and a hydrophobic tail. The almost unlimited variety in
monomers and polymerization methods makes it possible to
[
11–15]
[20]
precisely tune the properties of the polymersomes.
In
membrane. Herein, we describe a procedure to achieve this
general, polymersomes are less dynamic than liposomes,
aim. To demonstrate that our method is generic, three types of
enzymes were selected as candidates for inclusion, that is,
Candida antarctica lipase B (CALB), horseradish peroxidase
[
+]
[#]
[$]
[
*] Dr. P. M. L. Garcia, N. Sancho Oltra, Dr. N. S. Hatzakis,
S. M. Kuiper, Prof. R. J. M. Nolte, Prof. A. E. Rowan,
Prof. J. C. M. van Hest
(HRP), and glucose oxidase (GOX).
The previously reported procedure of encapsulating
CALB in the inner aqueous compartments of the polymer-
Institute for Molecules and Materials
Department of Organic Chemistry
Radboud University
Toernooiveld 1, 6525 ED Nijmegen (The Netherlands)
Fax: (+31)24-365-2929
E-mail: a.rowan@science.ru.nl
Homepage: http://www.science.ru.nl/orgchem
[
18]
somes was adopted for HRP and GOX.
Fluorescence
microscopy was used to confirm that the incorporation of the
enzymes labeled with Alexa Fluor in these compartments was
successful. Electron microscopy clearly showed that encap-
sulation of these enzymes did not disrupt the structures of the
polymersomes (Figure 1). The diameters of the enzyme-filled
polymersomes ranged from 50 to 1100 nm with an average
diameter of 517 nm, as determined by measuring the diam-
eters of polymersomes from a representative number of TEM
images (see Supporting Information). The possibility of using
the enzyme-filled polymersomes as nanoreactors was inves-
tigated by enzyme-activity assays that could be monitored
spectroscopically (Scheme 1).
The initial rate of substrate conversion by the enzyme-
filled PS–PIAT polymersomes after filtration was compared
with the initial rate of conversion of free enzymes in solution.
The concentration of the latter enzymes was identical to that
of the solution of enzyme used for the encapsulation, and
therefore the concentration of enzyme in the inner aqueous
compartments of the polymersomes was expected to be the
same as that of the free enzymes in solution. The effective
enzyme concentration in the total reaction volume (polymer-
somes and enzyme-free dispersion medium) was actually 1000
Dr. D. M. Vriezema
Encapson B.V.
Toernooiveld 100, 6525 EC Nijmegen (The Netherlands)
+
[
] Currentaddress:
Department of Medicinal Chemistry
N.V. Organon
Molenstraat 110, 5342 CC Oss (The Netherlands)
$
[
] Currentaddress:
Nano-Science Center
H.C. Ørsted Instituttet
Universitetsparken 5, Bygning D, 2100 Copenhagen (Denmark)
#
[
] Currentaddress:
Department of Synthetic Organic Chemistry
University of Groningen
Nijenborgh 4, 9747 AG Groningen (The Netherlands)
[**] We thank Dr. E. Pierson for guidance with the fluorescence
microscope and NWO-ACTS and Synthon B.V. for financial support.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2007, 46, 7378 –7382
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7379