A Block Copolymer for Functionalisation of Polymer...

S. F. M. van Dongen, M. Nallani, S. Schoffelen, J. J. L.M. Cornelissen, R. J. M. Nolte, J. C. M. van Hest
The cell-penetrating peptide Tat was conjugated to this
block copolymer and blended with non-derivatised
PBD-b-PEG. This mixture of block polymers was then used
for polymersome formation. [6] These polymersomes with
Tat on their surfaces were actively targeted to the
endocytic compartments of dendritic cells.
We have recently introduced polymersomes as
nanoreactors for multistep synthesis. It was possible to
selectively position the enzymes glucose oxidase (GOX)
and horse radish peroxidase (HRP) in either the water
pool or the membrane [8] of semiporous polymersomes
consisting of polystyrene 40 -block-poly[L-isocyanoalanine(2-
thiophen-3-yl-ethyl)amide] 50 (PS-PIAT). [9,12] As a third
enzyme Candida antarctica lipase B (CalB) was dissolved
in the bulk solution, enabling a three-step reaction sequence
to take place. This third enzyme was not tethered to the
polymersome itself, because the structure of PS-PIAT makes
it difficult to custom-functionalise the surface of the vesicles
it forms. Conjugation to the surface would be desirable, for
example to recover all catalytic entities from the solution by
filtration of the large polymersomes.
The direct conjugation of entire proteins to well-defined
block copolymers has been shown before, by e.g. Reynhout
et al., who synthesised PEG-b-PS polymers that were
functionalised with a heme-like cofactor. [13] Reconstitution
of this cofactor with the apoenzyme apo-HRP yielded a
biohybrid triblock copolymer. While these biohybrid polymers
did aggregate into vesicles, amongst other interesting
architectures, the HRP-analogue was not catalytically
active. In the work of Opsteen et al. the conjugation of
fluorescent proteins to an azide-functionalised polymersome
surface was shown. [14] Although PS-PAA and PS-PEG
have as obvious advantage that their hydrophilic termini
can be conveniently transformed into functional groups,
as shown in the previous two examples, [13,14] their application
as nanoreactors is hampered, since the polymersomes
that are fully constituted of these block copolymers
are not permeable for most organic substrates. In order to
develop a polymersome that combines both desired
aspects, namely porosity and ease of surface functionalisation,
we designed a specific block copolymer that can
be easily incorporated in a PS-PIAT bilayer via coaggregation
during vesicle formation, following the strategy
presented for liposomes by Reulen et al. [15]
Here, we report polymersomes with hybrid membranes,
composed of PS-PIAT and PS-PEG block copolymers with
functional handles. In order to present a bio-orthogonal
reactive group of high selectivity at the surface, we
introduced acetylene moieties at the end of a so-called
‘anchor’, a strategy that has been used for liposomes by
Cavalli et al. [16] and for polymersomes by Li et al. [17] For
coaggregation with the block copolymer PS 40 -PIAT 50 during
vesicle formation, the hydrophobic block of this anchor
was chosen as PS 40 , with PEG 68 as a hydrophilic block, the
length of which is long enough to bridge most hydrophilic
layers of conventional polymersome membranes.
Their functionality was demonstrated by conjugation of
azido-functionalised CalB [18] to the surface of PS-PIAT
polymersomes with 10 wt.-% of the PS-PEG anchor
embedded in their bilayers. The enzyme was shown to
retain catalytic activity when immobilised on the vesicle
surface.
Experimental Part
General Procedure for Polymersome Formation
A solution of either PS-PEG or PS-PIAT admixed with the appropriate
wt.-% of 3 in THF (0.5 mL, 1.0 g L 1 ) was gently injected into
ultrapure water (2.5 mL). After equilibration for 40 h the suspension
was transferred to an Amicon Ultra Free-MC centrifugal filter
with a 100 nm cutoff and centrifuged to dryness. The polymersomes
were redispersed in ultrapure water (600 mL) and
centrifuged again. This step was repeated six times. The resulting
vesicles were redispersed in ultrapure water (1 mL).
Typical ‘Click’ Reaction on Polymersome Surface
To a dispersion of polymersomes in ultrapure water (200 mL) an
aqueous solution of azido-functionalised CalB (33 mL, 75 10 6 M,
2 equivalents relative to 3) was added. Aqueous solutions of
Cu(II)SO 4 5H 2 O with sodium ascorbate (10 10 3 M each, 33 mL)
and bathophenanthroline ligand (10 10 3 M, 33mL) were premixed
and then added to the dispersion, which was left at 4 8C for
60 h. The mixture was then transferred to an Amicon UltraFree-
MC centrifugal filter with a 100 nm cutoff and centrifuged to
dryness. The polymersomes were redispersed in ultrapure water
(600 mL) and centrifuged again. This step was repeated until no
CalB activity could be detected in the filtrate. The resulting
biohybrid was redispersed in ultrapure water (200 mL).
Activity Assay for CalB
A stock solution of DiFMU octanoate in DMSO (1 10 3 M) was
prepared and stored at 18 8C. In a single well of a 96-well micro
titer plate a dispersion of polymersomes (98 mL) or an aliquot of
control solution (98 mL) was placed. To this, DiFMU octanoate stock
solution (2 mL, final concentration 20 10 6 M) was added, and
monitoring of fluorescence emission of the hydrolysis product at
460 nm (l ex ¼ 355 nm) was started immediately after mixing.
Results and Discussion
In order to be able to functionalise the surfaces of PS-PIAT
polymersomes, we designed the generic PS-b-PEGacetylene
diblock copolymer 3, which is shown in
Scheme 1. Although block copolymer 3 does not have a
suitable ratio of block lengths for this purpose, the
322
Macromol. Rapid Commun. 2008, 29, 321–325
ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/marc.200700765