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
Figure 2. Transmission electron microscopy (TEM) micrographs of polymersomes with or
without 3. The scale bars represent 500 nm. (A) PS-PIAT with 10 wt.-% 3; (B) PS-PIAT
with 10 wt.-% 3 and azido-functional CalB clicked on its surface; (C) PS-PEG; (D) PS-PEG with
10 wt.-% 3.
somes, the most uniform size distribution was obtained
when 10 wt.-% of 3 was used. In this case, vesicles sizes
ranging from 60 to 130 nm were measured, with an
average diameter of approximately 100 nm, as can be seen
in Figure 2 and in the Supporting Information.
To investigate the incorporation of anchor 3 into these
vesicles, polymersome dispersions were lyophilised, the
remaining polymers were dissolved in THF (0.5 mL) and
subsequently analysed using gel permeation chromatography
(GPC). Two peaks were observed, a major one
Figure 3. Activity of azido-functional CalB conjugated to a polymersome
surface (solid dots) compared to a blank where 3 was
not embedded in the polymersomes (hollow dots). The chemical
structure shows the substrate DiFMU octanoate.
corresponding to PS-PIAT
(RT ¼ 8.30 min) and a smaller
one at the elution time of 3
(RT ¼ 7.94 min), indicating that 3
was indeed present in the polymersomes.
To determine the
degree of incorporation, a calibration
curve was made (see Supporting
Information), using anchor (3)/
PS-PIAT mixtures of known
weight percentages. Repeated
measurements of PS-PIAT polymersomes
containing a theoretical
10.0 wt.-% of anchor were found to
have 8.5 wt.-% of 3 embedded in
their membranes, indicating an
incorporation efficiency of 85%.
To assay the availability of the
acetylene termini present on the
polymersome surface, functionalised
vesicles were treated with
Cu(II)SO 4 , sodium ascorbate and
bathophenanthroline ligand in
the presence of MeO-PEG 45 -N 3 .
Both PS 238 -PEG 17 and PS- PIAT
vesicles containing 10 wt.-% of 3
were reacted in this way, and were
subsequently washed, lyophilised and redissolved in THF.
In both cases, GPC analysis of the resulting solutions
showed a new peak at a retention time corresponding to
an M n of 8.31 kg mol 1 , which is the estimated M n of
anchor 3 with a coupled PEG 45 chain. This indicates that
the anchor is not only incorporated in PS-PIAT membranes
and PS-PEG bilayers, but also that its reactive moiety is
accessible in both these systems.
To demonstrate the utility of anchor 3, we immobilised
the model enzyme CalB, which is widely used in organic
synthesis. [30,31] The CalB used was functionalised by
replacing its methionine residues with the non-canonical
analogue azidohomoalanine during expression. Since four
of the five resulting azides are buried inside the protein,
the resulting CalB was site-specifically reacted at the only
solvent-accessible azide using Cu(I)-catalysed click chemistry.
[18]
PS-PIAT polymersomes containing the acetylene anchor
were incubated with azido-functionalised CalB (2 equivalents,
relative to 3) for 65 h at 4 8C in the presence of
Cu(II)SO 4 , ascorbate and ligand. The vesicles were then
washed until no CalB activity could be detected in the
flow-through, according to a DiFMU octanoate assay. [8] The
polymersomes in the supernatant were resuspended in
ultrapure water and then also assayed for CalB activity. As
can be seen in Figure 3, polymersomes with CalB clicked to
their surfaces indeed showed enzymatic activity, whereas
324
Macromol. Rapid Commun. 2008, 29, 321–325
ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/marc.200700765