A Block Copolymer for Functionalisation of Polymer...

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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
A Block Copolymer for Functionalisation of Polymersome Surfaces Scheme 1. Synthesis of functional block copolymer 3: (i) EDC (5 equiv.), 4-pentynoic acid (6.5 equiv.), DMAP (catalytic), CH 2 Cl 2 , RT, 14 h, 84%; (ii) NaN 3 (19 equiv.), DMF, RT, 12 h, 90%; (iii) CuBr (1 equiv.), PMDETA (1 equiv.), CH 2 Cl 2 ,N 2 atmosphere, RT, 70 h, 89%. amphiphilic block copolymer PS-PEG is known to form stable vesicles for certain block ratios; the ratio of block copolymer 3 was chosen in such a way that it was not able to yield polymersomes on its own. [19] It has been demonstrated that the PEG chain end can be conveniently functionalised. [20] Furthermore, the presence of the alkyne moiety allows selective and specific coupling with azides via the Cu(I)-catalysed [3 þ 2] Huisgen cycloaddition, colloquially known as ‘clicking’. [21–24] This method has found widespread use in the field of protein conjugation due to its general bio-orthogonality and efficiency in aqueous media. [14,23–26] A schematic representation of our strategy is shown in Figure 1. In our approach, 3 is admixed with PS-PIAT, and is intended to coaggregate during vesicle formation. When embedded in the membrane, its acetylene moiety should be exposed to the aqueous solution to enable conjugation via click chemistry. Figure 1. In our strategy, a percentage of acetylene-functionalised block copolymer is blended with regular vesicle forming polymers. The resulting polymersomes can then be functionalised at their surfaces using the Cu(I)-catalysed [3 þ 2] bipolar Huisgen cycloaddition. To synthesise 3, the path shown in Scheme 1 was followed, where atom transfer radical polymerisation [27] (ATRP) was used to produce PS 40 , which was subsequently functionalised with an azide by substitution of its v-bromide with NaN 3 , leading to 2. [20,23–26,28,29] The PEG block was functionalised at both termini via an EDCmediated esterification with 4-pentynoic acid, producing the a, v-acetylene bearing polymer 1. A Huisgen cycloaddition between 1 and 2, using a large excess of 1 to avoid triblock formation, led to the desired diblock copolymer 3. Inadvertently formed triblock was removed by a precipitation of 3 in Et 2 O, since the additional polystyrene block sufficiently solubilised PS-PEG-PS to avoid its precipitation along with 3 (see Supporting Information). To determine whether 3 had an influence on polymersome morphology, a variety of weight percentages of the functional block copolymer were blended with PS-PIAT, and these mixtures were dissolved in THF (0.5 mg, 1 g L 1 ). After injection in ultrapure water (2.5 mL) the samples were left to self-assemble for 60 h. Mixtures containing up to 20 wt.-% 3 were found to still form spherical polymersomes. The versatility of anchor 3 was indicated by similar tests performed with PS-PEG vesicles. For all anchor containing polymer- Macromol. Rapid Commun. 2008, 29, 321–325 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mrc-journal.de 323
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