Photo-Cross-Linking Polymersome Nanoreactors with Size-Selective Permeability

The design of stable, inert, and permeable nanoreactors remains a challenge due to the additives required to create a cross-linked network, limiting their potential for catalysis. Polymersomes are nanovesicles self-assembled from amphiphilic block copolymers that can act as nanoreactors by encapsulating catalysts. A major restriction toward their use is their stability and reduced permeability. In order to overcome this, polymersome membranes can be cross-linked to retain their shape and function. Here, we report the synthesis of a PEG-b-P(S-co-4-VBA) polymer, which can self-assemble into polymersomes and subsequently be cross-linked using UV light. We demonstrate that these polymersomes are stable over a long period of time in various organic solvents, that incorporation of functional handles on their surface is possible, and that they are able to undergo reactions. Additionally, we show that co-assembly with up to 40% PEG-b-PS present results in the formation of pores in the membrane structure, which allows for the structure to be used as a nanoreactor. By encapsulating a platinum nanocatalyst, we are able to catalyze the depropargylation of a small coumarin substrate, which was able to enter and leave the porous nanoreactor.


Materials, instrumentation and methods
All PEG polymers with different functional end groups were obtained from AV Chemistry. All other reagents and chemicals were purchased from Sigma Aldrich, unless and otherwise stated. All other reagents were obtained from commercial sources and were used without purification unless otherwise stated. Solvents were dried by passing over activated alumina columns in a MBraun MB SPS800 under a nitrogen atmosphere and stored under argon. Reactions were carried without the need for an inert atmosphere unless stated otherwise, in which case the reaction was performed under a dry atmosphere of argon. Standard syringe techniques were applied for the transfer of dry solvents and air-or moisture sensitive reagents. Styrene was passed over alumina to remove the inhibitor 4-tertbutylcatechol. The inhibitors in 4-VBC (TBC + ONP + 2-Nitro-p-cresol) were removed via extraction with diethylether and 0.5% NaOH in water, evaporating the organic layer. [

α-methoxy-poly(ethylene glycol) ATRP macromolecular initiator (5)
α-methoxy-ω-hydroxy-poly(ethylene glycol) (5.0 g, 2.5 mmol, 1 eq.) was dried by co-evaporation with toluene to remove excess water. The polymer was dissolved in distilled THF (20 mL) followed by addition of trimethylamine (1.04 mL, 7.50 mmol, 3 eq.) in a flame-dried Schlenk flask and the mixture was cooled to 0 °C. α-bromoisobutyryl bromide (616 µL, 5.00 mmol, 2 eq.) was added dropwise and the mixture was stirred for 24 h, slowly warming to 21 °C. After the reaction, the mixture was filtered and subsequently concentrated under reduced pressure. The polymer was precipitated in ice-cold diethyl ether (3x) and dried in vacuo overnight to yield 5 as a white powder

α-methoxy-ω-2-phenyl-2-(phenylcarbonothioyl)thio)acetate-poly(ethylene glycol) (2)
A Schlenk tube was flame-dried under vacuum, and loaded with magnesium turnings (292 mg, 12.0 mmol, 3 eq.), and evacuated for 15 min and refilled with argon (3x). Afterwards, dry THF (30 mL) and an I2 crystal were added. A solution of bromobenzene (1.88 g, 12.0 mmol, 3 eq.) in dry THF (30 mL) was added dropwise and the mixture was stirred at 50 ˚C for 1 h. Carbon disulfide (914 mg, 12.0 mmol, 3 eq.) was added, after which the mixture was stirred for another 30 min at 50 ˚C. A solution of 1 (8.7 g, 4.0 mmol, 1 eq.) in dry THF (20 mL) was added and the dark red solution was refluxed for 16 h. The reaction mixture was filtered to remove the left-over magnesium and concentrated under reduced pressure, and subsequently purified by column chromatography on silica gel using MeOH/DCM (gradient to 1:9 v/v) as eluent. The polymer was precipitated in ice-cold diethyl ether
When the required length was obtained, the polymerization was terminated by removing the Schlenk tube from the oil bath and diluting the mixture with DCM. The mixture was then concentrated under reduced pressure. The polymer was precipitated with ice cold methanol (3x) and dried in vacuo

α-methoxy-poly(ethylene glycol)-b-poly(styrene-co-4-vinylbenzyl acrylate) (PEG44-b-P(S138-co-4-VBA18)) (4)
A flame-dried Schlenk tube equipped with a stirring bar was loaded with K2CO3 (150 mg, 1.1 mmol, 27 eq.) in DMF (4 mL) and cooled to 0 °C. Acrylic acid (76 µL, 1.1 mmol, 27 eq.) was added and the mixture was stirred for 1 h at 0 °C. Then, 3 (0.80 g, 40 µmol, 1 eq.) was added and the mixture was stirred for 3 h at 80 °C. Afterwards, the reaction mixture was diluted with DCM and extracted with water (2x) and brine (2x), concentrated under reduced pressure and precipitated in ice cold  The polymersome suspension was irradiated for 5 minutes at 60% power with the full wavelength range. 8.0 mL of ultrapure water was then added to quench the polymersomes. The polymersomes were spun down using a centrifuge (10 min, 13.000 rpm) and washed with ultrapure water a total of three times.

General procedure of resuspending cross-linked polymersomes in organic solvent
The cross-linked polymersomes in ultrapure water were spun down using a centrifuge (10 min, 13.000 rpm) after which the supernatant was removed. The pellet was then resuspended in the organic solvent, and washed with organic solvent a total of three times.

Synthesis of cross-linked polymersomes with functional handles.
Similar procedure as 2.8.2, substituting cross-linkable polymer 4 (10 mg) for cross-linkable polymer 2.8.5 Click reaction of DBCO-handle and 3-azido-7-hydroxycoumarin 2 x 300 µL of THF suspended cross-linked polymersomes with 10% DBCO-handle were added to two Eppendorf tubes. To one of these tubes, 0.50 mg of 3-azido-7-hydroxycoumarin was added. The polymersomes in the other tube were washed with THF three times and washed back into water.
After this, 0.50 mg of 3-azido-7-hydroxycoumarin was added to the second tube. Both tubes were shaken using an Eppendorf thermomixer for 1 hour. The fluorescence was measured using an excitation wavelength of 485 nm (bandwidth 20 nm) and a detection wavelength of 535 nm (bandwidth 20 nm). [4] 2.8.6 Synthesis of cross-linked polymersomes with pores Similar procedure as 2.8.2, substituting cross-linkable polymer 4 (10 mg) for cross-linkable polymer 4 (10-x mg) and PEG-b-PS (x mg). The number x ranges from 1 to 8, forming the 90%-20% crosslinked polymersomes.

Synthesis of cross-linked polymersomes with Pt NP
Similar procedure as 2.8.2 or 2.8.6, with the modification of adding a dispersion of Pt NPs in ultrapure water instead of only ultrapure water. To remove the excess PtNPs, the polymersomes were washed three times using a 0.22 μm centrifugal filter (10 min, 13.000 rpm) after the standard washing procedure.          Relative fluorescence is given, as a relative comparison was all that was desired. 7 gives a slightly higher fluorescence compared to the blank, most likely due to residual amounts of fluorescence despite the propargyl group. Addition of cross-linked polymersomes with and without encapsulated Pt NPs give a similar amount of fluorescence, indicating no reaction takes place. A fivefold increase is measured for the addition Pt NPs as well as for the addition of 60% cross-linked polymersomes containing Pt NPs, indicating the availability of the Pt NP catalyst for the substrate in the 60% polymersomes.