Formation of non-spherical polymersomes driven by hydrophobic directional aromatic perylene interactions

Polymersomes, made up of amphiphilic block copolymers, are emerging as a powerful tool in drug delivery and synthetic biology due to their high stability, chemical versatility, and surface modifiability. The full potential of polymersomes, however, has been hindered by a lack of versatile methods for shape control. Here we show that a range of non-spherical polymersome morphologies with anisotropic membranes can be obtained by exploiting hydrophobic directional aromatic interactions between perylene polymer units within the membrane structure. By controlling the extent of solvation/desolvation of the aromatic side chains through changes in solvent quality, we demonstrate facile access to polymersomes that are either ellipsoidal or tubular-shaped. Our results indicate that perylene aromatic interactions have a great potential in the design of non-spherical polymersomes and other structurally complex self-assembled polymer structures.

spherical micellar-like structures formed by PEG43-b-P(NIPAM21-co-t-BocAEA9) after subjection to the "solvent-switch" process and incubation at 40 °C (a higher magnification image is shown inset). (C) Cloud point profile of PEG43-b-P(NIPAM21-co-t-BocAEA9). The cloud point or estimated LCST of PEG43-b-P(NIPAM21-co-t-BocAEA9), defined as the temperature that results in a relative transmittance of 50%, was determined to be 36.2 °C. In summary, these results above illustrate the importance of having aromatic perylenes on the polymer for non-spherical polymersome formation. Scale bars, 1 µm and 200 nm inset. from PEG43-b-P(NIPAM23-co-PDMI19) using the "solvent-switch" method. The estimated membrane thickness of ~10.1 nm suggests that these polymersomes have an interdigitated bilayer membrane structure too. Scale bar, 100 nm.

Materials and instrumentation
All chemicals were purchased from commercial sources and were used as received unless otherwise stated. N-isopropylacrylamide was recrystallized from hexane three times prior to use. Deuterated chloroform was filtered over anhydrous potassium carbonate and neutral aluminium oxide prior to use.
Gel Permeation Chromatography (GPC) analyses were performed on a Shimadzu modular system, comprised of a DGU-12A degasser, an LC-10AT pump, an SIL-10AD auto-injector, a CTO-10A column oven and an RID-10A refractive index detector. The system is equipped with a 50 x 7.8 mm guard column and four 300 x 7.8 mm linear columns (500, 10 3 , 10 4 , 10 5 Å pore size, 5 µm particle size). N,N-dimethylacetamide (DMAc, HPLC grade, 0.05% w/v BHT, 0.03% w/v LiBR) was used as the eluent at a flow rate of 1 mL min -1 . The system was calibrated with poly(methyl methacrylate) (PMMA) standards ranging from 500 to 10 6 g mol -1 . All samples were filtered with 0.22 µm syringe filters prior to analysis. UV-Vis spectroscopy and fluorescence spectroscopy measurements were recorded using a Varian Cary 50 Bio UV-Vis spectrophotometer and a Varian Cary Eclipse fluorescence spectrophotometer, respectively. Absorption and fluorescence spectra of samples were recorded in a 0.7 mL dual pathlength (1.0/0.2 cm) quartz cuvette.
Transmission electron microscopy (TEM) analyses were performed on a JEOL1400 TEM operating at an accelerating voltage of 100 kV. Samples were prepared by dropping 10 L of 0.1 mg mL -1 polymer solution onto a formvar/carbon-coated copper grid (n.b. a carbon onlycoated copper grid was used for the analysis of 0.1 mg mL -1 PEG43-b-P(NIPAM21-co-PDMI9) in tetrahydrofuran (refer to Fig. 1c) due to the incompatibility of formvar with tetrahydrofuran).
The droplet was left on the grid for at least 2 minutes before being blotted off with a filter paper. The grid was then left to air-dry overnight prior to analysis. No staining was used. TEM images were analyzed using ImageJ for the determination of minor/major axis lengths (ellipsoidal particles were measured using an elliptical region of interest (ROI) selection tool; tubular structures were measured using a segmented line tool) and membrane thicknesses of the self-assemblies obtained.

Cryogenic-transmission electron microscopy (cryo-TEM) analyses were performed on an FEI
Tecnai G2 TEM operating at an accelerating voltage of 200 kV. Images were acquired using a BM Eagle 2K CCD Camera and an in-built low-dose software. Samples were vitrified using a Leica EM GP vitrification robot following a general procedure, as follows: 6 L of aqueous 0.5 mg mL -1 PEG43-b-P(NIPAM21-co-PDMI9) solution was pipetted onto a 300 mesh copper grid with lacey formvar/carbon film support. The sample droplet was allowed to equilibrate for 30 seconds at room temperature and at 90% relative humidity, before being blotted from one side for 1.8 seconds. The blotted grid was subsequently plunged into liquid ethane held at ~-174 °C. Excess ethane was blotted away with a piece of pre-cooled filter paper. The vitrified grid was stored in a cryo grid box immersed in liquid nitrogen before finally being cryotransferred into the microscope using a Gatan 626 cryo-transfer holder.

Synthesis of perylene-3,4,9,10-tetracarboxylate tetrabutyl ester (PTE)
A 250 mL round-bottomed flask was charged with perylene-3,4,9,10-tetracarboxylic dianhydride (PDA) (10 g, 25.5 mmol), tetrabutylammonium hydroxide (30 mL, 0.12 mol) and 1-butanol (40 mL). The mixture was sonicated to aid the dissolution of PDA. After PDA was completely dissolved, 1-bromobutane (40 mL, 0.37 mol) and potassium carbonate (10 g, 72 mmol) was added and the mixture was refluxed for 3 h. The crude reaction mixture was allowed to cool to room temperature. The yellow precipitate in the cooled mixture was collected by 22 suction filtration and washed with copious amounts of methanol and water. The yellow precipitate was then redissolved in 100 mL of chloroform and dried over anhydrous magnesium sulphate before being passed through a silica plug, using chloroform as the eluent. The filtered solution was then concentrated in vacuo and the resulting yellow solid obtained was air-dried at room temperature overnight and further dried under high vacuum to yield perylene -3,4,9,10-tetracarboxylate tetrabutyl ester (PTE) as a yellow solid (15.8 g, 95%). 1

Synthesis and characterization of PEG 43 -DDMAT and t-BocAEA
Supplementary Figure 30. Synthetic route to obtain the t-Boc-protected monomer t-BocAEA.

Synthesis of N-(tert-butoxycarbonyl)aminoethyl acrylate (t-BocAEA)
N-(tert-butoxycarbonyl)ethanolamine (6.00 g, 37.2 mmol) and triethylamine (3.77 g, 37.3 mmol) were dissolved in 13 mL of tetrahydrofuran and stirred under nitrogen at 0 °C (ice bath) for 20 min. To the solution was then added acryloyl chloride (3.34 g, 36.9 mmol) dropwise over 1 h. Following the addition, the mixture was stirred at 0 °C for 2 h before being allowed to warm to room temperature, and stirred for a further 12 h, during which the formation of an off-white precipitate was observed. The precipitate was collected by suction filtration and purified by silica column chromatography (dichloromethane:ethyl acetate, 7:3, v/v) to yield an off-white oil that was further purified by recrystallization from hexane to yield N-(tert- 166. 2, 155.9, 131.4, 128.2, 79.8, 64.0, 39.8, 28.5 CDCl3.

Deprotection of PEG43-b-P(NIPAM21-co-t-BocAEA9)
A solution of PEG43-b-P(NIPAM21-co-t-BocAEA9) (100 mg) in 3 mL of dichloromethane was stirred at 0 °C (ice bath) for 10 min. 1 mL of trifluoroacetic acid was added dropwise to the solution over 2 min. Following the addition, the mixture was further stirred at 0 °C. After 2 h, the crude reaction mixture was concentrated in vacuo (repeated three times using dichloromethane to azeotropically remove trifluoroacetic acid). The resulting yellow oil was 36 then dissolved in minimal amounts of tetrahydrofuran and precipitated into hexane. The resulting precipitate was collected by centrifugation and dried under high vacuum to yield PEG43-b-P(NIPAM21-co-AEA9) as a light yellow solid. The number of NIPAM and AEA repeating units in the deprotected diblock terpolymer were determined by comparing the peak integrals in the 1 H NMR spectra as shown in Supplementary Fig. 37. Mn,NMR = 5,675 g mol -1 ; Mn,GPC = 4,700 g mol -1 ; Đ = N/A (GPC analysis gave an unreliable value of 1.01). (PEG43-DDMAT, 0.32 g, 0.14 mmol) and 2,2'-azobis(2-methylpropionitrile) (AIBN, 0.2 M in toluene) (4.57 mg, 27.9 µmol) were dissolved in 900 µL of N,N-dimethylformamide and degassed by purging with argon for 20 min. The degassed mixture was then placed in a preheated oil bath at 65 °C. After 170 min, the reaction vessel was exposed to air and quenched in an ice bath. The crude reaction mixture was diluted in minimal amounts of tetrahydrofuran and precipitated into cold hexane:diethyl ether (2:1, v/v). The resulting precipitate was collected by centrifugation, redissolved in minimal amounts of tetrahydrofuran and precipitated into cold hexane:diethyl ether (2:1, v/v) again. The purification process was repeated four times in total.

Supplementary
The purified product was collected and dried under high vacuum to yield PEG43-b-P(NIPAM22co-t-BocAEA5) as a light yellow solid (555 mg). The number of NIPAM and t-BocAEA repeating units in the diblock terpolymer and the number-average molecular weight by NMR (Mn,NMR) were not determined in this step due to overlapping peaks in the 1 H NMR spectrum as shown in Supplementary Fig. 39