Nanoporous Vesicular Membranes of Amphiphilic Polymers Containing Trans / Cis Isomers
2022; Chinese Chemical Society; Volume: 4; Issue: 8 Linguagem: Inglês
10.31635/ccschem.022.202201916
ISSN2096-5745
AutoresHui Chen, Yu Xia, Yujiao Fan, Xiangjun Xing, Sylvain Trépout, Min‐Hui Li,
Tópico(s)Metal-Organic Frameworks: Synthesis and Applications
ResumoOpen AccessCCS ChemistryRESEARCH ARTICLE5 Aug 2022Nanoporous Vesicular Membranes of Amphiphilic Polymers Containing Trans/Cis Isomers Hui Chen, Xia Yu, Yujiao Fan, Xiangjun Xing, Sylvain Trépout and Min-Hui Li Hui Chen Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Chaoyang District, Beijing 100029 Chimie ParisTech, PSL University Paris, CNRS, Institut de Recherche de Chimie Paris, UMR8247, 75231 Paris Cedex 05 , Xia Yu Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Chaoyang District, Beijing 100029 , Yujiao Fan Chimie ParisTech, PSL University Paris, CNRS, Institut de Recherche de Chimie Paris, UMR8247, 75231 Paris Cedex 05 , Xiangjun Xing *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Wilczek Quantum Center, School of Physics and Astronomy, Shanghai Jiao Tong University, Shanghai 200240 , Sylvain Trépout *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Institut Curie, PSL University, Université Paris-Saclay, CNRS UMS2016, Inserm US43, Multimodal Imaging Center, 91400 Orsay and Min-Hui Li *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Chaoyang District, Beijing 100029 Chimie ParisTech, PSL University Paris, CNRS, Institut de Recherche de Chimie Paris, UMR8247, 75231 Paris Cedex 05 https://doi.org/10.31635/ccschem.022.202201916 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Nanoporous membranes and vesicles are interesting systems with potential in applications offering channels for material exchange. Herein, nanoporous membranes and polymersomes are developed by self-assembly of trans- and cis-stereoisomers of amphiphilic polymers. Two polymers, PEG550-TPE-Chol and PEG550-SS-TPE-SS-Chol, containing a central tetraphenylethene (TPE), a cholesterol (Chol), and a poly(ethylene glycol) (PEG550) are studied. Their difference resides in the spacers connecting the TPE to the Chol and to PEG, where PEG550-SS-TPE-SS-Chol contains disulfide bonds (–SS–) with two longer and more flexible spacers compared to PEG550-TPE-Chol. For PEG550-TPE-Chol, a progressive transformation from standard vesicles to porous vesicles, networks, and cylindrical micelles is shown as the trans/cis ratio increases. A local, hexagonal structure of nanopores is observed in the membrane of PEG550-TPE-Chol (trans/cis = 50/50), while a two-dimensional crystalline hexagonal structure of nanopores with long-range order is obtained in that of PEG550-SS-TPE-SS-Chol (trans/cis = 50/50). This self-assembly is likely driven by the microphase separation between vesicle-forming trans-isomers and micelle-forming cis-isomers, where both kinetic effects and free energy minimization play important roles. The hexagonal pore organization is facilitated by higher molecular mobility due to the softer and longer spacers or higher temperature. All nanostructures exhibit cyan aggregation-induced emission fluorescence. Moreover, PEG550-SS-TPE-SS-Chol polymersomes can be destroyed using reducing agents, which may be useful for controlled release. Download figure Download PowerPoint Introduction Nanoporous vesicles (also called perforated vesicles or stomatosomes) were first clearly identified in mixtures of two small amphiphiles,1–4 where one amphiphile favors micelle formation, and the other favors vesicle formation. These vesicles are evidenced as intermediate structures in the transition from bilayer vesicles to micelles as the ratio of two amphiphiles varies.2 A typical example is a mixture of cetyltrimethylammonium chloride/egg phosphatidylcholine (C16TAC/EPC) in NaCl aqueous solution with a molar ratio of C16TAC/EPC = 50/50. Other examples include catanionic surfactant mixtures with an excess of anionic or cationic species, such as a mixture of sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium bromide (C12TAB, 35.6% in mole) in NaBr aqueous solution,5,6 and myristic acid (MA) and cetyltrimethylammonium hydroxide (C16TAOH, 43% in mole) in water.7 SDS, C12TAB, MA, or C16TAOH alone form micelles in solution, while the SDS/C12TAB pair (1/1 molar ratio) and the MA/C16TAOH pair (1/1 molar ratio) are vesicle-forming catanionic surfactants. A system of SDS/C12TAB with 35.6 mol % C12TAB yields a mixture of SDS/catanionic-pair with a 45/55 molar ratio, which formed porous vesicles. Interestingly, a system of MA/C16TAOH with 43 mol % C16TAOH results in a MA/catanionic-pair mixture with a 25/75 molar ratio; this mixture forms icosahedra with 12 pores in their 12 vertices. Here, icosahedra are observed instead of spherical vesicles because the MA/C16TAOH catanionic-pair formed a crystalline bilayer membrane with a two-dimensional (2D) crystalline structure (also called gel phase). Structuring pores into stable capsule membranes is extremely useful for material transportation or exchange. However, porous vesicles made of small molecular amphiphiles suffer from low stability because of the low molecular weight building blocks. Later, supramolecular capsules with nanopores were reported in dumbbell-shaped amphiphiles containing rod-core8 or dendritic amphiphiles with a fluorinated-core.9 In the first supramolecular capsule, it was possible to change the pore size or pore opening/closing by varying temperature5 but the mechanism of pore formation in these one-component amphiphiles has not been elucidated yet.8,9 Recently, our group reported nanoporous polymersomes self-assembled from a mixture of trans- and cis-stereoisomers of amphiphilic rod-coil polymer PEG550-TPE-Chol (Figure 1a).10 The trans- and cis-isomers and their mixtures show distinct self-assembly behaviors in water, where trans-PEG550-TPE-Chol forms classical vesicles, cis-PEG550-TPE-Chol self-assembles into cylindrical micelles, and trans/cis mixtures of PEG550-TPE-Chol (trans/cis = 60/40), either naturally synthesized without isomeric separation during synthesis or intentionally mixed using trans- and cis-isomers, yield vesicles with nanopores. trans-PEG550-TPE-Chol polymersomes were also light-gated, where the nanopores could be opened upon UV illumination. Figure 1 | (a) Chemical structures of trans- and cis-stereoisomers of PEG550-TPE-Chol. (b) Chemical structures of PEG550-SS-TPE-SS-Chol with a mixture of trans- and cis-stereoisomers (50/50). Download figure Download PowerPoint To gain further insight into the formation mechanism of these nanopores, herein, we have studied a complete series of mixtures of trans- and cis-isomers of PEG550-TPE-Chol (trans/cis = 95/5, 75/25, 60/40, 50/50, 40/60, 25/75, and 5/95). A progressive transformation from standard vesicles to porous vesicles, interwoven ribbon networks, and cylindrical micelles is observed. More interestingly, in the mixture with an equivalent trans/cis ratio (50/50), some local hexagonal structuring of nanopores is observed in an overall random organization. We also studied a similar amphiphilic polymer, PEG550-SS-TPE-SS-Chol, with longer and more flexible spacers between PEG-TPE and TPE-Chol moieties (Figure 1b). Remarkably, hexagonal pore organization with long-range well-ordered 2D crystalline structures is revealed in their membranes and vesicles. The formation of this hexagonal superstructure is believed to be driven by microphase separation between vesicle-forming trans-isomers and micelle-forming cis-isomers, where both kinetic effects and free energy minimization play important roles. Finally, the aggregation-induced emission (AIE) and reduction-responsive properties of disulfide-containing PEG550-SS-TPE-SS-Chol polymersomes were also investigated. Experimental Methods Materials 4-Methoxybenzophenone (≥98%), titanium tetrachloride (TiCl4, >98%), boron tribromide (BBr3, >98%), 4-dimethylaminopyridine (DMAP, >99%), Zn powder (2.5 cm, >99%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, >98%), lithium hydroxide (LiOH, ≥98%), cholesterol (Chol, >95%), poly(ethylene glycol) methyl ether (MeO-PEGn-OH, n = 12, Mn = 550 Da, Mw/Mn = 1.01), and 3,3ʹ-dithiodipropionic acid were purchased from Alfa Aesar (Bejing, China), without further purification. Instruments and measurements 1H NMR and 13C NMR spectra were recorded on Bruker AV 400 spectrometers (Bruker, Billerica, MA, United States). Chemical shifts δ are given in ppm and referenced to tetramethylsilane in CDCl3 (0 ppm). UV spectra were measured on a Milton Ray Spectronic 3000 Array spectrophotometer (Milton Roy Company, Davidson, NC, United States). Photoluminescence (PL) spectra were recorded on Horiba FluoroMax spectrofluorometers (Horiba, Kyoto, Japan). Hydrodynamic diameters of self-assemblies and their distributions in pure water were measured at 25 °C by dynamic light scattering (DLS, Malvern zetasizer 3000HS; Malvern Panalytical, Malvern, United Kingdom) with a 633 nm laser. A 90° scattering angle was used for all measurements. Self-assembled morphologies were observed by cryo-electron microscopy (cryo-EM). Cryo-EM images were acquired on a JEOL 2200FS (JEOL Ltd., Tokyo, Japan) energy-filtered (20 eV slit) field emission gun electron microscope operating at 200 kV using a Gatan US1000 camera. For sample preparation, a total of 5 μL of samples were deposited onto a 200-mesh holey copper grid (Ted Pella Inc., Redding, CA, United States) and flash-frozen in liquid ethane cooled to liquid nitrogen temperature using a Leica EM-CPC system (Leica, Wetzlar, Germany). Giant polymersomes were observed by epifluorescence microscopy using a Leica DMR (Leica, Wetzlar, Germany) upright microscope equipped with a Retiga Exi charge-coupled device (CCD) camera (Teledyne Technologies, Thousand Oaks, CA, United States) and light-emitting diode (LED) Lamp (λ = 365 nm). Samples (10 μL) were deposited between a glass slide and cover slip with spacers. Then, observations were performed with a 100x objective lens (N.A. 1.3). Images were acquired using Image-Pro Plus (Media Cybernetics, Rockville, MD, United States) and processed/analyzed using ImageJ. Synthesis of TPE-SS-Chol-OH A mixture of TPE-2OH (2.75 mmol, 1.0 g), SS-Chol (2.60 mmol, 1.5 g), (3-dimethylaminopropyl)ethyl-carbodiimide monohydrochloride (EDCI, 3.30 mmol, 0.63 g), and DMAP (3.30 mmol, 0.40 g) was dissolved in 50 mL of dichloromethane, to which trimethylamine (5.50 mmol, 0.56 g) was then added. The synthesis of SS-Chol is described in Supporting Information. The resultant solution was stirred at room temperature overnight. Then the reaction mixture was washed with saturated NaCl aqueous solution three times. After solvent evaporation, the crude product was purified by flash chromatography (petroleum ether (PE)/ethyl acetate (EA) = 5:1). Yield: 53%. 1H NMR (400 MHz, CDCl3): δ = 7.10–6.98 (m, 12H, ArH), 6.91–6.81 (m, 4H, ArH), 6.57 (t, 2H, J (H,H) = 8.00 Hz, ArH), 5.38 (s, 1H, C = CH), 4.66 (m, 1H, COOCH), 3.04–2.91 (m, 6H, CH2CH2SSCH2), 2.71 (t, 2H, J (H,H) = 2.00 Hz, CH2COO), 2.34 (d, 2H, J (H,H) = 8.00 Hz, CH2), 2.02–0.68 (m, 45H, alkane). Synthesis of HOOC-SS-TPE-SS-Chol A mixture of TPE-SS-Chol-OH (1.08 mmol, 1.00 g), 3,3ʹ-dithiodipropionic acid (1.08 mmol, 0.23 g), EDCI (1.30 mmol, 0.20 g), and DMAP (1.30 mmol, 0.16 g) was dissolved in 50 mL of dichloromethane, to which trimethylamine (2.16 mmol, 0.22 g) was then added. The resultant solution was stirred overnight at room temperature. Then the reaction mixture was washed thrice with saturated NaCl aqueous solution. After solvent evaporation, the crude product was purified by flash chromatography (PE:EA = 1:1). Yield: 40%. 1H NMR (400 MHz, CDCl3): δ = 7.14–7.00 (m, 14H, ArH), 6.88–6.83 (m, 4H, ArH), 5.38 (s, 1H, C=CH), 4.66 (m, 1H, COOCH), 3.02–2.93 (m, 12H, COOCH2CH2SSCH2), 2.82 (t, 2H, J (H,H) = 8.00 Hz, CH2COOH), 2.74–2.70 (m, 2H, CH2COO), 2.34 (d, 2H, CCH2C), 2.05–0.68 (m, 45H, alkane). Synthesis of PEG550-SS-TPE-SS-Chol The mixture of TPE-SS-Chol (0.36 mmol, 0.40 g), poly(ethylene glycol) methyl ether (MeO-PEG12-OH, Mn = 550 Da, Mw/Mn = 1.01) (0.32 mmol, 0.18 g), EDCI (0.43 mmol, 67 mg), and DMAP (0.43 mmol, 53 mg) was dissolved in dichloromethane. The reaction solution was stirred overnight at room temperature. The organic phase was first washed with 1 mol/L HCl aqueous solution, then dried with Na2SO4 and filtered. After solvent evaporation, the product was purified by flash chromatography (Dichloromethane: MeOH = 30:1). Yield: 70%. 1H NMR (400 MHz, CDCl3): δ = 7.14–6.97 (m, 14H, ArH), 6.88–6.81 (m, 4H, ArH), 5.37 (s, 1H, C=CH), 4.67–4.59 (m, 1H, COOCH), 3.70–3.53 (m, H for PEG), 3.37 (s, 3H, OCH3) 3.01–2.92 (m, 12H, CH2CH2SSCH2), 2.79–2.69 (m, 4H, CH2COO), 2.33 (d, 2H, CHCH2C, J (H,H) = 8.00 Hz), 2.07–0.67 (m, H for alkane). Self-assembly by film hydration Typically, 5 mg of stereoisomer mixture of amphiphilic polymer was dissolved in 1 mL of chloroform (CDCl3). The solution was stirred at room temperature for 30 min to achieve complete dissolution. Then, about 200 μL of this solution was uniformly deposited on the surface of a roughened Teflon plate (2 cm × 2 cm). The sample was dried in vacuum for at least 5 h to remove all solvent and to obtain a polymer film. The plate with the amphiphilic polymer film was placed into a bottle, and about 12 mL of deionized water was gently added. After sealing the bottle, the thin polymer film was hydrated at 65 °C for 48 h. The resultant sample solution was cooled to room temperature for DLS and cryo-EM measurements. Self-assembly by nanoprecipitation 2.5 mg of the stereoisomer mixture of amphiphilic polymer was dissolved in 1 mL of dimethylformamide (DMF; concentration: 2.5 mg/mL). 2 mL of deionized water was slowly injected at a rate of 200 μL/h with slight shaking. The whole process of nanoprecipitation was carried out at room temperature. The obtained turbid mixture was dialyzed against water over 2 days to remove the DMF using a Spectra/Por regenerated cellulose membrane with a molecular weight cutoff of 3500 Da (water was frequently freshened). Results and Discussion Syntheses Both amphiphilic rod-coil polymers, PEG550-TPE-Chol and PEG550-SS-TPE-SS-Chol, have a hydrophobic part composed of two rigid cores, a tetraphenylethene (TPE) group, a Chol moiety, and a hydrophilic part made of poly(ethylene glycol) (PEG550, Mn = 550 Da) (see Figure 1). The molecular weight of PEG550-TPE-Chol is Mn = 1510 Da and that of PEG550-SS-TPE-SS-Chol is Mn = 1650 Da. The only difference between the two molecules resides in the flexible spacers: one disulfide bond –S–S– is inserted in the spacers between PEG and TPE and between TPE and Chol, respectively, for PEG550-SS-TPE-SS-Chol (Figure 1a) compared to PEG550-TPE-Chol (Figure 1b). The TPE core is a stilbene-type moiety which has structurally distinct and thermally stable trans- and cis-stereoisomers.10 Both trans- and cis-isomers of PEG550-TPE-Chol (Figure 1a) were synthesized according to published procedure,10 and from them, different isomer mixtures were then prepared. The PEG550-SS-TPE-SS-Chol was naturally synthesized without special separation of trans and cis forms during the synthetic process (see Experimental Methods section and Supporting Information Scheme S1 and Figures S1–S3 for detail), and the final products contained trans and cis-isomers in equivalent molar rate (50/50). Self-assemblies of different mixtures of trans- and cis-isomers of PEG550-TPE-Chol Self-assembly was performed using the thin-film hydration method at high temperature (T = 65 °C) to accelerate the sample hydration and molecular self-organization, as described in the Experimental Methods section. The self-assembly behaviors of trans-PEG550-TPE-Chol, cis-PEG550-TPE-Chol, and their mixtures with different ratios (trans/cis = 25/75, 40/60, 50/50, 60/40, and 75/25) were carefully studied. The morphologies and sizes of self-assemblies of all mixtures were characterized by cryo-transmission electron microscopy (cryo-TEM), as shown in Figure 2. As discussed in a previous paper,10 the synthesized trans-PEG550-TPE-Chol contained 5% cis-isomer (trans/cis = 95/5), and the cis-PEG550-TPE-Chol contained 5% trans-isomer (trans/cis = 5/95). Nevertheless, this trace opposite isomers did not influence the self-assembly behaviors of both the trans- and cis-PEG550-TPE-Chol.10 As shown in Figures 2a and 2b, trans-PEG550-TPE-Chol self-assembled into normal vesicles whereas cis-PEG550-TPE-Chol assembled into cylindrical micelles. This is expected, since it has already been reported that giant liposomes immersed in a solution of micelle-forming lysolipids may incorporate the lysolipids into their lipid bilayers, up to 10 mol %.11 Qualitatively following the theory of Israelachvili et al.,12,13 the packing parameter p = v/al of trans-PEG550-TPE-Chol in aqueous solution should be around 1 for vesicle formation, while the p value of cis-PEG550-TPE-Chol decreases and approaches 1/2 for cylindrical micelle formation (where v is the hydrophobic volume, a the optimal interfacial area, and l the length of the hydrophobic block normal to the interface). A schematic representation of their self-assembly and packing is given in Figures 2a and 2b. Figure 2 | Cryo-EM images of self-assemblies obtained from trans- and cis-PEG550-TPE-Chol mixtures with different ratios trans/cis = 95/5 (a), 75/25 (c), 60/40 (d), 50/50 (e), 40/60 (f), 25/75 (g), 5/95 (b). The two samples with trans/cis = 95/5 and 5/95 behave like trans-PEG550-TPE-Chol and cis-PEG550-TPE-Chol, respectively (see main text). The molecular packing models of vesicles and cylindrical micelles are shown in (a) and (b), respectively. The red ellipsoid represents the Chol moiety, and the green trefoil represents the TPE group. The red line represents the aliphatic spacer, and the blue line represents the PEG550 chain. In the cylindrical micelle model, empty red ellipsoids are used to represent the Chol moiety to highlight molecular organization along the cylinder long axis. For the cylindrical micelles formed by cis-PEG-TPE-Chol, along the cylindrical axis the Chol moieties turn around this axis to form the cylindrical micelles. Scale bars are 100 nm. Download figure Download PowerPoint The membrane thickness of the vesicles and the diameter of cylindrical micelles (all for hydrophobic parts) were evaluated from the full width at half maximum of the density profile perpendicular to the membrane through statistical analysis of about 30 different vesicles in the cryo-EM images. The thickness of the trans-PEG550-TPE-Chol vesicle was measured as 7.5 ± 0.5 nm, and the diameter of cis-PEG550-TPE-Chol cylindrical vesicles was 6.5 ± 0.5 nm. The plausible molecular models of the hydrophobic parts for trans- and cis-PEG-TPE-Chol in the bilayer membranes and cylindrical micelles have an interdigitated organization since (1) the thickness of the vesicle membrane (e = 7.5 nm) is between the extended lengths of ltrans (4.54 nm) and 2ltrans (9.08 nm), and (2) the diameter of cylindrical micelles (dc = 6.5 nm) is also between the extended lengths lcis (4.05 nm) and 2lcis (8.10 nm). Note that Chol is a versatile building block that can support the formation of bilayer membranes14 and fibril structures15,16 due to its molecular rigidity, self-assembling nature, asymmetric carbons, and so on. The most representative morphologies of self-assembly observed by cryo-EM for the five mixtures intentionally prepared from trans- and cis-PEG550-TPE-Chol (trans/cis = 75/25, 60/40, 50/50, 40/60, 25/75) are displayed in Figures 2c–2g, and supplemental images are present in Supporting Information Figures S4–S8. As shown in Figure 2c, the 25% content cis-isomer is sufficient to introduce nanopores in the membranes and vesicles, but the pores are not homogeneous in size. 20% of the pores have a diameter 15 nm (see also Supporting Information Figure S4). The self-assembly process is believed to be driven by microphase separation between trans- and cis-isomers: cis-rich domains form the hemicylindrical structures of the pores in porous vesicles and of the edges of interwoven ribbons. The coexistence of pores of very different sizes suggests that the kinetic effect may be dominant in the microphase separation. When the contents of trans- and cis-isomers are more balanced, such as trans/cis = 60/40, 50/50, and 40/60, perforated membranes and vesicles (including small and giant ones) are the main morphologies (Figures 2d–2f and Supporting Information Figures S5–S7). At such ratios, the pore sizes become more homogeneous at around 12–13 nm in diameter. However, note that the pores are not round, but more like a convex polygon. Therefore, kinetic effects may also exist. Interestingly, in the case of trans/cis = 50/50, pores in the membrane tend to organize locally in the hexagonal structure (see structure inside the red frames in Figure 2e, and Supporting Information Figure S6). When cis-isomers become dominant as in trans/cis = 25/75, many cylindrical micelles and a few interwoven ribbons (meshes) are observed (Figure 2g and Supporting Information Figure S8). Finally, in cis-isomers, only cylindrical micelles are observed (Figure 2b). Table 1 summarizes the values measured by cryo-EM for bilayer membrane thickness (e), cylindrical micelle diameters (dc), pore diameter (dh), and the minimal wall thickness (ew) between two pores (see Figure 3c for the definition) for trans-PEG550-TPE-Chol, cis-PEG550-TPE-Chol, and their mixtures. The thicknesses of porous membranes (e ∼ 7.2–7.4 nm) remain nearly the same as that of classical membranes without pores of trans-isomer (e = 7.5 nm). The minimal wall thickness (ew ∼ 6.8–7.0 nm) is close to (slightly higher than) the cylindrical diameter (dc = 6.5 nm = 2 × hemicylinder radius). Therefore, there is very little space for the bilayer structure between the hemicylindrical edges of two adjacent pores. Table 1 | Size Characterization of Different Morphologies of trans-PEG550-TPE-Chol, cis-PEG550-TPE-Chol, and Their Mixtures by Cryo-EM. Self-Assembly Method Is Film Hydration Sample Membrane Thickness e (nm) Cylindrical Micelle Diameter dc (nm) Pore Diameter dh (nm) Pore Wall Thickness ew (nm) trans-PEG550-TPE-Chol 7.5 ± 0.5 – – – Mixture I (trans/cis = 75/25) 7.4 ± 0.6 – 5.2–100a – Mixture II (trans/cis = 60/40) 7.2 ± 0.5 – 13.0 ± 1.7 6.9 ± 0.7 Mixture III (trans/cis = 50/50) 7.3 ± 0.6 – 11.5 ± 1.2 7.0 ± 0.7 Mixture IV (trans/cis = 40/60) 7.2 ± 0.6 – 11.9 ± 1.2 7.0 ± 0.6 Mixture V (trans/cis = 25/75) – – 11.3 ± 3.0b 6.8 ± 0.6b cis-PEG550-TPE-Chol – 6.5 ± 0.5 – – aThe pore size is very heterogeneous. 5.2 nm is the lowest measurable value for visible holes. Big holes, up to 100 nm, are located in loose mesh parts. bThe pore sizes were measured in the parts of the meshes. Figure 3 | The molecular packing models of perforated membrane and interwoven network formed by trans/cis mixtures of PEG550-TPE-Chol. (a) Schematic illustration of first appearance of pores when the micelle-forming cis-isomer is added in a bilayer membrane of trans-isomer. (b) Schematic illustration of morphological transformation with an increasing fraction of cis-isomer. (c) Schematic illustration of porous structures to indicate the pore diameter (dh), pore wall thickness (ew), and membrane thickness (e). Download figure Download PowerPoint The observed morphological transitions in the mixtures of trans- and cis-PEG550-TPE-Chol can be heuristically understood as follows (Figures 3a and 3b). Cylindrical micelle-forming cis-isomers added to a bilayer membrane tend to impose a positive spontaneous curvature on the constituent monolayers (leaflets). Due to the phase separate tendency between the two isomers, the monolayers may remain as a flat bilayer only up to a critical concentration (cc) of the micelle-forming cis-isomers, here cc > 5% in molar ratio. Above this critical concentration, microphase separation takes place, and the micelle-forming cis-isomers assemble into hemicylindrical structures with a negative curvature (the pores), which results in perforation of the vesicle membrane (Figure 3a). The formation of nanopores, their shapes and sizes, and their distributions in the bilayer membrane are likely determined jointly by free energy minimization and by the kinetics of microphase separation process between trans- and cis-isomers. With further increases of micelle-forming cis-isomers, hemicylindrical edges grow and the flat bilayer areas shrink. Consequently, a transformation sequence was observed from bilayers to perforated bilayers with increasing numbers of pores, to an interwoven ribbon network, and finally cylindrical micelles (Figure 3b). A similar scenario has previously been proposed for mixtures of small amphiphiles like the diglycerol monodecanoate (DGMD)/monoglycerol monodecanoate (GMD)/water system.2 Self-assemblies of PEG550-SS-TPE-SS-Chol with trans/cis = 50/50 We are especially interested in the local hexagonal pore organization in the trans/cis = 50/50 mixture. To understand the relationship between the nanopore structure and molecular properties, we also studied polymers with similar chemical structures to PEG550-TPE-Chol, but with longer and more flexible spacers connecting PEG-TPE and TPE-Chol. Among them, PEG550-SS-TPE-SS-Chol with trans/cis = 50/50, which was naturally synthesized without specific separation of trans and cis forms during the synthetic process, showed extraordinary self-assembly properties. Perforated membranes and vesicles were the only morphologies observed, and the pores self-organized into a well-ordered hexagonal superstructure on the surfaces of membranes and vesicles (Figure 4a–4e and Supporting Information Figure S9). The long-range order of nanopores can be directly seen from the sharp Bragg peaks in the Fourier transforms (FT) of cryo-EM images (see FT insets in Figure 4c). In some locations, darker membranes were observed (Figure 4c left) and their FT contained 12 bright peaks which turned out to be two hexagonal lattices rotated 15° (in plane rotation) relative to one another, showing that those dark membranes were not new structures but rather a superposition of two membranes. Figure 4 | Cryo-EM images of self-assemblies obtained from PEG550-SS-TPE-SS-Chol with trans/cis ratio of 50/50 at magnifications of 30000x (a–c) and 60000x (d–e). The method of thin-film hydration was used to perform the self-assembly. (a) Cryo-EM image of vesicles. (b) Cryo-EM image of flat membranes. (c–e) Observation and characterization of the hexagonal lattice in flat membranes. (f) and (g) are high-resolution images obtained after image analysis used to recover the high-resolution information of the hexagonal lattice. (h) Schematic representation of hexagonally organized porous membrane. The insets in (c) show the FT of a single membrane (blue region) and of the superimposition of two membranes (red region). Download figure Download PowerPoint Table 2 summarizes the values of bilayer membrane thicknesses (e), pore diameters (dh), and minimal wall thicknesses (ew) for PEG550-SS-TPE-SS-Chol (trans/cis = 50/50), together with those of PEG550-TPE-Chol (trans/cis = 50/50). Measurements show that the membrane thickness (e = 8.4 nm) and minimal wall thickness (ew = 7.9 nm) of PEG550-SS-TPE-SS-Chol (trans/cis = 50/50) are higher than those of PEG550-TPE-Chol (trans/cis = 50/50). This is expected because PEG550-SS-TPE-SS-Chol has longer spacers with two disulfide bonds (–S–S–) inserted in the middle. The counter lengths of hydrophobic parts are ltrans = 5.26 nm and lcis = 4.77 nm for \PEG-SS-TPE-SS-Chol, higher than those of PEG550-TPE-Chol (ltrans = 4.54 nm and lcis = 4.05 nm). Comparing these values to the e = 8.4 nm and ew = 7.9 nm, we can deduce that the molecular packing models in Figures 2a and 2b are also applicable to trans- and cis-PEG500-SS-TPE-SS-Chol. Surprisingly, the longer PEG550-SS-TPE-SS-Chol has much smaller pore diameter (dh = 7.0 nm), compared with that of PEG550-TPE-Chol (dh
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