Light-gated nano-porous polymersomes from stereoisomer-directed self-assemblies

Capsules with holes in the walls exist in natural systems like virus capsids, and in biomimetic systems like immune-stimulating complexes vaccines and liposomes of phospholipids/surfactants mixtures. Structuring pores into stable membrane and controlling their opening are extremely useful for applications that require nano-pores as channels for material exchange and transportation. Polymersomes, which are stable and robust vesicles made of amphiphilic block copolymer, are good candidates for drug carriers or micro/nanoreactors. Engineering structure-inherent and light-gated nano-porous polymersomes is especially appealing, but still unexplored. Here we present these polymersomes made from a polymer including a tetraphenylethene (TPE) in its center. TPE is an emblematic uorogen with aggregation-induced emission, which possesses two stereoisomers sensitive to photoisomerization. Trans-isomer of the polymer forms classical vesicles, cis-isomer forms cylindrical micelles, while trans/cis mixtures construct perforated vesicles with nano-pores. Under UV light, the classical polymersomes of trans-isomer can be perforated by its cis-counterpart generated from the photoisomerization.


Introduction
Capsules with regularly spaced holes in the walls exist in Nature. For example, many virus capsids have such a structure, where protein units form a rigid frame with holes. 1 In the biomimetic systems like ISCOM (immune-stimulating complexes) vaccines, cage-like structures exhibit also regular holes, but they are formed from small amphiphilic molecules including saponins, phospholipids and cholesterol 2 .
Perforated vesicles (also called stomatosomes) made from small amphiphilic molecules were then clearly identi ed in two-amphiphiles mixtures, 3,4,5,6 such as in cetyltrimethylammonium chloride/egg phosphatidylcholines (C16TAC/EPC) in NaCl aqueous solution. All these two-amphiphiles systems have a common feature: the amphiphile 1 tends to form micelles, whereas the amphiphile 2 favors vesicle formation. The perforated vesicles are evidenced as intermediate structures in the transition from bilayer vesicles to cylindrical micelles with the increase of the ratio of amphiphile 1 over amphiphile 2. 4 Later, supramolecular capsules with nanopores were reported in dumbbell-shaped amphiphiles containing rodcore 7 and in dendritic amphiphiles containing uorinated-core. 8 In contrast to the multicomponent systems of small amphiphilic molecules, only one amphiphilic dendrimer is necessary to form porous membrane in these systems. The mechanism of their pore formation is more complicated and seems to be closely related to the membrane rigidity. 7,8 It was possible to change the pore size or pore opening/closing in supramolecular capsules by external stimuli like temperature variation. 7 Structuring pores into stable membrane of capsules and controlling their opening are extremely useful for applications that require nano-pores as channels for material exchange and transportation, such as organ-on-chip tissue engineering. 9,10 However, the number of experimental and theoretical researches in perforated vesicles is still limited.
Here we report on the perforated vesicles with nanopores formed from a mixture of stereoisomers of an amphiphilic polymer PEG550-TPE-Chol (M n = 1510 Da), where the hydrophilic part is poly(ethylene glycol) (PEG550, M n = 550 Da), and the hydrophobic part is composed of two rigid cores, a tetraphenylethene (TPE) group and a cholesterol (Chol) moiety, and two aliphatic spacers serving as PEG-TPE and TPE-Chol connections (see Fig. 1A and B). From the point of view of materials, capsules with higher molecular weight and rigid cores are more stable and robust than those made from small molecular amphiphiles.
The cholesterol moiety is introduced because it is a biocompatible and bio-sourced lipophilic molecule very popular for the core-construction of self-assemblies 11,12 . The role of TPE is three-fold. Firstly, TPE core is a stilbene-type moiety which has structurally distinct and stable trans and cis stereoisomers (Fig.   1A). 13,14 Thanks to the shape difference between the trans and cis isomers, their packing parameter is different and stereoisomer-directed self-assembly has been realized: normal vesicles for trans-PEG550-TPE-Chol (Fig. 1C), cylindrical micelles for cis-PEG550-TPE-Chol (Fig. 1D), and perforated vesicles for trans/cis mixture (Fig. 1B). Secondly, the stilbene-type TPE can undergo trans-cis photo-activated isomerization under UV illumination. 15 This photo-isomerization ( Fig. 2) can make the self-assembly photo-responsive. Under UV illumination of high intensity (15 mW/cm 2 ), both normal vesicles and cylindrical micelles tend to transform into porous vesicles (Fig. 5), and the design of smart nanostructures is possible. Finally, TPE is a typical aggregation-induced emission (AIE) uorophore and highly emissive in aggregate states. 16,17 The self-assembled nanomaterials containing TPE exhibit tuneable brightness, high sensitivity and photostability. [18][19][20] All PEG550-TPE-Chol self-assemblies in this work are highly uorescent under normal intensity of UV for observation and imaging (typically < 0.5 mW/cm 2 ). Therefore, the porous polymersomes reported here can provide a deeper insight in this intriguing perforated membrane structure. They may have interesting potential applications as nanoreactors with selective permeability or membranes of separation with selective transport, etc. The AIE uorescence polymersomes and cylindrical polymer micelles may also be advantageous uorescent systems for application such as bio-imaging and in vivo study of drug bio-distribution. This paper is organized as follows. The synthesis of PEG550-TPE-Chol (without separation of transand cis-isomers during the synthesis), trans-PEG550-TPE-Chol and cis-PEG550-TPE-Chol will rst be described, followed by their characterization including the study of AIE uorescence property and transcis isomerization. Then the self-assembly behaviors of trans-PEG550-TPE-Chol, cis-PEG550-TPE-Chol, and mixtures of transand cisisomers (with trans/cis ratio of 60/40) will be discussed in detail. The studied trans/cis mixtures include PEG550-TPE-Chol obtained inherently by synthesis without isomers separation (trans/cis ratio = 60/40) and the mixture prepared intentionally by mixing trans and cis isomers with the same ratio. Finally, the AIE uorescence of these self-assemblies including cylindrical micelles, nano-porous vesicles and smooth vesicles will be characterized and their morphological changes in response to UV irradiation will be discussed.

Results
Synthesis and characterization of PEG550-TPE-Chol and their stereoisomers The synthesis of amphiphilic molecules PEG550-TPE-Chol was performed through the synthetic route as shown in Supplementary Scheme S1. Initially, Ti-catalyzed McMurry coupling reaction was employed to provide TPE-2OMe trans/cis mixture (Fig. S1). Because transand cisisomers of TPE-2OMe have nearly identical retardation factor (R f ) on thin-layer chromatography (TLC), they cannot be separated easily by column chromatography. If no separation between transand cis-isomers was made during the whole synthetic procedure, the nal product PEG550-TPE-Chol contained nearly the equivalent rate of transand cis-isomers (see Fig. S3 for 1 H NMR spectrum, calculated ratio trans/cis = 60/40, calculation detail will be discussed below).
The transand cis-PEG550-TPE-Chol were then prepared with the separated intermediate derivatives trans-TPE-2COOMe and cis-TPE-2COOMe, respectively ( Fig. 2A and Scheme S2), where two big and polar substituents methyl 6-hexanoate replaced the methyl groups in TPE-2OMe. Indeed, the two isomers of TPE-2COOMe showed different R f , 0.52 and 0.44, respectively, on TLC using petroleum ether and ethyl acetate (10:1 volume ratio) as eluent solvents (Fig. S10). They were successfully separated by careful column chromatography. The single crystal of the pure isomer with R f = 0.52 was acquired by slow evaporation of solvents. Its crystal structure was characterized by X-rays diffraction (see Fig. S11 and Table S1). The results clearly show that the functionalized phenyl groups are located on the opposite sides of the central double bond. Unambiguously, the isomer collected rstly in column chromatographic experiment with R f =0.52 corresponded to the trans-TPE-2COOMe. The isomer collected later with R f =0.44 was cis-TPE-2COOMe. Both isomers are very stable and can be stored at room temperature. 1 H NMR spectra ( Fig. 2A) show clearly the difference of chemical shifts between H a (H b ) for trans-isomer and H a ' (H b ') for cis-isomer at δ around 6.6 ppm (6.9 ppm). The transand cis-TPE-2COOMe were then used as starting materials to synthesize corresponding amphiphilic stereoisomers, respectively (see SI and Scheme S2), via hydrolysis reaction to get trans and cis-TPE-2COOH followed by a rst esteri cation reaction with cholesterol and a second esteri cation with methoxy-poly(ethylene glycol) (mPEG550-OH).
The structures of all the compounds were con rmed by 1 H NMR, 13 C NMR and high-resolution mass spectrometry (HRMS) ( Fig. S4-S9, Fig. S12). Transand cisisomers were stable and maintained their geometry along the synthetic process from TPE-2COOMe to PEG550-TPE-Chol (see Fig. S13 and S14).
For the nal amphiphilic stereoisomer trans-PEG550-TPE-Chol, the 1 H NMR analysis detected tiny cissignals of Ha' next to the main trans-signals of H a ( Fig. S15 and Fig. 2B). Careful peak integration revealed that the trans-PEG550-TPE-Chol contained around 5% of cis isomer. The same result was also observed for the cis-PEG550-TPE-Chol ( Fig. S15 and Fig. 2C), which contained about 5% of trans isomer. This small imperfection can be explained as follows. In order to obtain enough nal compounds of transand cis-PEG550-TPE-Chol, TPE-2COOMe have been synthesized in great quantity. The large-scale separation of transand cis-TPE-2COOMe by manual column chromatography are not perfect because their R f values are too close (see Fig. S10) and it is di cult to separate them completely. Nevertheless, we will show hereinafter this trace impurity will not in uence the self-assembly behaviors of both transand cis-PEG550-TPE-Chol.
The molecular weight of transand cis-PEG550-TPE-Chol were then analyzed by size exclusion chromatography (SEC) (Fig. S16). Their SEC peaks are well separated, and the elution time of transisomer is longer than that of its cis counterpart. This observation is completely coherent with the fact that the shapes of transand cis-PEG550-TPE-Chol are quite different, even though their molecular weight are the same (M n = 1510 Da). Because of the presence of TPE moieties, both transand cis-PEG550-TPE-Chol exhibit aggregation-induced emission under UV light around 365 nm. As shown in Fig. S17, they are not uorescent when they are solubilized in solution of acetone, but highly uorescent in aggregated states formed from acetone/water mixture.
The possibility to activate the isomerization between two stereoisomers by heating and by UV illumination has been checked. Both isomers are rather thermally stable as shown by 1 H NMR spectra in Fig. S18 and Fig. S19. Heating their solution in DMSO up to 70℃ cannot induce trans-to-cis or cis-totrans isomerization. When heating at 120℃ for 24h, only 12-14% of isomerization occurs. Under weak UV light (365 nm, < 0.5 mW/cm 2 ), both isomers in solution are also stable during normal observation time (in the order of minutes), as shown by 1 H NMR spectra in Fig. S20. Typically, the UV light (365 nm) for TLC revealing is ≤ 0.5 mW/cm 2 , and that in Spectro uorometer is 0.25 mW/cm 2 . After 2 min of illumination by UV light (365 nm, 0.5 mW/cm 2 ), no photoisomerization can be detected (Fig. S20). In contrast, photoisomerization of isomers in solution takes place readily under high UV light as shown by 1 H NMR spectra in Fig. 2B and 2C. Trans and cis-PEG550-TPE-Chol exposed to UV light (365 nm, 15 mW.cm -2 ) for 2 min undergo already 19% and 25% of isomerization, respectively. After 10 min of illumination, both trans-tocis and cis-to-trans isomerization reach a stationary state with trans/cis ratio of 60/40. The trans-isomer is in a little excess compared to cisone, because the trans-PEG550-TPE-Chol is more photo-stable than the cis one and the cis-isomer has a faster isomerization rate than that of trans-isomer. No signi cant photo-cyclization was observed for both cis and trans-PEG550-TPE-Chol after 10 min of illumination (nearly no 1 H NMR signals at 6.7 -6.8 ppm). 15,21 Self-Assembly of transand cis-PEG550-TPE-Chol and their mixtures: vesicles, perforated vesicles and cylindrical micelles Considering the molecular weight (M n = 1510 Da) of PEG550-TPE-Chol, which is higher than traditional small amphiphilic molecules like lipids EPC or DHPC, we prepared the vesicles using thin-lm hydration method at high temperature (T = 65 °C) to accelerate the sample hydration and polymer self-organization. Typically, the chloroform solution of PEG550-TPE-Chol or their stereoisomer (0.5 wt%) was uniformly deposited on the surface of a roughened Te on plate, followed by drying in vacuum to remove all solvent and to get a thin polymer lm. Then the thin lm sample was hydrated with deionized water at 65 °C for 48 h in a sealed bottle. We have shown in the above section that the stereoisomers are stable at 70°C for at least 30 min of heating. Here a long heating duration (48h) being used, we rst checked if there were any trans-cis isomerization of amphiphiles during the self-assembling process. For this purpose, the selfassemblies of transand cis-PEG550-TPE-Chol recovered by freeze-drying was dissolved in deuterated DMSO and analyzed by 1 H NMR, respectively. As shown in Fig. S21, the trans/cis ratios of both transand cis-PEG550-TPE-Chol do not show any change and keep being 95/5 and 5/95, respectively, after the selfassembling process, which prove the absence of the isomerization.
The morphologies and sizes of the self-assemblies were characterized by cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS). PEG550-TPE-Chol (trans/cis = 60/40) self-assembled into perforated vesicles, while trans-PEG550-TPE-Chol self-assembled into normal vesicles and cis-PEG550-TPE-Chol into cylindrical micelles (Fig. 1B, C and D, Fig. S22-24). Their statistical sizes distributions by DLS are shown in Fig. S25, which indicate multimodal size distributions, especially for vesicles of transisomer with sizes from 80 nm to 5 mm. Indeed, different sizes of vesicles were observed as shown in Fig.   S23. Because the DLS analysis is based on spherical models and gives hydrodynamic diameters (D h ) of spherical particles, the data for cis-PEG550-TPE-Chol do not re ect the real size of long cylindrical micelles. The length of cylindrical micelles reaches several hundreds of nanometers until several micrometers, and some closed loops are also visible (see Fig. S24). Note that the trace amount of cisisomer (5%) in the trans-PEG550-TPE-Chol did not in uence the formation of vesicles, which represent the absolute majority of the morphologies. Meanwhile, cis-PEG550-TPE-Chol formed only cylindrical micelles despite the presence of 5% trans-isomer according to the collected cryo-EM images. The membrane thickness of vesicles and the diameter of cylindrical micelles were evaluated from the FWHM (Full Width at Half Maximum) of the electronic density pro le perpendicular to the membrane through statistical analysis of about 30 different vesicles in the cryo-EM images. The thickness of vesicle membrane for trans-PEG550-TPE-Chol was measured as 7.5 ± 0.5 nm, the diameter of cylindrical micelles for cis-PEG550-TPE-Chol as 6.5 ± 0.5 nm and the thickness of the membrane of perforated vesicles for PEG550-TPE-Chol as 7.3 ± 0.5 nm.
When the stereo-structure changes from transto cis-type, the morphological transition of self-assemblies from vesicles to cylindrical micelles should be driven by the minimization of the interfacial energy.
Following qualitatively the theory of Israelachvili et al. 22,23 , 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 should be smaller and approach 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). Here, the chemical structures are the same for both isomers, so their steric shapes dictate their packing parameter in the self-assemblies. A schematic representation of their self-assembly and packing is given in Fig. 3A and 3B. The plausible molecular models of the hydrophobic part for transand cis-PEG-TPE-Chol in the bilayer membranes and cylindrical micelles are illustrated in Fig. S26, with the lengths of l trans = 4.54 nm and l cis = 4.05 nm. The thickness of vesicle membrane (e = 7.5 nm) is between l trans (4.54 nm) and 2l trans (9.08 nm), the diameter of cylindrical micelles (d c = 6.5 nm) is also between l cis (4.05) and 2l cis (8.10 nm). Therefore, both transand cis-PEG-TPE-Chol in the bilayer membrane and in the cylindrical micelles should be packed in the interdigitated way as shown in Fig. 3A and 3B.
Cholesterol is a versatile building block which supports the formation of bilayer membranes 24 and bril structures 25,26 due to its molecular rigidity, self-assembling nature, asymmetric carbons, etc. For the cylindrical micelles formed by cis-PEG-TPE-Chol, along the cylindrical axis the cholesterol moieties turn around this axis to form the cylindrical micelles, whose cross-section is round as shown in Fig. S24.
Interestingly the PEG550-TPE-Chol forms perforated membranes and vesicles (see Fig. 1B and Fig. S22), similarly to the mixture of small molecular amphiphiles, e.g., C16TAC and EPC. 3 The novelty here is the PEG550-TPE-Chol is a stereoisomers-mixture synthesized as one compound, simply and inherently without the tedious separation of intermediates transand cis-isomers during the synthetic procedure. Further, we prepared arti cially the mixtures of both stereoisomers to get trans/cis ratio of 60/40, the same as inherently synthesized PEG550-TPE-Chol, and then performed the self-assembling by lm hydration in the identical conditions. Fig. 4 shows the representative morphologies of its self-assemblies observed by cryo-EM, which are the same as those obtained with inherently synthesized PEG550-TPE-Chol (Fig. 1B).
The scenario of porous membrane formation can be explained as follows. Micelle-forming cis-isomers in a bilayer membrane try to impose a positive curvature on the constituent monolayers (lea ets). The monolayers may remain united to a at bilayer only up to a critical concentration of the micelle-forming cis-isomers. Above this critical point, edges may form, which would contain predominately the micelleforming cis-isomers and assemble into hemicylindrical structures with a negative curvature (the holes). With trans/cis ratio of 60/40, the hole part and strand part have similar proportion in the mesh-like membrane. Table 1 summarizes the measured values for bilayer membrane thickness (e), cylindrical micelle diameters (d c ), pore diameter (d h ) and the minimal wall thickness (e w ) between two pores (see Fig. 4 for the de nition). We found e w ≥ d c , which means the narrowest place of the wall is almost composed of the cross-section of two face-to-face hemicylinders. Since both transand cisisomers can undergo trans-to-cis and cis-to-trans isomerization under UV illumination, we have also studied the morphological transformation of their self-assemblies under UV light of higher intensity. Typically, the aqueous dispersions of self-assemblies of transand cis-PEG550-TPE-Chol were exposed to UV light (365 nm, 15 mW.cm -2 ) for 30 min, respectively. One part of the irradiated sample was investigated by cryo-EM, and the rest was rst freeze-dried and then solubilized in CDCl 3 for analysis by 1 H NMR. 1 H NMR spectra (Fig. S27) show that after UV illumination, the trans/cis ratios in both samples become 60/40, in agreement with the results obtained in solution samples exposed to UV light ( Fig. 2B and 2C). It is also notable that photocyclization of TPE moiety begin to occur as a trace of new peak emerged at 6.7 -6.8 ppm for both cis and trans-PEG550-TPE-Chol after 30 min illumination (Fig. S27). Cryo-EM images of both UV treated samples are shown in Fig. 5C and 5D as well as in Fig. S28 and S29. Vesicles of trans-PEG550-TPE-Chol initially with homogeneous and smooth surface were perforated by the cis-isomers resulted from the photoisomerization, while cylindrical micelles of cis-PEG550-TPE-Chol under UV interweaved to form meshes and perforated membranes because of the appearance of trans-isomers. The perforated membranes obtained from cylindrical micelles were less structured (Fig. 5D) than those from smooth vesicles, probably for the kinetic reason, i.e., because of the slow rate of the fusion between separated cylindrical micelles. As expected, perforated vesicles and membranes of PEG550-TPE-Chol did not show signi cant change upon UV light exposure (see cryo-EM images in Fig. S30), because of simultaneous trans-to-cis and cis-to-trans isomerization.
Big morphologies like giant vesicles and large membranes also co-existed with nanostructures in PEG550-TPE-Chol self-assemblies as shown in Fig. 4 (and Fig. S22-23, S28-S30). Cryo-EM allows to detect the nanostructures in detail, but it cannot visualize the giant structures wholly. Therefore, epi uorescence optical microscopy was also used for observation of the giant vesicles formed by PEG550-TPE-Chol (Fig. 6). Under UV light, giant vesicles are cyan uorescent as shown in Fig. 6. With the increase of UV illumination time, the membrane becomes uctuating, probably because the hole parts and strand parts interchange continuously resulting from the simultaneous trans-cis and cis-trans isomerization in PEG550-TPE-Chol. Even with this high intensity UV of 15 mW/cm 2 , the uorescence intensity only decreased, but not quenched, after 2 min of illumination ( Fig. 6 and Fig. S31), demonstrating good photostability of AIE polymersomes. Further, the uorescence will be greatly decreased after 60 min illumination due to the cyclization 21 of the TPE moiety ( Fig. S31-32).
All these self-assemblies exhibited AIE uorescence under UV illumination of normal intensity for observation and imaging (typically < 0.5 mW/cm 2 ). With stronger UV intensity (15 mW/cm 2 ) and longer illumination time (2 -30 min), photo-responsive features were recorded due to trans-to-cis and cis-totrans photo-isomerization. Smooth vesicles of trans-PEG550-TPE-Chol were perforated by the cis-isomers stemmed from the photoisomerization, while cylindrical micelles of cis-PEG550-TPE-Chol interweaved to form meshes and perforated membranes. Nano-porous vesicles of PEG550-TPE-Chol upon strong UV light exposure did not show signi cant change in nanoscale, but in micrometer scale membrane uctuation was observed by epi uorescence microscopy.
The vesicles of trans-PEG550-TPE-Chol can be considered as light-gated polymersomes, where pores of 9-27 nm were generated under strong UV illumination (15 mW/cm 2 ). It can be used to perform traced transportation under weak UV light and controlled release of small molecules (< 30 nm) under stronger UV light. Nano-porous polymersomes and polymer membranes are also promising for applications that require nano-pores as channels for material transportation. We speculate that the nano-porous polymersomes may be used as capsules for the culture of multicellular spheroids which are considered as good in-vitro models of micro-tissues like tumors or neurons 10,27 . All nutrients (proteins and oxygen) required for cell division can diffuse freely through the capsule membrane and allow proliferation of the encapsulated cells. Meanwhile, since PEG is by nature cell-repellent, cells form cellular aggregates, i.e., multicellular spheroids. Organ-on-chip tissue engineering may be possible using these nano-porous giant polymersomes.   The morphologies models of polymersomes (A) and cylindrical micelles (B) formed by trans-isomer and cis isomer, respectively. The red ellipsoid represents the cholesterol moiety, and the green trefoil the TPE group. The red line represents the aliphatic spacer, and the blue line the PEG550 chain. In the cylindrical micelle model, empty red ellipsoids are used to represent the cholesterol moiety in order to highlight molecular organization along the cylinder long axis.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. LightgatednanoporouspolymersomesfromstereoisomerdirectedselfassembliesSIsubmission.docx