Biomimetic Pulsating Vesicles with Both pH-Tunable Membrane

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Biological and Medical Applications of Materials and Interfaces

Biomimetic Pulsating Vesicles with both pH-Tunable Membrane Permeability and Light-Triggered DisassemblyReassembly Behaviors Prepared by Supra-Amphiphilic Helices Tengfei Yan, Fei Li, Jun Tian, Liang Wang, Quan Luo, Chunxi Hou, Zeyuan Dong, Jiayun Xu, and Junqiu Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09632 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 5, 2019

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ACS Applied Materials & Interfaces

Biomimetic Pulsating Vesicles with both pH-Tunable Membrane Permeability and LightTriggered Disassembly-Reassembly Behaviors Prepared by Supra-Amphiphilic Helices Tengfei Yan, Fei Li, Jun Tian, Liang Wang, Quan Luo, Chunxi Hou, Zeyuan Dong, Jiayun Xu, and Junqiu Liu* State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012 (P. R. China).

Abstract: The reversible unfolding-refolding transition is considerably important for natural elastomeric proteins (e.g. titin) to fulfil their biological functions. It is a great importance of developing synthetic versions by borrowing their unique stretchable design principles. Herein, we present a novel pulsating vesicle by means of the aqueous self-assembly of supraamphiphilic helices. Interestingly, this vesicle simultaneously features dynamic swelling and shrinkage movements in response to external proton triggers. Titin-like unfolding-refolding transformation of the artificial helices was proved to play a crucial role in this pulsatile motion. Moreover, vesicular membrane of this vesicle has exhibited tunable permeability during the reversible expansion and contraction circulation. Meanwhile, light can also be used as a driving force to further regulate the disassembly-reassembly transformation of the pulsating vesicle. In addition, drug delivery system was also employed as an investigating model to estimate the permeability variation and disassembly-reassembly behaviors of the pulsating vesicles, which displayed unique dual quick- and sustained-release behaviors towards anti-cancer agents. It is anticipated that this work opens an avenue for fabricating novel stretchable biomimetics by using the exclusive unfoldingrefolding nature of artificial foldamers. KEYWORDS: pulsating vesicle, self-assembly, supra-amphiphilic helix, unfolding-refolding, sustained-release

INTRODUCTION One of the most fascinating characteristics of natural proteins is their unfolding-refolding transition abilities under specific physiological stimuli.1 As a representative example, the protein titin (2 MDa, c.a. 1 mm long) provides muscle with passive elasticity via reversible unfolding-refolding of the linear array between the tandem domains of immunoglobulin and fibronectin-III types.2,3 Which usually plays important roles in diverse biological activities ranging from microscopic cell adhesion, muscle contraction to macroscopic events like insect flight, frog jumping or jellyfish swimming.4 Considerable efforts have been devoted to uncover the design principles and working mechanism of natural elastomeric proteins.5-10 However, engineering their biomimetics by employing the unfolding-refolding characteristic of those stretchable elastin proteins, thus to further achieve specific function or application still remains a great challenge and rarely explored so far.1113

Until recent years, the dramatic development of artificial foldamers provided possibilities to mimic the intriguing unfolding-refolding transition of muscle protein titin at molecular level and a number of seminal works have been reported in this field.14,15 For instance, Hecht and co-workers presented a series of photoswitchable aromatic foldamers fabricated by azobenzene linked oligo (meta-phenylene ethynylene) helices.16-18 Lehn et al. provided several pyridine-pyrimidine helical codons with ion-triggered reversible extension and contraction motions.19-21 Our group also established some

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quinoline- or pyridine-derived foldamers whose tubular interiors possessed reversible collection and triggered-release behaviors towards alkali metal ions, resulting from the acid-triggered unfolding-refolding transition of the artificial helices.22,23 Nevertheless, these works mainly focused on the primary structural control of individual helix at one dimensional level, which appeared too simple to compare with sophisticated natural occurring systems.24 Till now, only few attempts were reported on fabricating foldamer-based supramolecular vesicles.25,26 While, how to prepare novel stretchable nano-architectures via employing the unfolding-refolding characteristic of artificial helix is always highly desired and still remains a difficult task for biochemical researchers.27

Figure 1. Chemical structures and schematic representations of a) α-CD, b) 2, R-2, and S-2 with proton-sensitive unfolding-refolding transition abilities. c) Cartoon illustration of the proton-triggered pulsating vesicle self-assembled by supra-amphiphilic helices (α-CD/2) in aqueous solution. Following this, herein we present a de novo biomimetic pulsating supramolecular vesicle which was self-assembled by a kind of stretchable supra-amphiphilic helix in water. Interestingly, this vesicle performed periodic expansion and contraction movements in response to external proton stimuli, as we named pulsating behaviors. The acid-sensitive reversible unfolding-refolding structural transformation of the supra-amphiphilic helices was proved to play a crucial role in this pulsatile motion. Moreover, membrane permeability of this helix-based vesicle also revealed significant variation along with its pulsating circulation (Figure 1). Besides, this vesicle also manifested dynamic disassembly-reassembly behaviors when exposed to alternate UV and visible light. Additionally, drug delivery system was adopted as an investigating model to validate the dual-responsibilities of the pulsating vesicles, exhibiting preferable controlled-release capabilities at acidic tumor environment compared with the UV-induced quick-release behavior. To the best of our knowledge, such kind of bio-inspired vesicle synchronously qualified with dynamic size-jumping, tunable membrane


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permeability, light-triggered disassembly-reassembly behaviors and potential drug-delivery applications was never reported so far.

Figure 2. a) TEM image, b) cryo-TEM image, and c) DLS profile of the helix-based vesicles at neutral state (pH = 7). d) TEM image, e) cryo-TEM image, and f) DLS profile of the expanded vesicles at acidic condition (pH = 4).

RESULTS AND DISCUSSION An azobenzene-containing aromatic foldamer, Azo-(mQQ)2-MBA (denoted as 2, Q: 8-amino-2-quinoline carboxylic acid; mQ: 8-aminomethyl-2-quinoline carboxylic acid, MBA: methylbenzylamine), was adopted as our basic building block whose conformation was considerably rigid and well-defined, which made them incline to proceeding intermolecular stacking or self-assembly (Figure 1b and 1c). Synthetic procedure and characterization results of 2 were elaborately described in the second part of Supporting Information. The helical orientation of (mQQ)n sequence with phosphonate side chains has been undoubtably collaborated through single crystal analysis by Huc and coworkers,28 and intramolecular electrostatic repulsion and hydrogen bonds (NH···N) play pivotal roles in stabilizing its folding tendency (Figure S1). To prepare supra-amphiphilic building block, a size-suitable host molecule with preferable water-solubilities is also necessary.29,30 Hence, α-cyclodextrin (α-CD), a six α-D-glucopyranoside linked cyclic oligosaccharide, was selected to serve as our host to recognize trans-Azo group of 2, thus to form the primary supra-amphiphilic building block (Figure 1a and Figure S2). The self-assembly behaviors of this supra-amphiphilic helix (α-CD/2) were subsequently investigated after adding equimolar 2 into α-CD solution at the concentration of 0.05 mM in water. The morphology of these assemblies was firstly characterized by transmission electron microscopy (TEM) and cryo-TEM instruments (Figure 2a and 2b), which were confirmed to be typical supramolecular vesicles with uniform spherical surface and well-defined cavities. From the cryoTEM image (inset of Figure 2b), membrane thickness of the helix-based vesicles was clearly visualized and measured to be (25.6 ± 0.5) nm at frozen state, implying a multiple-layers vesicular membrane structure on the basis of theoretical



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simulation of α-CD/2 (Figure S2). Moreover, dynamic light scattering (DLS) measurement was subsequently carried out and revealed that overall diameter of the supramolecular vesicles was about 164.2 nm in aqueous solution (Figure 2c), agreeing with the cryo-TEM results. Furthermore, critical aggregation concentration (CAC) of the supramolecular vesicles was determined to be approximate 0.028 mg mL-1 by means of measuring ultraviolent (UV) absorption values of the supraamphiphilic complex (α-CD/2) at a range of different concentrations by using previously reported methods (Figure S3).31 The host-guest complexation behaviors of α-CD and 2 was further investigated by 1H NMR spectroscopy via employing trans-1 as an investigating objective due to the relatively poor water-solubility of 2. As we can see from Figure 3a and 3b, the resonance protons peaks (Ha, Hb, Hc, Hd, and He) of trans-1 experienced a upfield-shift at the presence of equimolar αCD in a mixed solution of D2O and DMSO-d6 (1/2, vol/vol). Upfield shift variations of the proton signals (Δδ) were calculated to be -0.021, -0.020, -0.018, -0.015, and -0.022 ppm for Ha, Hb, Hc, Hd, and He, respectively. The upfield-shifting of proton signals provided an implication for the inclusion between trans-1 and α-CD in this solution. 2D-NOESY NMR measurement were further employed to give a direct evidence for the host-guest complexation occurrence, and strong NOE correlation between aromatic protons of trans-1 (δ, 7.55-8.15 ppm) and α-CD’s inner protons (δ, 3.3-3.8 ppm) can be clearly observed. Besides, the association constant between α-CD and 2 was subsequently assessed by using modified Benesi-Hildebrand equation32,33 through gradually increasing the concentration ratio of α-CD and 2 in the mixed solution of water and acetonitrile (50/1, vol/vol), which was calculated to be (530 ± 30) M-1 (Figure S4).


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Figure 3. Partial 1H NMR spectra of a) 5 mM trans-1. b) 5 mM trans-1 and 5 mM α-CD. c) 5 mM trans-1 and 5 mM αCD after irradiation at 365 nm for 30 min. (d) 5 mM trans-1 and 5 mM α-CD after further irradiating by visible light for 30 min. e) 2D-NOESY NMR spectrum of trans-1 and α-CD at the same concentration of 5 mM. All these measurements were tested in a mixed solvents of D2O and DMSO-d6 (1/2, vol/vol). As aforementioned, some aromatic oligoamide foldamers have already shown acid-sensitive unfolding-refolding capabilities.22,23 Thereby, we wondered whether we could control the unfolding-refolding tendency of supra-amphiphilic helix via proton stimuli, and thus to further manipulate the size or morphology of the supramolecular vesicles. Correspondingly, we tried to observe the morphological variation of this helix-based vesicle at acidic conditions in water. According to DLS analysis, the hydrodynamic diameter of the vesicles did not show obvious change immediately when the pH value of was decreased to 4. Interestingly, as time went by, the hydrodynamic diameter significantly increased from 164.2 to 342.0 nm (increased by 108.3%) within 120 min in water (Figure 2f and Figure 4c). Furthermore, detailed structural information of the acid-treated supramolecular vesicles was also clearly detected by cryo-TEM instrument (Figure 2e), showing larger vesicles with intact vesicular morphologies. Whose membrane thickness (inset of Figure 2e) was measured to be about (3.9 ± 0.2 nm), which was much thinner than initial thickness (i.e. 25.6 ± 0.5 nm) of the



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supramolecular vesicles (inset of Figure 2b). In addition, scanning electron microscopy (SEM) and TEM instruments were also adopted to reconfirm the expansion and contraction movements of the helix-based pulsating vesicles, sharing the same results with cryo-TEM and DLS analysis (Figure 2d and Figure S5). On the contrary, we found the expanded vesicles gradually shrank back to original size (~ 164.2 nm) after the acid was adequately neutralized within 120 min. The pHstimuli responsive expansion and contraction variation of the vesicles were named as pulsating behaviors correspondingly. The reversible pulsatile motion of the supramolecular vesicles could be recycled several times via repeatedly modulating the pH values between 4 and 7 in water, according to the test results of DLS instruments (Figure 4a).

Figure 4. a) Reversible size variation of the pulsating vesicles detected by DLS via alternately regulating the pH values between 7 and 4. b) UV-vis titration of the vesicles at different pH levels in water (c = 0.025 mM). c) The diameter variation of the vesicles at different times (pH = 4), detected by DLS instrument. d) CD spectra of 2, R-2, and S-2 at the concentration of 0.5 mM in CH2Cl2. e) CD titration of R-2 (c = 0.5 mM) by alternately injecting diluted TFA and TEA in CH2Cl2. f) Zeta potential variation of the pulsating vesicles at different pH values in water. Furthermore, UV-vis titration experiment was also carried out to confirm this pulsating process of the helix-based vesicles. As depicted in Figure 4b, the characteristic absorption band (at about 375 nm) of the vesicles dropped by approximate 30% within 2 hours at the pH value of 4, which gradually recovered to its original position after the acid was totally neutralized by injecting alkali solution. The reversible fluctuation of the UV-vis absorptance was contributed to the unfolding-refolding structural transition of supra-amphiphilic helix (α-CD/2). Except for aqueous solution, UV-vis and fluorescence titration experiments of 2 were also carried out in organic solvents by means of iterative addition of trifluoroacetic acid (TFA) and triethyl amine (TEA) in the solution of 2 which was previously dissolved in CH2Cl2, exhibiting similar proton-dependent variation characteristics with the supramolecular vesicles in water (Figure S6 and S7). In addition, circular dichroism (CD) titration was further performed to provide a direct characterization to the dynamic unfolding-refolding transition of 2. Firstly, chiral-induced 2 (i.e. R-2 and S-2) were acquired by replacing racemic MBA with R- or S-MBA groups on the C terminus of 2 through straightforward synthetic procedures, whose reaction routes and characterizations were clearly described in the second part of Supporting Information (Figure 1b and Scheme S1). As depicted in Figure 4d, strong Cotton effect was distinctly observed at 360 nm from the CD spectra of R-2 and S-2 at the same concentration of 0.5 mM in CH2Cl2, which offered an additive proof for the helical sense of 2 in solution state. Then,


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R-2 was selected to evaluate the unfolding-refolding behavior of (mQQ)n sequence by alternately adding organic acid and base. As shown in Figure 4e, the characteristic peak of R-2 at 360 nm declined significantly and gradually disappeared when supplied with enough TFA, indicating the secondary structure of (mQQ)n sequence was destroyed by organic acid (i.e. TFA). On the contrary, the chiral signal of R-2 reappeared and recovered back to its original state when sufficient TEA was gradually injected for neutralization. The reversible fluctuation of chiral signals could be repeated many times by the alternate addition of diluted TFA and TEA in CH2Cl2, implying the dynamic unfolding-refolding transformation of (mQQ)n sequence could be easily governed by proton triggers. In order to reveal detailed structural transformation during the unfolding-refolding process of (mQQ)n sequence, analogues of Q and mQ units were designed and synthesized to perform 1H NMR spectroscopy, respectively (Scheme S2). It was noted that a novel protonated hydrogen resonance peak of the TFA-treated mQ-analog appeared from the 1H NMR spectra at 9.0 ppm. While there wasn’t any new peak about the Q-analog was detected at the same conditions (Figure S8), which implyed only mQ rather than Q segments on (mQQ)n sequence were protonated at acidic state (Figure 1b). As far as we know, Q unit and Qn serial foldamers are exceptionally stable in essentially any nonprotic or protic solvents according to reported literatures.34,35 Additionally, the protonated surface of the vesicles was further validated by zeta potentiometer and calculated to be 27.1 mV after the supramolecular vesicles were injected into acidic solution (pH = 4) and settled for enough time (about 2 hours), which was much higher than the initial measured value (only about 3.5 mV) at neutral states (Figure 4f). Thereby, the size-jumping mechanism of the pulsating vesicles can be explained as follows. Firstly, at acidic condition (pH = 4), mQ segments of 2 were partially protonated with a degree about 88.8% which was deduced from the acidity coefficient (pKa = 4.9) of quinoline groups.36 As a result, the unfolded supra-amphiphilic helices were equipped with positive charges, which enabled them to repel their neighboring building blocks through the intermolecular electrostatic force, leading to a bigger vesicle with much thinner membrane wall. Reversely, the opposite deprotonation process of the mQ units on 2 subsequently occurred after the acid was totally neutralized, resulting in a synchronous decrease of electrostatic force among the charged building blocks and the size-contraction of the supramolecular vesicles (Figure 1c).



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Figure 5. a) Light-induced trans-cis structural isomerization of 2. b) UV-vis spectra variation of 2 consecutively irradiated by UV and visible light. c) Reversible absorbance changes of trans- and cis-2 (c = 10-5 M) measured by UV-vis spectrometer. d) TEM image of irregular aggregates after the supramolecular vesicles were irradiated under a UV lamp for 30 min. e) TEM image of the reassembled vesicles when the mixed solution was further the irradiated by visible light for another 30 min. It is well-known that α-CD is a frequently-used host molecule which is capable of recognizing trans-isomer but excluding the bulky cis-isomer of azobenzene groups owing to the mismatched spatial conformation according to previous reported literatures.37-39 Given the azobenzene moiety on 2, it is conceivable that morphological structure of the vesicles might be further modulated by transforming the trans-2 and cis-2 isomers via using light as a driving force (Figure 5a). The UV-vis spectroscopy was firstly employed to examine the photo-isomerizing capabilities of 2. As shown in Figure 5b, we noticed the characteristic absorption band of 2 decreased significantly at the wavelength of 325 nm when the solution of 2 was exposed to UV light for 120 seconds. At the same time, a new band at around 440 nm has increased slightly (inset of Figure 5b), indicating that trans-2 has transformed to cis-2 totally within 2 min. Reversely, on exposure to visible light for the same time, the absorption band at 325 nm and 440 nm exhibited increased and decreased tendency simultaneously (Figure 5b). The absorption bands at 325 nm and 440 nm can be contributed to the typical π–π* and n–π* transitions of the azobenzene segment of 2, respectively.38 And the reversible isomerization between trans-2 and cis-2 could be reversibly achieved more than five times through repeatedly exposing 2 to UV and visible light in the solution of N,Ndimethylformamide (DMF) (Figure 5c). TEM measurement was subsequently carried out to monitor the morphology variation of the supramolecular vesicles when alternately treated with UV and visible light irradiation. Irregular aggregates were immediately visualized after the


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supramolecular vesicles were irradiated under a UV lamp for 30 min (Figure 5d) with the overall diameter about 120 nm, which shared the same results with SEM and DLS measurements (Figure S9 and S10). Reversely, well-defined vesicular structures were re-detected after the mixed solution was exposed to visible light for another 30 min (Figure 5e). The lighttriggered dynamic disassembly and reassembly process of the supramolecular vesicles could be recycled more than four times according to the DLS analysis (Figure S11). At the same time, new resonance peaks (denote as Ha*, Hb*, Hc*, Hd*, and He*, respectively) of cis-1 were distinctly observed from the 1H NMR spectra after UV irradiating for 30 min, which were considered to be transformed from trans-1 isomer (Figure 3c). When exposed to visible light for another 30 min, the new resonance peaks of cis-1 (Ha*, Hb*, Hc*, Hd*, and He*) decreased, and the original resonance peaks of trans-1 (Ha, Hb, Hc, Hd, and He) has increased simultaneously, implying the opposite structural transformation from cis-1 to trans-1 was achieved by using visible light trigger (Figure 3d). In consideration of the pH-related membrane thickness variation, we wondered whether the membrane permeability of this vesicle likewise changed along with its pulsatile motion. To address this question, fluorescence experiment was firstly carried out to examine the membrane permeability of the pulsating vesicles by monitoring the leakage of 5(6)carboxyfluorescein (CF) from CF-loaded vesicles on the basis of previously reported methods.40,41 As depicted in Figure 6a, there was only small quantity of CF (less than 5%) leakage within 100 min at neutral state. By contrast, most of CF (more than 75%) was leaked out within 70 min, after the CF-loaded vesicles were previously exposed under a UV lamp for 30 min, which was mainly due to the light-induced fracture of this supramolecular vesicle (Figure S11). Interestingly, at weak acidic condition (i.e. pH = 4), the CF-loaded vesicles manifested significant controlled-releasing tendency and only about 40% of CF was leaked out within 100 min, implying that the thinner membrane of the expanded vesicles at acid condition has much better permeability in contrast to the releasing profile of contracted vesicles and UV-treated vesicles. (Figure S12).



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Figure 6. a) CF-releasing profile of the CF-loaded vesicles at different conditions. b) In vitro concentration-dependent cell viability of MCF-7 upon incubation with free vesicles, DOX, DOX-loaded vesicles with and without and UV light irradiation, respectively. c) MTT assay of MCF-7 when exposed to UV light for 30 min as control experiments. d) CLSM images of MCF-7 cells before and after cell internalization of RhB-loaded vesicles. With desire to further evaluated the quick- and sustained-release abilities of this pulsating vesicle, drug delivery system was correspondingly performed as an investigating model (Figure S13). As is known, there is a lower pH profile in tumors than normal cells or tissues which mainly resulted from the superfluous lactic acid generated by rapid proliferation of cancerous cells.42 Except for that, UV light was also one of the most frequently-used maneuvers to achieve remote optically controlled release of anti-cancer drugs in drug delivery systems.43,44 Therefore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was adopted to assess the anti-cancer abilities of the drug-loaded vesicles against MCF-7, wherein free vesicles, doxorubicin (DOX), DOX-loaded vesicles with and without UV light irradiation were incubated, respectively. The drug-loading efficiency was measured to be about 42.1% by using UV-vis spectroscopy measurements by using previously reported methods (Figure S14).45 As we can see from Figure 6b, the concentration-dependent cell death of free DOX and the UV light was clearly observed, and more than half of the MCF-7 was killed at the concentration of 10 μg mL-1, resulting from the light-induced rupture of the supramolecular vesicles which enabled loaded-DOX of the vesicles released quickly. By contrast, it was noteworthy that the cell viability increased slightly when MCF-7 was incubated with the DOX-loaded vesicles without UV irradiation. Through comparison with quick-release treatment of the UV-treated vesicles, the pulsating vesicle displayed preferable sustained-releasing abilities with higher cell viability. Meanwhile, the supramolecular vesicles also exhibited desirable low cytotoxicity within the given concentrations on benefit of the satisfactory low-toxicity of CD families46,47 and quinolines derivatives.48,49 And the cell viability was not affected by the irradiation of UV light within the given concentrations (Figure 6c). DOX-loaded vesicles were further validated by TEM and confocal laser scanning microscopy (CLSM) images, manifesting dark and red interiors, respectively (Figure S15 and S16). Additionally, cellular uptake of the vesicles was also demonstrated by CLSM after encapsulation of a fluorescent dye, rhodamine B (RhB). And the internalized RhB-loaded vesicles were obviously visualized as red dots with excitation wavelength of 507 nm after incubating with MCF-7 for 12 hours (Figure 6d).

CONCLUSIONS In summary, we have presented a new kind of pulsating vesicle prepared by aqueous self-assembly of stretchable supraamphiphilic helices. Interestingly, this vesicle manifested periodic swelling and shrinkage movements under external proton stimuli. Titin-inspired unfolding-refolding structural transformation of the supra-amphiphilic helices was proved to play a crucial role in this reversible pulsatile motion. At the same time, membrane of the helix-based vesicle also showed tunable permeability along with this pulsating motion, resulted from wall thickness variation during expansion and contraction movements of the vesicle. Moreover, this vesicle also displayed reversible disassembly-reassembly process when it was alternately exposed to UV and visible light in virtue of the photo-isomerization behaviors of the azobenzene moiety on guest molecules. In addition, drug delivery system was also employed as an investigating model which has confirmed the pulsating vesicles with dual sustained- and quick-release of anti-cancer drugs, induced by dual proton and light triggers, respectively. And we believe that this line of research has opened a new researching field for chemical


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scientists to fabricate novel stretchable biomimetic materials through employing the exclusive unfolding-refolding nature of artificial foldamers. EXPERIMENTAL SECTION General Information. N, N-dimethylformamide (DMF), triethylamine (TEA) and dichloromethane (CH2Cl2) were dried by distillation with CaH2 for one week. Distilled water was polished by ion exchange and filtration. Tetrahydrofuran (THF) was dried by distillation with sodium-benzophenone prior to use. α-CD and other organic reagents were purchased from commercial vendors and used without further purification. All reactions were monitored by thin layer chromatography (TLC) visualizing with UV light, and column chromatography purifications were performed by using silica gel (SiO2, 200300 mesh). All the purifed products were clearly characterized by 1H NMR, 13C NMR, and HR-MS (ESI) instruments. Scanning Electron Microscopy (SEM). The sample films on a silica wafer surface were prepared by evaporation of one drop sample solutions (V = 2.5 µL, c = 0.05 mM) in the air at room temperature. The measurements were performed on FESEM 6700F from JEOL operating at the voltage of 10 kV at room temperature (~ 25oC). Transmission Electron Microscopy (TEM). The sample was prepared by dropping the supramolecular vesicles solution (V = 2.5 µL, c = 0.05 mM) on carbon-coated copper grid (200 mesh) and the solution was allowed to evaporate in the air at room temperature. And the dried specimen was observed by using JEM-2100F instrument operating with the operating voltage of 200 kV. Cryo-TEM. The sample was prepared by dropping the supramolecular vesicles solution (V = 2.5 µL, c = 0.05 mM) on carbon-coated copper micro grid (200 mesh) and the drop was allowed to froze in liquid helium immediately. And the frozen specimen was observed by using FEI Talos 200c from FEI company, operating at the voltage of 120 kV. Liquid nitrogen was used to keep the temperature at a low level. Dynamic Light Scattering (DLS). The sample was prepared by dropping the supramolecular vesicles solution in a disposable cell and measured for three times after equilibrium for 120 seconds. The pH values were regulated by adding HCl and NaOH solutions. The data were collected by instrument of Zetasizer Nano Series from Malvern Company. Zeta Potential Measurements. The sample was prepared by dropping the supramolecular vesicles in a disposable cell and measured for three times after equilibrium for 120 seconds at the concentration of 0.05 mM. The pH value was set to be 4 and 7, respectively. Zetasizer Nano Series equipped with a He–Ne laser (633 nm, 4 mW) and an avalanche photodiode detector from Malvern Company was used to collected data. UV-visible Spectroscopy. The measurement was carried out by UV-2450 from Shimadzu Corporation. The sample solution of supramolecular vesicles was prepared by dissolving the supramolecular vesicles in aqueous solution at the concentration of 0.05 mM. The pH values were regulated by adding HCl and NaOH. Fluorescence Spectroscopy. The samples were prepared by dissolve 2 in CH2Cl2 solution with the concentration of 0.01 mM at room temperature (~ 25oC). Dilute TFA and TEA were gradually added to perform the protonation and deprotonation process. The measurements were performed at 5301PC from Shimadzu, with excitation wavelength of 350 nm. Raw data were analyzed and treated by the software of Origin (version 8.5).



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Circular Dichroism (CD) Spectroscopy. The solution sample (300 µL) was prepared by dissolve chiral induced helices in CH2Cl2 solution at the concentration of 0.5 mM at room temperature (~ 25oC). Diluted TFA and TEA were utilized to devastate and restore the intramolecular hydrogen bonds. The measurements were performed by using PMS-450 from biologic company. Raw data were analyzed and treated by software of Origin (version 8.5). Cell Culture and MTT Assay. MCF-7 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 100 IU mL-1 penicillin, 100 mg mL-1 streptomycin and were maintained at 37oC in a humidified atmosphere of 5% CO2. The cells were collected by using trypsin-EDTA solution and were seeded onto 96-well plates at a density of 1.5 × 104 cells per well, cultured for 24 h. Then further incubated with DOX for 6 h. 20 μL of MTT solution (5 mg mL-1 in PBS) was added to each well, and the plates were incubated for an additional 4 h. The MTT solution was then removed, and 150 μL of DMSO was added to dissolve the formazan crystals. The plates were incubated for an additional 10 min, after which the absorbance at 492 nm was recorded using a BIO-RAD iMark microplate reader from Japan. Confocal Laser Scanning Microscopy (CLSM). The sample was prepared by dropping the target solution (~ 50 μL) to a glass slide and covered by a cover glass. And the CLSM images were acquired by using FV1000 from Olympus Company. Nuclear Magnetic Resonance (NMR) Spectroscopy. Proton nuclear magnetic resonance (1H NMR) spectra and carbon nuclear magnetic resonance (13C NMR) spectra were recorded on Bruker AVANCEIII 500. Chemical shifts were reported in ppm relative to the residual solvent peak (CDCl3 = δ 7.26 ppm DMSO = δ 2.50 ppm for 1H NMR spectrum; CDCl3 = δ 77.16 ppm, DMSO = δ 39.52 ppm for 13C NMR spectrum). Multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad singlet). High-Resolution Mass Spectroscopy (HR-MS). HR-MS of our compounds was recorded by ESI-MS Agilent 1290microTOF Q II from Bruker Company, which were dissolved in MeOH or MeCN at a very low concentration previously.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Detailed information including the synthetic routes and characterizations of our target molecules, schematic representation of (mQQ)n sequence or the supra-amphiphilic helix, UV-vis spectra of 2 and 2/α-CD, SEM images of the supramolecular vesicles at different pH values. UV-vis and fluorescence spectra of 2 alternatively treated by TFA and TEA. SEM and DLS measurements of the irregulates. schematic representation and DLS analysis of disassembly-reassembly process of the vesicles. cartoon illustration of drug delivery process of this supramolecular vesicle. TEM and CLSM of the DOX-loaded vesicles were clearly presented in second part of the Supporting Information.

AUTHOR INFORMATION Corresponding Author


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*E-mail: [email protected] ORCID Tengfei Yan: 0000-0003-4797-0808 Quan Luo: 0000-0002-3231-2039 Zeyuan Dong: 0000-0001-6509-9724 Junqiu Liu: 0000-0002-0533-171X Author Contributions J. L. conceptualized the project. T. Y. have performed most of the experiments described in the manuscript. T. Y. and J. L. wrote the overall manuscript and supporting information. F. L. carried out the MTT essay. J. T. performed HR-MS measurements. L. W. performed cryo-TEM measurements. C. H., Q. L., Z. D. and J. X discussed the results and comments on the manuscript. Notes The authors declare no conflict of interest.

ACKNOWLEDGES This work was supported by the National Natural Science Foundation of China (nos. 21420102007, 21574056, and 91527302).

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TOC figure


Biomimetic Pulsating Vesicles with Both pH-Tunable Membrane

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