Selective Metal-Ion-Mediated Vesicle Adhesion Based on Dynamic

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Selective metal-ion mediated vesicle adhesion based on the dynamic self-organization of a pyrene-appended glutamic acid Pengyao Xing, Yajie Wang, Minmin Yang, Yimeng Zhang, Bo Wang, and Aiyou Hao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04279 • Publication Date (Web): 21 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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ACS Applied Materials & Interfaces

Selective metal-ion mediated vesicle adhesion based on the dynamic self-organization of a pyreneappended glutamic acid Pengyao Xing,† Yajie Wang,† Minmin Yang,† Yimeng Zhang,† Bo Wang† and Aiyou Hao*† †Key Laboratory of Colloid and Interface Chemistry of Ministry of Education and School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China. E-mail: [email protected]

Abstract Vesicles with dynamic membranes provide ideal model system for investigating biological membrane activities, whereby the vesicle aggregation behaviors including adhesion, fusion, fission, and membrane contraction/extension have attracted much attention. In this work we utilize an aromatic amino acid (PGlu) to prepare nanovesicles which aggregate together to form vesicle clusters selectively induced by Fe3+ or Cu2+, and the vesicles would transform into irregular nanoobjects when interacting with Al3+. Vesicle clusters have better stability than the pristine vesicles, which hinders the spontaneous morphological transformation from vesicles into lamellar nanosheets with long incubation period. The difference between complexation of Fe3+ and Al3+ to vesicles was studied by using various techniques. On the basis of the metal ion-vesicle interactions, this self-assembled nanovesicle system also behaves as the effective fluorescent sensor to Fe3+ and Al3+, which cause fluorescence quenching and enhanced excimer emission respectively. Keywords: aromatic amino acid • vesicle • structural evolution • vesicle fusion • cation sensor

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1 Introduction Having a similar topology and structure to cell membranes, vesicles represent a branch of membranous hollow spheres from self-organized synthetic amphiphiles.1,

2

Due to the unique

hollow structure as well as enhanced permeability and retention (EPR) effect of vesicles, they could be utilized in various areas such as drug carrier for cancer therapy.3 Among those applications, cyto-mimetics which stands for the mimicking of cellular activities has attracted considerable attention.4, 5 The reason why people choose vesicle as a platform to mimic the cell membrane activity is on the basis of dynamic properties of vesicle membranes. Vesicle membranes, unlike other assemblies like nanoparticles or nanofibers with high aggregation number, are composed of loosely packed building blocks which shall undergo several dynamic processes spontaneously.6 For example, the vesicle membranes are capable of lateral diffusion, flip-flop as well as exchanging building blocks with bulky solution. These dynamic processes result in the meta-stability of vesicle, allowing us to well control the vesicle membrane movement. Therefore, like cell membrane activities including membrane trafficking, budding and fusion, vesicles exhibit similar aggregation, adhesion, fusion and fission activities, providing an ideal membrane biomimetic platform.7-9 Groups like Zhou, Ravoo, Webb contributed many excellent works to this field.4-13 Utilizing various non-covalent interactions like host-guest, metal-ligand coordination and hydrogen bonding, the molecular recognition can be realized on the vesicle membranes, which allows for the occurrence of membrane fusion or vesicle adhesion.10-13 Among these strategies, embedding metal receptors in the vesicle membranes to fabricate metal ionresponsive vesicle membrane fusion/aggregation system is a convenient and cost effective pathway.14-17 Furthermore, metal-coordination induced vesicle aggregation could help people better understand the role of metal ions in cell membrane movement due to the fact that phospholipids in cell membranes are capable of forming complexes with some specific metal ions like calcium ion.4 Through rational design of building blocks, the vesicle aggregation can be triggered by selective metal ions. Therefore, the vesicle-aggregation system might behave as favorable metal ion sensor platforms, which have been seldom referred. Actually, design of vesicle-based sensors is essential in many applications. In spite of biomimetic aspect, many vesicle systems were designed to be sensors to facilitate the afterward applications.18-22 For instance, although single molecular fluorescent sensors without selfassembly capability can be designed into biocompatible and be applied in sensing analyses in living cells or tissues, it is quite hard to achieve the theranostic functions or combined therapy.23, 24 However, drug release behaviors of vesicular drug carrier with sensing function in vivo can be visually guided according to the optical changes in specific environments.25 During the last decade, self-assembled vesicles have been designed to detect pH, biomolecules like saccharide or adenosine triphosphate (ATP), biohazardous agents like reactive oxygen species (ROS), and so on.18-22 Synthetic vesicles or liposomes have also been developed for detecting metal ions.26


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Building blocks of vesicles for this purpose are mainly from polydiacetylene (PDA) derivatives. PDA-based building blocks are capable of self-organizing into bilayer liposomal vesicles of which bilayer packing array would be strengthened via UV irradiation-triggered polymerization.23 After embedding metal ion receptors covalently or noncovalently within the bilayer arrays, the bulky colors or fluorescent emission colors shall be altered upon interacting with selective metal ions due to vesicle aggregation or metal ion-luminophore interaction. Utilizing this strategy, metal ions such as Hg2+, Pb2+, Al3+ and even K+ have been detected selectively by Kim and some other groups.27-30

Scheme 1. Chemical structure of PGlu and schematic representation of the topological transformation from self-assembled vesicles from PGlu to 2D nanoplates, vesicle aggregates and irregular particles via spontaneous aging, Cu2+/Fe3+ induction and Al3+ induction. In this work, we aim to develop nanoscale vesicular self-assemblies that response to highly selected metal ions via aggregation behavior, in which process, metal ions can be detected. The compound (PGlu) shown in Scheme 1 acts as the building block in vesicle formation, containing pyrene, alkyl spacer as well as glutamic acid moieties. These segments are basic units for ensuring the self-assembly31-33 as well as the fluorescent sensing. Using a solvent displacement method in tetrahydrofuran (THF)/water mixture, fluorescent nanovesicles were prepared, which would transform into lamellar nanoplates with long incubation period. When Fe3+ was added, vesicles shall stick with each other to form vesicle aggregates with greatly quenched emission. Al3+, however, transformed vesicle into irregular particles with enhanced excimer emission. Fluorescent



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and UV-vis absorption studies indicate that, both of Fe3+ and Al3+ can be detected quantitatively. At the same time, the vesicle aggregation into vesicle clusters hinders the aging process, and improves their stability in turn.

2 Experimental section Materials

PGlu was synthesized according to a previous report.31 All other reagents were of AR grade and were purchased from Country Medicine Reagent Co. Ltd., Shanghai, China. All reagents were used without further purification. Characterizations

The samples for transmission electron microscopy (TEM) were measured on a JEM100CX II electron microscope (100 kV). Atomic force microscopy (AFM) images were recorded under ambient condition by using a Veeco Nanoscope Multimode III SPM, and operated in tapping contactmode. In the preparation of AFM sample, we dropped the solution (about 30 µL) on the surface of a silicon wafer. After probably ten minutes, filter paper was used to remove most of the solution on the silicon wafer while the minimal residue absorbed on the surface was tested by AFM. Copper grids and polished silicon wafers were used to prepare samples for TEM and AFM characterizations respectively, which were air-dried. The average diameter of vesicles was recorded by dynamic light scattering (DLS) measurement with a Wyatt QELS Technology DAWN HELEOS instrument, which used a 12-angle replaced detector in scintillation vial and a 50 mW solid-state laser. FT-IR measurements were observed using an Avatar 370 FT-IR Spectrometer. KBr was used in the process of sample disks preparation. The FT-IR measurements were carried out at room temperature. Samples for FTIR characterization were collected by drying precipitates of PGlu assemblies. X-ray diffraction (XRD) experiments were performed on a German Bruker/D8 ADVANCE diffractometer with Cu Kα radiation (λ= 0.15406 nm, 40 KV, 40 mA). The textures of nanoplates were characterized using an Olympus BX63 polarized optical microscope. Sonication experiments were performed on a KQ-100 bath-type sonicator (Kunshan Ultrasonic Instrument Co. Ltd) with 40 KHz frequency and 100 W power at room temperature.

Optical microscopy (OM) was performed on an Olympus CX31RTSF-5

Biological microscope with polarized light accessory. Methods

For the preparation of vesicles, at first, concentrated solutions (10 mM) of PGlu in THF were prepared. Then certain amount of concentrated solutions was transferred into small vials


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by pipette, followed by adding THF and water with certain volume ratios. Then the solution was sonicated for 10 mins, followed by the stabilization for several hours before characterization.

3 Results and discussion In order to trigger the vesicular self-assembly from PGlu, a solvent displacement or nanoprecipitation method34 was employed. Employing water as the major phase and a watermiscible solvent (THF) as a good solvent, nanoaggregates were prepared. After injecting THF solution of PGlu into aqueous phase, followed by the sonication, a homogenous solution with slight opalescence was obtained. The emission properties during the self-assembly process were evaluated in Figure 1. Although pyrene moiety belongs to planar aromatic segment, the emission of PGlu showed a gradual increase with the increase in water volume fraction (fw, vol%). Some compounds that have similar emission phenomenon have been classified as aggregation-induced-emission

luminophores

(AIEgen),

including

the

famous

hexaphenylsilole, tetraphenylethene and stilbene.35, 36 In spite of this, PGlu cannot be regarded as an AIEgen. Its fluorescent enhancement is quite similar to our previous observation of the fluorescence behavior of another planar aromatic amino acid building block.33 The suppressing of intramolecular vibration and motions combined with the loosely packed selfassembly array (less self-quenching) contributed much to the fluorescence enhancement. After PGlu’s self-aggregation in THF/water mixture (3×10-4 M, fw = 90 %), excimer emission formed. The monomer and excimer emission intensity ratio (inset of Figure 1a) was compared, exhibiting a mutation increase when the fw was higher than 50 % which could be assigned as the critical aggregation solvent ratio. The concentration dependent fluorescence was also conducted (Figure 1b), where displayed a gradual decreasing tendency though the monomer/excimer intensity ratio increased with the increase in concentration (fw = 90 %). Compared with monomeric state in pure THF, self-assembled PGlu exhibited decreasing absorbance as well as slight red-shift in UV-vis spectra comparison (Figure 1c), suggesting that PGlu adopted a J-type π π-stacking during self-aggregation. The slight opalescence due to the self-aggregation reflected on the variation of absorbance at visible region. For instance, the absorbance at 400 nm showed a sudden increase caused by the appearance of nanostructures in solution, giving a critical aggregation concentration (CGC) at around 0.25 mM in the case of fw = 90 %.



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Figure 1. Water fraction-dependent fluorescent emission spectra (a, from 0 vol% to 90 vol%; concentration was fixed at 3×10-4 M). Concentration-dependent emission spectra (b, from 0.1 mM to 0.6 mM; fw was fixed at 90 vol%). UV-vis spectra comparison between molecular free and aggregated state (c) at a same concentration. Concentration-dependent UV-vis spectra (d, fw was fixed at 90 vol%). Subsequently, we carried out morphological studies on the self-assemblies from PGlu. After casting the solution (3×10-4 M, fw = 90 %) on silicon wafer which was then dried in air, AFM characterization with tapping mode was employed. The results were displayed in Figure 2a, b and Figure S1, S2 in the supporting information (SI), where independent spherical nanoparticles could be observed. The size of the obtained nanoparticles was polydispersed, and the diameter was varied from tens of nanometer to hundreds of nanometers. The statistic height and diameter profiles were shown in Figure 2c calculated from AFM data. Most of the nanoparticles were around 20 nm or less in height while around 100 nm in diameter. The diameter result is in good agreement with the 3 dimensional (3D) AFM image shown in Fig. 2b and dynamic light scattering size distribution presented in Figure 2d. These results indicate that, the nanoparticles in dried state, have high aspect ratio of ca. 5 with a flat shape. So it is reasonable to speculate the nanoparticles might be hollow inside. In order to verify the assumption, TEM was employed (Figure 2e-f, Figure S3 in the SI). Like AFM images, we


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observed the dispersed nanoparticles (Figure 2e) with similar diameters. However, the enlarged TEM image suggested that the nanoparticles were actually nanovesicles. Clear boundary between interior parts and the vesicle membrane exhibited in vesicles (Figure 2e, S3). Some vesicles own thick wall with thickness of 10 to 20 nm, indicating that they are multi-walled vesicles (Figure 2e). However, single wall nanovesicles (unilamellar) of which membrane thickness was around several nanometers (Figure 2f) also presented. The multidispersity of diameter and wall thickness distribution might be resulted from the vesicle preparation method we employed.34 The formation of membranous vesicles is contributed not only by the intrinsic amphiphilicity of PGlu, but also by the particular co-solvent environment. It is known that, THF and water are highly miscible (the Flory−Huggins interaction parameter of THF and water is around 17), and once the THF solution is injected into water phase (a poor solvent to PGlu), THF with PGlu would generate nanospheres (emulsion-like nanodroplet) to lower the contact interface areas.34 Due to the high affinity between THF and water, water shall permeate into the nanospheres to interact with THF, at which moment, PGlu molecules were repelled and squeezed to the phase interface, forming hollow interior and membranes eventually (a solvent-etching process).37,

38

The mechanism is slightly

different with the traditional direct dissolution method from bulk materials. Consequently, the membrane thickness as well as the diameter may adopt a wider distribution. An advantage of the soft matters prepared by the solvent displacement is that they are more dynamic, and triggered by external stimulus, the aggregates are capable of transforming into other nanostructures in a controlled manner.7



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Figure 2. Representative 2D AFM image (a, 5µm × 5µm) and 3D AFM image (b) of vesicles. Height and diameter profiles of vesicles from AFM data (c). DLS size distribution of vesicle (d). Representative TEM images of vesicles with different magnifications (e, f), where (e) displays the multi-lamellar vesicles and (f) displays the unilamellar vesicles. As elucidated above, the vesicles were dynamic and metastable. Actually, for vesicle samples with relative high concentration values (like 0.4 mM, 0.5 mM), precipitation


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occurred after aging for several days. That means, the vesicles are kinetically stable while the precipitates are thermodynamically stable. By increasing the PGlu concentration to about 0.8 mM or higher, both vesicles and precipitates generated without aging period. Then the morphology of the precipitate from high concentration was studied by optical microscopy (OM), TEM, and AFM experiments, shown in Figure 3a-f. Under natural light of OM, the precipitate exhibited thin, transparent flakes, and they showed colorful textures when the natural light was altered to polarized light. The results indicate that, the molecular packing within the flakes is long-range ordered.39 TEM image with higher magnification verified the morphology. These flakes possess regular and neat edges thanks to the ordered molecular arrangement. A representative AFM image of a flake is displayed in Figure 3d. Its size is up to several macrons while thickness is only tens of nanometers, the high aspect ratio of which is about 40, suggesting the 2 dimensional feature of the nanoplate. Notably, though nanplates formed at high concentration own neat edges and well-defined appearance, the nanoplates transformed from vesicles possess rather irregular edges accompanied by some tubular structures (see arrows in Fig. 3e) which means that a vesicle fusion-directed morphological transformation could be expected. Meanwhile, some vesicles could also be observed adjacent to nanoplates as displayed in Fig. 3f probably due to the incomplete transformation. FT-IR that can provide information about hydrogen bonding was then employed (Figure 3g). Peak at 1724 cm-1 can be assigned to C=O stretching vibration of hydrogen bonded carboxylic acid, indicating the presence of inter-carboxylic acid hydrogen bonding.31-33 In contrast to that, the free C=O stretching vibration peak appeared at 1690 cm-1 is comparatively small. In addition, peaks located at 1630 cm-1 as well as 1543 cm-1 are contributed by the amide I and amide II bands, confirming the existence of amide-amide hydrogen bonding.40 The inter-amide hydrogen bonding is further supported by the peak at 3351 cm-1 as a appearance of an N-H stretching vibration (amide A band). The high directionality of hydrogen bonding existing in the nanoplates facilitates the ordered molecular arrangement and the regular shapes. The molecular packing of PGlu was further characterized by XRD (Figure 3h). Sharp peaks indicated the good crystallinity and long-range ordered molecular organization. Peaks located at 2theta degree of 2.45, 5.02, 7.58, 10.23, 12.79, 16.03, 18.09, 20.56, 22.7 and 25.3 corresponded to the d-spacing of 3.60 nm, 1.73 nm, 1.17 nm, 0.87 nm, 0.69 nm, 0.55 nm, 0.49 nm, 0.43 nm, 0.39 nm and 0.35 nm respectively. These d-spacing value ratio is consistent with 1:1/2:1/3:1/4:1/5:1/6:1/7:1/8:1/9:1/10, suggesting that, PGlu adopted a highly ordered lamellar molecular packing in nanoplates (Figure 3i). The first order of d-spacing of 3.6 nm is the inter-layer distance, which is in accordance with the double length of PGlu (ca. 1.8 nm according to the energy optimized geometry). The lamellar structure transformed from vesicles are slightly different from our previous study in pure water (d-spacing = 3.3 nm)31 may be due to the loosely stacked molecular array in the presence of good solvents.



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Figure 3. Optical microscopy images of nanoplates under natural (a) and polarized light (b). TEM (c) and AFM images (d) of the nanoplates from high concentration sample. TEM images of nanoplates from aging samples (e, f). Right arrows of (e) stand for the vesiclefusion-constituted nanoplate edges and inset of (f) indicates the incompletely transformed vesicles. FT-IR spectra (g, red line from aged low concentration sample while black one from high concentration sample) and XRD pattern (h) of the nanoplate. (i) Proposed molecular packing arrays of vesicles (upper image) and nanoplates (down image). Aging-triggered morphological transformation occurs in some supramolecular selfassembled systems. A similar example reported by us is the spontaneous transformation of a self-assembled fluorenyl-glutamic acid building block from nanotubes into lamellar crystals.32 Some other aromatic amino acid or short peptide-based building blocks also showed nanofiber bundle or dispersion phenomenon with increasing incubation time.41 Vesicle, as a kind of thermodynamic unstable colloidal particle, has been reported in some cases that would undergo self-aggregation into other well-defined nanostructures or microstructures. For example, Huang synthesized some pillar[n]arene-based amphiphiles that afforded into vesicle assembly in aqueous media immediately, and these vesicles were capable of re-organizing into lamellar packed nanotubes or microtubes spontaneously via necklace-like or nanosheet-


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like intermediates without external stimuli induction.42, 43 In our case, kinetically controlled formation of nanovesicles immediately occurs when PGlu molecules enter in aqueous media. However, the nanovesicles are not stable enough due to the carboxylic acid moieties on vesicle surface has strong tendency to form intermolecular hydrogen bonding (a thermodynamic process). Ascribed to the spontaneous Brownian motion, vesicles could contact and collide with each other to allow the happening of inter-carboxylic acid hydrogen bonding formation. Thus, after a longer incubation period, nanovesicles gradual transformed into hydrogen bonded bilayer arrays (lamellar packing).

Figure 4. (a) I/I0 (I stands for the fluorescent intensity after adding metal ions while I0 stands for the initial fluorescent intensity) as well as IE/IM (IE and IM stand for the flurescent intensity of excimer and monomer respectively) vaules with different metal ions (CPGlu = 3×10-4 M, Cmetal ion = 10-4 M, fw = 90 %). (b) Digital photographs with the addition of different metal ions (from left to right: control group, Na+, K+, Mg2+, Fe3+, Co2+, Ni2+, Al3+, Cu2+, Zn2+ respectively) under natural light and UV lamp (365 nm). (b) Emission spectra of self-assembled PGlu with the addition of Fe3+ (CPGlu = 3×10-4 M, concentration of Fe3+ from 0 to 0.25 mM, fw = 90 %). (c) I/I0 value (monomer emission) as a function of the concentration of Fe3+. (d) I/I0 value as a function of the concentration of Al3+.



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Figure 5. (a) UV-vis spectra of self-assembled PGlu with the addition of Fe3+ (CPGlu = 3×10-4 M, concentration of Fe3+ from 0 to 0.3 mM, fw = 90 %); inset shows the macroscopic change under natural light after the addition of Fe3+. (b) The change of absorbance at 400 nm with the addition of Fe3+ and Al3+ (CPGlu = 3×10-4 M, fw = 90 %). Then the responsiveness to selective metal ions was carried out. Due to the presence of pyrene moiety, the variation of nanoaggregates upon interacting with metal ions can be reflected on the fluorescent properties. Several common metal ions were added into the selfassembled vesicle system, allowing for the change of luminescent properties, as shown in Figure 4a, b and S4 in the SI. It was found that, Fe3+ could greatly quench the emission of PGlu while Al3+ was capable of elevating the excimer emission of PGlu. Therefore, the selfassembled nanovesicle behaved as a selective metal ion-sensing supramolecular platform where both of Fe3+ and Al3+ can be detected. When different amount of Fe3+ interacted with the nanovesicles, the fluorescence was quenched gradually (Figure 4c, S5). The emission of PGlu was totally quenched when 0.8 equiv or more Fe3+ existed in the system. The change of relative fluorescence intensity (I/I0) with Fe3+ concentration showed a nonlinear fit. The detection limit which can be defined as the change of 10 % of fluorescence intensity was calculated to be ca. 5 µM for Fe3+.44 Another remarkable fluorescence change is aroused by the addition of Al3+. Different from other metal ion influences, Al3+ enhanced the excimer emission of PGlu, which enabled the easy visual detection. As displayed in Figure S6 and 4d, the excimer emission increased with the addition of Al3+, exhibiting a moderate linear fit. However, the monomer emission decreased first followed by an increasing tendency. The detection limit of Al3+ was determined to be 30 µM according to the above-mentioned method. Utilizing Benesi-Hildebrand method, the 1/(I0-I) value as a function of 1/[Fe3+] showed a good linear fit (R2 = 0.996: a 1:1 binding mode) on the basis of the fluorescence intensity changes and the binding constant (K) between Fe3+ and PGlu was determined to be 1.47×104 M-1 (Figure S7 in the SI). Similarly, a K value of 4.13×103 M-1 was obtained for Al3+-PGlu complex (Figure S8). In order to probe the interaction between PGlu molecules and metal


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ions, UV-vis spectroscopy was employed (Figure 5a, b, S9 in the SI). Upon interacting with Fe3+, the absorbance of vesicle system decreased with red shifts, suggesting the occurrence of a secondary self-assembly. Fe(III) is capable of forming complexes with pyrene via cation-π interaction which has been proven by the gas phase analysis.45 In the presence of amine or amide substituent, cation-π might be strengthened. In aqueous media, pyrene moieties can also form complexes with Fe3+, which also contributed to the observed red-shift. The insertion of iron ions normally could solvate the inner core of nanoaggregates and disrupt the π πstacked arrays, resulting in the quenching of monomer and excimer emission. The addition of Al3+ into the vesicle system would not bring about any obvious shift in UV-vis spectra though the absorbance decreased slightly due to further aggregation. Therefore, Al3+ may not interact directly with pyrene moieties like Fe3+. Instead, it should mainly interact with amide or carboxylic acid groups via metal-ligand coordination interaction. From Fig. 4a-b, it can be found that Cu2+ also arouses emission quenching, indicating the assemblies may be altered. On this account, we then further evaluated the influence of Cu2+ on the self-assembly behaviors of PGlu. Slight turbidity (Fig. 4b) appeared after the addition of Cu2+ (0.2 mM) into the vesicle system (CPGlu = 3×10-4 M, v/v = 1/9), indicating the possibility of morphological transformation from vesicle to the structures with larger size. Under TEM observation, we found that, similar to Fe3+, vesicles aggregated together to form large clusters (Figure S10). Nevertheless, the vesicles barely showed fusion tendency and no tubular or other 1 dimensional nano-objects have been observed. Upon interacting with Cu2+, vesicle membranes no longer stay intact, though the spherical shape was remained. It is suggested that, Cu2+ is less favored to induce the vesicle adhesion and fusion compared to that of Fe3+. Fluorescent emission of vesicles was quenched with the addition of Cu2+, as shown in Figure S11. However, the ability of emission quenching for Cu2+ is less than that of Fe3+. The addition of Cu2+ caused the absorption decrease of some main peaks while enhanced the absorption in visible areas, in good agreement with the formation of larger vesicle clusters (Figure S12). After the Fe3+-PGlu and Al3+-PGlu complexes were dried, they were subjected to FT-IR characterization. As shown in Figure S13, peaks located at 630 cm-1 are assigned to metaloxygen (M-O) band, indicating that the carboxylic acid might be coordinated by the metal ions.46 In addition, peaks at 1438 cm-1 or 1468 cm-1 are ascribed to the stretching of -COOwhich did not appear in free PGlu samples (Figure 3g), further confirming the metal-ligand coordination interaction. Another evidence for the presence of metal-carboxylic acid coordination is the shrinkage of peak at 1724 cm-1, and a new peak appeared at 1616 cm-1 alternatively, suggesting the disappearance of the inter-carboxylic acid hydrogen bonding. Thus, Fe3+ interact with PGlu via coordinating pyrene’s π-surface and carboxylic acid



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moieties while Al3+ only interact with carboxylic acid groups. As we can see from UV-vis spectra after adding metal ions, the absorption of the self-assembled system at visible region was enhanced significantly, whereby the solution turned turbid. From the bulky photographs (Figure 4b), Fe3+ and Al3+ increased the turbidity and opalescence of the solution. Clearly, under the same condition, Al3+ is more favorable to trigger the turbidity than that of Fe3+, as shown in Figure 5b. It means that, the size of aggregates in Al3+ treated vesicle system is bigger than that of Fe3+.

Figure 5. (a-c) TEM images of aggregated vesicle with different magnifications (CPGlu = 0.3 mM, CFe(III) = 0.1 mM, fw = 90 %). (d-h) AFM images of aggregated vesicles with different magnifications where (f) is a 3D image. The scales are 10 µm × 10 µm, 5 µm × 5 µm, 5 µm × 5 µm, and 2 µm × 2 µm for (d), (e), (g), and (h) respectively. (i) and (j) are diameter and height distribution profiles based on AFM data. We further examined the morphologies after the addition of metal ions. It was found that, instead of nanovesicles from self-assembly of PGlu, Fe3+ induced the formation of entangled networks and clusters (Figure 5a-c). The enlarged TEM images showed that, these networks and clusters actually consisted of spherical joints with diameter about 100 nm. It implies the clusters may be derived from the crosslinking of pristine vesicles. We also observed the


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existence of tubular structures (Figure 5c), which may originate from the vesicle fusion. In order to better understand the parental relationship between vesicles and the Fe3+-induced cluster/network, AFM characterization was performed. A tapping mode AFM image with scale of 10 macrons (Figure 5d) exhibited scattered irregular nanoclusters with diameters of about hundreds of nanometers. Some of aggregates formed network or flocculent shapes (Figure 5e, S14 in the SI). The magnified 3D or 2D AFM images shown in Figure 5f-h and S11, revealed that the nanoclusters as well as the networks were constituted by spherical nanoparticles. A cross-section height profile in Figure 5h indicated the diameter of basic unit in the nanoclusters is around 50 nm, which is much less than their parent nanovesicles with diameter of 100 nm, suggesting that vesicle fusion may take place. Moreover, the phase AFM images in Figure S15 showed that the basic vesicle units exhibited obvious flat and compressed center areas (a typical vesicle morphology on silicon wafer). After vesicle aggregation, the diameter of nanoclusters was mainly distributed around 300 nm based on AFM data (Figure 5i), which is in accordance with the light scattering result (Figure S16 in the SI). The vesicle aggregation also aroused the increase in height of aggregates. The original vesicles were only ca. 20 nm in height, which was elevated to about 70 nm after aggregation (Figure 5j). Thus, the increase in both diameter and height values indicated that, the contacting and fusion were non-directional, though some linear tubular structures were generated. Normally, there are two main mechanisms for the membrane contacting and fusion, namely protein-assistance and perturbation-induction.7 For biomembranes, they prefer adopting the protein assistance pathway while most of artificial membrane movements would be induced by perturbation. For this system, the membrane contact and fusion were caused by the perturbation from the addition of Fe3+. As mentioned above, Fe3+ would interact with PGlu by coordination, further diminishing the colloidal stability of vesicles and causing the deformation of vesicles sequentially. Meanwhile, assisted by the inevitable Brownian motion, the impaired vesicles would attach into each other randomly due to the vanishing protection of intact membranes. The interactions that support the vesicle adhesion also include hydrophobic interaction, π π-stacking as well as hydrogen bonding, part of which may be destroyed by the coordination interaction of Fe3+. The aggregated vesicles have better colloidal stability than that of pristine vesicles, and after a long period, no apparent vesiclenanoplate transformation was observed. Compared to the vesicle aggregation caused by Fe3+, the addition of Al3+, however, gave rise to irregular structures. As displayed in Figure S17, S18 in the SI, brick-like structures with size from hundreds of nanometers to several macrons were obtained, and the vesicle structures were destroyed totally even at a moderate concentration of Al3+. We speculated that, Al3+ led to the destruction of bilayer vesicles and then generated amorphous nanostructures where π-π stacking interaction was remained and enhanced, facilitating the excimer emission. In contrast to Fe(III), Al(III) possesses relatively



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weaker coordination capability to PGlu, which shall coordinate to glutamic acid moiety instead of polyaromatic moiety (probed by UV-vis and FT-IR spectra). The coordination to amide and glutamic acid moieties disturbs the directional hydrogen bonding interaction, arousing the destruction of bilayer membranes to form nanoparticles. It is known that, excimer formation requires closely packed pyrene. Thus, when more monomers involve into the π-stacked arrays with a high aggregation number, excimer emission would be enhanced. In our chemistry, due to the loosely packed PGlu within flexible membrane structure, vesicular solution barely exhibits excimer emission. When being transformed into irregular nanoparticles with solid state, more PGlu molecules are involved into self-assemblies with greatly increased aggregation number (could be speculated from the aggregate size), giving rise to excimer emission.

Conclusions In summary, an amphiphile bearing pyrene and glutamic acid moieties was utilized as building block to prepare vesicles in aqueous phase. The vesicles are metastable and can transform into nanoplates with lamellar molecular packing with long incubation time. The addition of Fe3+ triggered the adhesion of vesicle to form vesicle clusters with quenched emission as well as higher colloidal stability. Al3+, however, induced the formation of irregular aggregates with enhanced excimer emission. Therefore, the supramolecular self-assembled system was capable of detecting the presence of both Fe3+ and Al3+ with good selectivity. The specific interaction of metal ions with the building block was then revealed by studying the topological morphology transformation.

Supporting Information UV-vis and emission spectra of samples with different metal ions, as well as the additional TEM and AFM images. This material is available free of charge via the internet at http://pubs.acs.org.

Corresponding Author *E-mail: [email protected].

Acknowledgement This work is supported by the research foundation, Department of Science & Technology of Shandong province, China. We thank Dr. Tao Sun for his contribution in the explanation of some key questions from reviewers, and the involvement in some data analysing works.

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