Self-Assembly and Disassembly of Amphiphilic Zwitterionic

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Self-assembly and Disassembly of Amphiphilic Zwitterionic Perylenediimide Vesicles for Cell Membrane Imaging Yong Ye, Yang Zheng, Chendong Ji, Jie Shen, and Meizhen Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15592 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 21, 2017

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ACS Applied Materials & Interfaces

Self-assembly and Disassembly of Amphiphilic Zwitterionic Perylenediimide Vesicles for Cell Membrane Imaging Yong Ye,a Yang Zheng,b Chendong Ji,a Jie Shen,b* and Meizhen Yina* a

State Key Laboratory of Chemical Resource Engineering, Beijing Laboratory of Biomedical

Materials, Beijing University of Chemical Technology, 100029 Beijing, China b

Department of Entomology, China Agricultural University, 100193 Beijing, China

KEYWORDS: perylenediimide, amphiphilic, zwitterionic, assembly, cell membrane

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ABSTRACT: Animal cells have complicated dynamics of cell membrane structures which require desirable dyes for in vivo imaging. Here, an asymmetric amphiphilic zwitterionic perylenediimide (ZP) derivative has been constructed by introducing an octyl chain and a zwitterionic head to each imide position of perylenediimide chromophore. ZP could selfassemble into vesicles in aqueous solution. The aggregated ZP vesicles have been explored to image cell inner or surface membrane structures by controlled disassembly process. After uptaken into cells, ZP vesicles disassemble into monomers and then incorporate into cell inner membranes. The vesicles can also disassemble in acid food and incorporate into cell surface membrane of gut cells. The research provides a new tool to label the complicated cell membrane structures with up to 3 days long-term labeling for life science applications.

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INTRODUCTION Cells have complicated membrane structures involved in a variety of cellular processes. Cell membrane consists of phospholipid bilayer and serves as the attachment surface for several extracellular structures.1 To visualize the structure of cell membrane, fluorescent dyes with specific staining effects are desirable.2 Commercial dyes, such as DiI and FM, have poor photostability and are easily to be bleached under long term laser excitation in a confocal microscope.3-4 To reveal long-term dynamics of cell membrane tracking in vivo, novel fluorescent probes with high photostability and long-term retention are of great need. Although perylenediimide (PDI) with a rigid and planar structure has strong red fluorescence and excellent photostability in bioimaging, it exhibits poor water solubility and weak fluorescence in aqueous solution due to easy aggregation of perylene chromophore.5 Therefore, modified PDIs with enhanced water solubility are of great interest and have been widely reported as in vivo markers and sensors with stable and strong fluorescence signals.6-12 For example, Zimmerman's group reports the water-soluble polyglycerol-dendronized PDIs serving as highly specific protein labels on the surface of living bacterial and mammalian cells.10 These modified PDIs could avoid the interference from biological autofluorescence when used as intravital dyes. In addition, asymmetric amphiphilic PDI13 and symmetric cationic PDI14 were investigated as membrane markers because of their excellent photostability and biocompatibility. So far, there is no report about asymmetric amphiphilic zwitterionic PDI compound as cell membrane probe. Combination of biocompatible zwitterionic head and photostablity perylene core could be promising in long-term cell membrane labelling.

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In this study, an asymmetric amphiphilic zwitterionic PDI derivative (ZP, Scheme 1A) with hydrophilic zwitterionic head and hydrophobic aliphatic tail was designed and synthesized according to the literature.15-16 The central perylene core was asymmetrically linked by one hydrophobic octyl chain and a zwitterionic group, giving rise to ZP. The zwitterionic group endowed ZP with biocompatibility and resistance to non-specific protein adsorption.16 Due to the amphiphilic characteristic, ZP could self-assemble into water-soluble, neutral and biocompatible vesicle,17 and could be explored as a marker to label either cell inner or cell surface membrane depending on the disassembly process.

Scheme 1. (A) Synthesis and self-assembly of the amphiphilic zwitterionic ZP. (B) Schematic illustration of disassembled ZP incorporating into cell membrane.

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RESULTS AND DISCUSSION As shown in Scheme 1A, asymmetric amphiphilic perylenediimide ZP was synthesized in three steps from commercially available 3,4,9,10-perylenetetracarboxydianhydride (PDA). Firstly, PDA was treated with n-octylamine in refluxing methanol to yield asymmetric perylene monoimide monoanhydride 1. As a flexible hydrophobic tail, octyl chain improves the solubility of perylene core.18 Then, compound 1 further reacted with N,N-dimethylethylenediamine to produce 2. The tertiary amino group in compound 2 provided reactive site for further quaternizing with propanesultone to generate desired amphiphilic ZP. With zwitterionic head and octyl chain, the designed amphiphilic zwitterionic ZP might incorporate into cell membrane with the help of hydrophobic and electrostatic interactions. More details of synthetic process and material characterizations were given in the Supporting Information (Figure S1-S3). As shown in Figure 1A, the obtained ZP dye exhibited good water dispersity. The absorption spectrum of ZP (Figure 1B) shows three main peaks in the ultraviolet-visible range at 495 nm, 466 nm, and 529 nm, respectively, with a detectable shoulder at 647 nm. The 0-1 (495 nm) transition was more intense than the 0-0 (529 nm) transition, which indicated that in water the Franck-Condon factors might favor the higher (0-1) excited state and H-type π-π stacking aggregates were formed.19-20 Fluorescence spectrum of ZP shows a maximum emission peak at 542 nm and a shoulder at 584 nm in aqueous solution (Figure 1B), which could avoid the interference from biological autofluorescence.21 The fluorescence quantum yield (Øf) of ZP dye was 0.10 in water, measured with Edinburgh Instruments' FLS 980 spectrofluorimeter at room temperature (Table S1). The Øf of ZP in water was lower than that in DMSO (Øf=0.67), which was explained by the aggregation of ZP in water. Therefore, the ZP dye could aggregate in

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aqueous environment and enhance its fluorescence when combined with hydrophobic biocomponents, such as lipids.13, 22-23

Figure 1. (A) Colors of aqueous ZP under natural and UV light; (B) absorption and fluorescence spectra of ZP in aqueous solution (7.0×10 -5 M); (C) UV-Vis absorption vs concentration of ZP; (D) fluorescence spectra vs concentration of ZP in aqueous solution (Concentration: 1.0×10-5 M to 1.0×10-4 M, λex = 485 nm, slit 5 nm). The concentration-dependent absorption and emission spectra of ZP in aqueous solution were investigated to further evaluate the influence of aggregation. Both absorbance and fluorescence intensities of ZP increased with the increase of concentration and no significant aggregation-induced quenching was observed (Figures 1C and D), indicating that ZP aggregates

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has good water dispersibility in the whole experimental concentration range. Additionally, compared with commercially available dye (DiI), ZP exhibited excellent photostability (Figure S4). Therefore, ZP has great potential in bioimaging with water dispersity and photostability.

Figure 2. (A) Transmission electronic microscopic images of the vesicles; (B) dynamic light scattering analyses of the vesicles; (C) absorption and (D) fluorescence spectra of ZP/DPPC ([ZP] = 70 µM). To give further insight into shape, size and size distribution of the ZP aggregates in aqueous solution, transmission electron microscopy (TEM) and dynamic light scattering (DLS) were carried out. ZP aggregates were relatively uniform vesicles (Figure 2A). 24 The hydrodynamic size of ZP was 215 nm (Figure 2B), which is larger than the hard core size of ZP because the

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outside hydrophilic zwitterionic groups could form a hydration layer.25 In general, it is assumed that particles up to about 100-200 nm can be internalized by endocytosis, while larger particles have to be taken up by phagocytosis. Therefore, we speculate that ZP is internalized by cells through both endocytosis and phagocytosis.26 The formation of well-defined vesicles of ZP may be attributed to π-π stacking between the perylene cores along one-dimensional long axis, and stabilized by surrounding hydrophilic zwitterionic group as shown in Scheme 1A. We calculated the geometry of ZP by gauss B3LYP/6-31g (d) (Figure S5). Due to the steric hindrance of zwitterionic group, ZP is wedge in geometry, which favors the formation of vesicle architecture.27 Therefore, we speculate that ZP vesicle is resulted from π-π stacking between perylene cores and the spontaneous curvature caused by steric hindrance effect of the zwitterionic group. Similar mechanism for the control of self-assembly of asymmetric amphiphilic molecule have been reported by Frank Würthner et.al.27 Furthermore, the nanoparticle possess nearly neutral surface charge after overnight aging (Table S1), which suggested the stability of ZP aggregates. Additionally, the neutral nanoparticle with zwitterionic terminal groups showed high resistance to non-specific protein adsorption,28-29 thus, avoiding quick recognition by the immune system and exhibit delayed blood clearance in vivo, which is desirable for in vivo bioimaging.30 The phospholipid bilayer of the cell membrane is hydrophilic on the surface and hydrophobic inside. The ZP vesicles, covered with abundant cationic and anionic charges on the surface could interact with the polar outer surface of cell membrane and then be internalized into cells. Due to acidic circumstance of lysosome, vesicles disassemble and incorporate into the lipid membrane with the help of hydrophobic alkyl groups (Scheme 1B). The Gauss length of ZP (2.79 nm, Figure S5) matched the thickness of lipid monolayer, which is suitable for membrane-

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incorporation of ZP.31 Above all, with proper size, ZP could anchor among the membrane by electrostatic pair and hydrophobic interactions.32 In order to confirm the assumption, dipalmitoyl phosphatidylcholine (DPPC), one of the biomembrane components, was chosen to stimulate cell membrane to interact with ZP vesicles. ZP aqueous solution (70 µM) was prepared as blank. When 200 µM DPPC was titrated to aqueous ZP vesicles, both the absorption and fluorescence intensity of ZP increased remarkably (Figure 2C and 2D) due to synergistic effect of electrostatic and hydrophobic interaction. The zwitterionic group of ZP could form electronic pair with the polar surface of DPPC micelles, and the hydrophobic moiety of ZP could be rapidly absorbed onto DPPC. These ZP vesicles could disassemble in the presence of DPPC micelles, indicating the presence of strong interactions such as hydrophobic effect and electrostatic forces between ZP and mimic membranes.33 In addition, the absorption and fluorescence intensity of ZP increased in acid aqueous (Figure S6), indicating slowly disassemble of ZP aggregation. We try to study the degree of disassemble by comparing the fluorescence quantum yield in water, acid aqueous (pH 3), DPPC micelle and “good” solvent (DMSO). ZP molecules aggregate in water (Øf =0.1) and free in DMSO (Øf =0.67). The Φf of ZP in DPPC micelle and acid aqueous (pH 3) were 0.42 and 0.16, respectively, which is obviously larger than the Φf of ZP in water. The increased Øf of ZP suggests the disassembly process of ZP aggregates in acid or in DPPC micelle. Thus, we speculate that the ZP vesicle could slowly disassemble in acid and lipid environment. To further test the imaging ability of ZP dye, ZP-labelled mimic membrane was observed by fluorescence microscope. When water droplets coated with DPPC34 was added to aqueous ZP (1 µM), ZP was rapidly adsorbed onto the surfaces of DPPC-coated droplet. Uniform fluorescent signal was observed

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around the circumference of the droplets, which was easily distinguished from the surrounding environment (Figure S7). Then a series of biological experiments were designed to explore the labelling effects of cell membrane structure. First, the cell viability assay was conducted to test the cytotoxicity of ZP. A commercially available membrane marker DiI was employed as control. After 48 h of incubation, cell viabilities of the two dyes were shown in Figure S8. ZP showed much higher cell viability than DiI, especially in the case of high concentration. At working concentration of Dil (10 µM, recommend by the manufacture), the viability of treated cells was 76%. While the cell viabilities of ZP treatments were all higher than 90% at all tested concentrations, suggesting excellent biocompatibility of ZP. Next, the labelling effects of ZP vesicles were studied in vitro. HeLa cells were selected to incubate with ZP and DiI solutions, respectively, for 24 h. Both Dil and ZP showed the same labeling patterns with enrichment in membrane-vesicles inside the cells (Figure 3A and B). Excitingly, ZP showed much higher fluorescence intensity compared with DiI (Figure 3C). In order to study the long term tracking ability of ZP and DiI in live cells, the incubation duration of ZP or DiI was extended up to 48 h. Within 0.25 h, the fluorescence intensity of ZP inside the cells was already stronger than that of Dil (Figure 3C), indicating ZP was more sensitive over DiI. The fluorescence of DiI inside cells increased along with the incubation duration from 0.25 h to 3 h, but then reduced gradually and finally quenched after 72 h of incubation (Figure 3C, red curve). On the contrary, the fluorescence intensity of ZP dye inside cells exhibited an increasing trend along with the incubation duration (Figure 3C, blue curve), indicating a continuous accumulation of ZP and good stability. After 3 h of incubation, the fluorescence intensity of ZP was much higher than that of DiI. At the working concentration of DiI (10 µM) and ZP (0.1 µM),

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ZP showed much less consumption and higher fluorescence intensity compared with DiI, therefore, ZP dye had great potential as long-term membrane tracker to visualize the biological membrane dynamics. The distribution of ZP fluorescence indicates that ZP vesicles could interact with cell membrane and then are internalized into cells by endocytosis. Due to the acid circumstance of lysosome, ZP vesicles disassemble into single molecules and incorporate into cell inner membrane. The inner membrane of HeLa cells labeled by ZP need to be further confirmed in more complicated tissue cells.

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Figure 3. The fluorescent images of HeLa cells incubated with (A) DiI (10 µM, 0.5 h) and (B) ZP (0.1 µΜ, 24 h). The red fluorescence of (A’) DiI and (B’) ZP inside cells is shown and then quantified by Image-J program. (C) Dynamic curves are presented to show the fluorescence intensity of each dye inside cells along with the incubation duration. Subsequently, we explored the labeling effect of ZP on tissue cells. Drosophila larval fat body tissues, containing complicated inner membrane structure, were dissected for the test. As

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expected, the inner membrane network of live fatbody cells was clearly visualized (Figures 4A and A’) when incubated with ZP solution at a low concentration of 1 µM for 1 h.

Figure 4. (A) Ex vivo and (B) in vivo membrane of Drosohpila tissues labeled by ZP. (A) Fluorescent images of Drosophila larval fat body tissue cultured with ZP vesicles; (A’) The separated channel (red, ZP) shows the inner membrane structures of fat body cells; (B) Fluorescence images of Drosophila larval gut when fed with artificial diet containing ZP vesicles; (B’) The separated channel (red, ZP) shows the cell surface membrane of gut cells.

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Finally, we performed in vivo experiment to further explore the labeling ability of ZP vesicles. The vesicles were simply mixed into artificial diet. Then Drosophila 3rd instar larvae were fed with the artificial diet containing ZP vesicles for 3 days. The mid gut tissues were dissected and observed with a fluorescence microscope. Excitingly, the gut cell outline was clearly labeled by ZP with red fluorescence (Figures 4B and 4B’), indicating that the surface membrane of gut cells is marked by ZP. Because that the artificial diet of Drosophila food is acid, according to the food formulation, ZP vesicles disassemble into single molecules and thereby incorporate into the cell surface membrane of gut cells. Therefore, unlike above cell and tissue culture environmental, ZP now can label the cell surface membrane, but not the cell inner membranes. While the control dye, Dil, showed no such ability to label cell membrane in vivo (data not shown). CONCLUSIONS Amphiphilic ZP with a zwitterionic head and octyl tail, having fluorescence emission above 584 nm, was synthesized, which could minimize the autofluorescence in bioimaging. Amphiphilic ZP aggregated and formed nanosized vesicles in aqueous solution. ZP vesicles could be internalized into cells through interaction with plasma cell membrane via opposite charge pairs, and subsequently disassemble into single molecules in acid lysosomes to mark the inner membrane structures of cultured cells and tissues through incorporation into phospholipid bilayer via hydrophobic interaction. When applied in vivo, the acid circumstance of food and/or gut (stomach) leads to ZP vesicles disassemble and directly label the surface membrane of gut cells. Moreover, ZP shows a long-term, up to 3 days, membrane tracking ability to monitor the cellular dynamics. Together with the excellent photostability and low-toxicity property, ZP has a great potential in diagnosis and therapy in the biomedical applications.

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ASSOCIATED CONTENT Supporting Information Experimental procedures, characterization of the macromolecules, supporting figures and text. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author *Meizhen Yin: [email protected] *Jie Shen: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (21574009, 51521062 and 51573012), the Beijing Natural Science Foundation (2142026), the Beijing collaborative innovative research center for cardiovascular diseases, and the Higher Education and High-quality and World-class Universities.

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839x359mm (72 x 72 DPI)


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Self-Assembly and Disassembly of Amphiphilic Zwitterionic

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