Controllable Aggregation-Induced Exocytosis Inhibition (CAIEI) of

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Controllable Aggregation-Induced Exocytosis Inhibition (CAIEI) of Plasmonic Nanoparticles in Cancer Cells Regulated by MicroRNA Ruo-Can Qian, Jian Lv, and Yi-Tao Long Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00465 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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Molecular Pharmaceutics

Controllable Aggregation-Induced Exocytosis Inhibition (CAIEI) of Plasmonic Nanoparticles in Cancer Cells Regulated by MicroRNA Ruo-Can Qian, Jian Lv, and Yi-Tao Long* Key Laboratory for Advanced Materials & School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China; KEYWORDS: Exocytosis Inhibition, Plasmonic nanoparticles, MicroRNA, Cancer Cells ABSTRACT: Recent advances of nanotechnology have produced plenty of intracellular drug delivery systems based on various functional nanoparticles. Although much progress has been achieved in improving the cellular uptake efficiency, the retention time of these engineered nanoparticles in living cells has not yet received wide attentions. Here, we report the controllable exocytosis of plasmonic gold nanoparticles (GNPs) based on a microRNA-21 (miRNA-21) targeted binary system. Rapid intracellular accumulation of GNPs was observed in miRNA-21 positive MCF-7 breast cancer cells, which blocked the exocytosis of the GNP aggregates. Under near-infrared (NIR) irradiation, MCF-7 cells were successfully killed due to the far-red and NIR absorption of the GNP aggregates. In contrast, in miRNA-21 negative cells, the dispersive GNPs escaped from the cells after 6 h. The traces of GNPs could be conveniently captured under the dark field microscope. This work provides a promising platform for the study of controllable aggregation-induced exocytosis inhibition (CAIEI) of nanocarriers, which is inspiring for the design of more effective nanodrugs for the treatment of cancer.

INTRODUCTION Engineered nanoparticles designed for drug delivery into the living cells have showed great promise for the diagnosis and treatment of various diseases, especially cancer.1-7 Despite the advantages on cellular targeting ability and improved uptake efficienty achieved by these drug delivery systems, there has been limited effort to consider the intracellular retention time of the nanoparticles.8,9 Besides effective cellular uptake, the sustained long-term retention time of nanoparticles in the cells is a key factor for the improvement of the therapeutic effect.10 The long retention times avoid the rapid removal of the nanoparticles via the exocytosis thus increased the duration of the nanoparticle-mediated treatment.11,12 Therefore, it is necessary to establish a feasible strategy to investigate the exocytosis of nanoparticles and their retention times. Cellular delivery systems based on plasmonic nanoparticles have been extensively studied due to their unique plasmon resonance scattering properties, easy preparation and surfacefunctionalization.13,14 Among them, the engineered gold nanoparticles (GNPs) attract special attention because of their high scattering intensity and excellent biocompatibility.15-18 Consequently, GNPs have been widely applied for the construction of various cell delivery systems.19,20 However, lack of study about how long these GNPs stay inside the cells has impeded their clinical application. Therefore, in this work, we describe a practical approach to regulate the exocytosis of GNPs by the microRNA-induced intracellular accumulation. MicroRNAs (miRNAs) are a class of noncoding short RNAs widely expressed in eukaryotic cells, usually 18-25 nucleotides.21,22 Recent researches show that the expressions of miRNAs play key roles in the genesis of cancer.23,24 In this work, we chose miRNA-21, which is overexpressed in many cancer cells,25 as the key to induce the formation of GNP

assemblies in cancer cells and then investigate the regulated exocytosis situation of the intracellular GNPs. As shown in Figure 1a, we designed a binary system composed by a pair of molecular beacon (MB)-functionalized GNPs, GNP-1 and GNP-2.26 The GNPs were readily taken into the cells by endocytosis.27 In MCF-7 breast cancer cells, rapid accumulation of GNPs occurred in the presence of intracellular miRNA-21, and the exocytosis of the GNP assemblies was greatly inhibited. The assemblies owned a prolonged retention time of 24 h, and we named this controllable retention “CAIEI” (Controllable Aggregation-Induced Exocytosis Inhibition). To achieve the CAIEI system, bare GNPs of 60 nm were modified with two different MB sequences (MB-1/MB-2 for GNP-1/GNP-2). With the thiollabeled 5′ end and the Cy5-tagged 3′ end, the MBs formed haipin structures on the GNP surface without showing Cy5 fluorescent signals initially due to the fluorescence resonance energy transfer (FRET) effect of GNP cores.28 With the loop of MBs designed to be complementary with miRNA-21, the hairpin would be opened in the presence of miRNA-21, thus turning on the Cy5 fluorescence. The far ends of the opened MB-1 and MB-2 were complementary with each other, thus a cross-linkage between GNP-1 and GNP-2 was induced, forming large GNP assemblies (Figure 1b). The miRNA-21 induced aggregation was firstly confirmed in solutions. After the mixture of GNP-1 and GNP-2 was incubated with miRNA-21, the color of the solution changed from red to dark purple (Figure 1c), indicating the formation of GNP assemblies, and the corresponding TEM images clearly showed the accumulation (Figure 1d). Moreover, the miRNA-21 induced GNP accumulation was observed under dark-field microscope (DFM). GNPs have the capability to absorb and scatter the incident light resonantly, and the strong

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Figure 1. MiRNA-controlled exocytosis of plasmonic nanoparticles. (a) Inhibition of GNP exocytosis by the miRNA-21 induced assembly. (b) MiRNA-21 caused GNP aggregation and fluorescence “on”. (c) GNP-1 (0.5 mL, 0.28 nM) and GNP-2 (0.5 mL, 0.28 nM) mixed solution before (i) and after (ii) treated with miRNA-21 (150 nM) for 15 min. (d) TEM images and (e) Dark-field images of GNP-1and GNP-2 mixed solution before and after the treatment of miRNA-21 corresponding to (c). (f) Average fluorescence intensity of the dots in (e). From left to right: red, green, and blue channel; i: left image in (e); ii: right image in (e). Scale bar in (d): 50 nm; Scale bar in (e): 5 µm. The results are presented as mean ± SD (n = 6). *P < 0.05, **P < 0.01 using a Student’s t test).

scattering light of GNPs could be conveniently observed by DFM.29-31 In the presence of miRNA-21, the GNP aggregates emerged and their color under dark-field microscope changed from green to orange (Figure 1e), showing stronger scattering light intensity. The average fluorescence intensity of the dots in dark-field images was counted by different color channels (red, green, and blue channel). MiRNA-21 treated GNPs showed obviously higher red channel intensity and lower green channel intensity (Figure 1f), indicating the assembly of GNPs. The related UV-Vis and fluorescence spectroscopy characterizations further confirmed the miRNA-21 induced aggregation of GNPs (Figure S1-3), which was used for the following investigation of the CAIEI in cancer cells. We demonstrate that the designed system could remain in MCF-7 cells for 24 h without obvious exocytosis, and could be used for selectively killing cancer cells.

EXPERIMENTAL SECTION Chemicals and Materials. Chloroauric acid (HAuCl4•4H2O) and Glutathione (GSH) were obtained from Sigma-Aldrich Inc. (USA). LysoTracker Green was obtained from Invitrogen China Ltd. Annexin V-FITC/propidium iodide (PI) apoptosis detection kit, 3-(4,5-dimethylthiazol-2-yl)2-diphenyltetrazolium bromide (MTT), MCF-7 cells, and MCF-10A cells were purchased from KeyGen Biotech. Co. Ltd. (Nanjing, China). Phosphate buffer saline (PBS, pH 7.4) contained 136.7 mM NaCl, 2.7 mM KCl, 8.72 mM Na2HPO4, and 1.41 mM KH2PO4. All other reagents were of analytical grade. All aqueous solutions were prepared using ultrapure water (≥ 18 MΩ, Milli-Q, Millipore).

The DNA sequences were obtained from Sangon Biological Engineering Technology & Co. Ltd. (Shanghai, China) with the following sequences: (a) MB-1: 5’-HS-(CH2)6CCGTTCTA TCA ACA TCA GTC TGA TAA GCT A TAGAACGG-Cy5-3’; (b) MB-2: 5’-HS-(CH2)6-TAGAACGG TCA ACA TCA GTC TGA TAA GCT A CCGTTCTA-Cy53’. The RNA sequences were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China) with the following sequences: (a) miRNA-21: 5’-UAG CUU AUC AGA CUG AUG UUG A-3’; (b) anti-miRNA-21: 5’-UCA ACA UCA GUC UGA UAA GCU A-3’; (c) mismatched miRNA-21: UAG CUU AUC AGA CAG AUG UUC A. Apparatus. The transmission electron microscopic (TEM) imaging was performed on a JEM-2010 high-resolution transmission electron microscope (JEOL Ltd., Japan). The dark-field images were obtained by an inverted microscope (eclipse Ti-U, Nikon, Japan) equipped with a dark-field condenser (0.8 < NA < 0.95) and a 40× objective lens (NA = 0.8), and a white light source (100 W halogen lamp) was used to excite the plasmon resonance scattering light of GNPs. The scattering light of GNPs was split by a monochromator (Acton SP2300i, PI) equipped with a grating (grating density 300 lines/mm; blazed wavelength 500 nm), and the scattering spectra were recorded by a spectrometer CCD (CASCADE 512B, Roper Scientific, PI).The fluorescence images were obtained on the same microscope using a mercury lamp (100 W Epi illuminator) as the excitation light source. MTT assay were performed on a microplate reader (Synergy 5, Biotech, USA). Coupled plasma atomic emission spectroscopy (ICP-


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AES, Agilent 725ES, USA) was used to analyze the intracellular amount of Au. The near-infrared (NIR) irradiation was generated by a 680 nm laser (Laserwave Optoelectronics Technology Co., Ltd., China). Nanoparticle diameters and zeta potentials were measured by Malvern dynamic light scattering and zeta potential integrated analyzer. Synthesis of MB-Functionalized GNPs (GNP-1 and GNP-2). Firstly, 60 nm GNPs were synthesized by a traditional seed-growing method.32 Briefly, the 13 nm seed GNPs were obtained by heating 50 mL of HAuCl4 (0.01%) solution to 100 °C, quickly added 5 mL of trisodium citrate (38.8 mM), and kept stirring at 100 °C for 30 min until the color turned to red. The obtained seed solution was then used to prepare 60 nm GNPs. Briefly, 100 µL of NH2OH•HCl (0.2 M) was added into 1 mL of the seed solution and diluted to 25 mL by pure water. Then 3.0 mL of HAuCl4 (0.1%) was added into the mixture dropwise under continuous stirring for 30 min until the color changed to dark red, and the solution was concentrated to 1 mL. For the preparation of MB-functionalized GNPs, Cy5-tagged MB (10 µL, 100 µM) was mixed with bare GNP solution (1 mL) and then stirred overnight. After that, 0.1 mL PBS buffer (containing 2 M NaCl) was added to the mixture dropwise for stabilizing the obtained MB functionalized GNPs (MB-1 modified GNP-1 and MB-2 modified GNP-2). The above solution was centrifuged and washed with PBS twice, then suspended in 1 mL PBS (0.28 nM for in vitro tests). Cell Culture. Breast cancer cells (MCF-7) cells were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS, Sigma), streptomycin (100 µg mL-1), and penicillin (100 µg mL-1) in a humid atmosphere with 5% CO2 at 37 °C. Cell number was calculated by a Petroff-Hausser cell counter. Normal breast epithelial cells (MCF-10A) were cultured in DMEM/F12 (1:1) medium supplemented with 5% equine serum, 10 µg mL-1 insulin, 0.5 µg mL–1 hydrocortisone, 20 ng mL-1 epidermal growth factor, 2 mM l-glutamine, 100 µg mL–1 streptomycin and 100 U mL-1 penicillin at 37 °C in a humidified incubator containing 5% CO2 and 95% air. Dark-field/Fluorescence Imaging and Scattering Spectroscopy. For DFM analysis of GNPs, the culture dishes were ultrasonic treated for 1 h in ethanol and then 1 h in pure water. The GNP solution was dropped on the bottom of a culture dish for DFM observation. For cell observation, cells (1 mL, 1×106 mL-1) were seeded on culture dishes overnight and then incubated with GNP in culture medium (with FBS) for various times at 37 oC. Then, the cells were washed and immersed in PBS for the microscopic observation. For exocytosis observation, the cells were treated with GNPs for 6 h and then washed and immersed in PBS. The scattering light of GNPs was split by a monochromator and recorded by a CCD spectrometer to obtain the scattering spectra. Quantification of Cellular Uptake of GNPs by ICP-AES. GNP-treated cells (1 mL, 1×106 mL-1) were digested by aqua regia (1 mL) and diluted to 5 mL by pure water for ICP-AES detection. The number of the Au atoms (N) of each GNP was calculated by N = (D / dAu)3 (D = diameter of GNP; dAu = diameter of Au atom). Thus, the amount of GNPs per cell (n) could be calculated by n = M / (N × C) (M = number of Au atoms determined from ICP-AES results; C = number of cells). Six parallel samples were tested.

RESULTS AND DISCUSSION

Intracellular behavior of the binary system (GNP-1 and

Figure 2. Cellular uptake and intracellular behavior of the binary system. (a) Time course microscopic images of MCF-7 cells incubated with the binary system (mixed solution of GNP-1 (10 µL, 0.28 nM) and GNP-2 (10 µL, 0.28 nM)), from up to down: bright-field images, DFM images, Cy5 fluorescence images). (b) Cell images showing the bounds (red dashed lines) and the entrapped GNPs (black dots) corresponding to the DFM images in (a). (c) Average fluorescence intensity of the dots in the DFM images in (a). From left to right: red, green, and blue channel. (d) Corresponding scattering spectra of GNPs in the DFM images in (a), pointed by the white arrows. (e) Corresponding color-coded fluorescence intensity of the Cy5 fluorescence images in (a). (f) The average red channel intensity in the cell areas corresponding to the Cy5 fluorescence images in (a). (g) Amount of intracellular GNPs after the cells were treated with the binary system by different times (calculated by ICP-AES). The results are presented as mean ± SD (n = 6). *P < 0.05, **P < 0.01 using a Student’s t test). Scale bar in (a), (b)&(e): 10 µm. The average channel brightness intensity was read by Adobe Photoshop software. Color scale bar in (e): 0-234 from up (black) to down (red).



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Figure 3. Cellular uptake and exocytosis of GNPs. (a) Time course microscopic images of MCF-7 cells incubated with only GNP-1or GNP-2 (20 µL, 0.28 nM for each respectively), from up to down: bright-field images, DFM images, Cy5 fluorescence images). (b) Cell images showing the bounds (red dashed lines) and the GNPs (black dots) corresponding to the DFM images in (a). Amount of intracellular GNPs after the cells were treated with (c) GNP-1 or (d) GNP-2 by different times. (e) Amount of intracellular GNPs after the cells were incubated with (A) GNP-1, (B) GNP-2 or (C) the binary system by 24 h. The results are presented as mean ± SD (n = 6). *P < 0.05, **P < 0.01 using a Student’s t test). Scale bar in (a)&(e): 10 µm.

GNP-2) in MCF-7 cells. Agglomerated forms of nanoparticles influence their intracellular behavior and exocytosis patterns.33,34 Before cell experiments, the stability of the MBmodified GNPs were monitored in both serum free and serum containing medium at 37 oC for 24 h (the incubation time in the cell experiments). The UV-Vis spectra, fluorescence spectra and dark-field images showed little change (Figure S4-6), indicating no aggregation or nonspecific desorption, which confirmed the good stability of the MB functionalized GNPs. In addition, the mixed GNP solution was treated by a mismatched miRNA-21 sequence and the GSH solution respectively. Negligible fluorescence was detected, and the GNPs in dark-field images maintained the green color after incubation, confirming the specific recognition between the GNPs and miRNA-21 (Figure S7). MTT assay confirmed the low cytotoxicity of the binary system (Figure S8), which was favorable for the following cellular experiments. Afterward, the internalization of the binary system and the exocytosis situation were examined in living cancer cells. MCF-7 cells were incubated with 20 µL of the binary system solution (10 µL GNP-1 and 10 µL GNP-2). After incubation, the internalized GNPs increased with the incubation time, and then the exocytosis behavior of these nanoparticles was analyzed by different methods. As shown in Figure 2a, the DFM images confirmed that

the GNPs were taken in to the cells rapidly within 1 h, and the amount of GNPs increased with the incubation time. At the same time, the fluorescence of Cy5 emerged in the cytoplasm, indicating the miRNA-21 induced opening of the hairpin structure (MB) on the surface of the GNPs. In addition, from the DFM images, efficient accumulation of the binary system composed of GNP-1 and GNP-2 was observed, as the color of GNPs changed from green to orange-red. In Figure 2b, the cell bounds were marked, from which we can see that the GNPs aggregates were entrapped within the cell membrane even after 24 h. The red channel intensity of the agglomerated GNPs increased while the green channel intensity dropped obviously compared to single GNPs (Figure 2c), and the scattering spectra of the aggregated GNPs showed an obvious redshift with stronger scattering intensity (Figure 2d). The miRNA-21 caused fluorescence recovery of Cy5 within cell areas were shown in Figure 2e,f. The amount of elemental Au in cells was evaluated by ICP-AES measurements, which further verified the efficient uptake of the binary system, and the amount of the GNPs per cell was estimated (Figure 2g). LysoTracker Green was used to show the localization of the GNPs after endocytosis. As shown in Figure S9, most of the GNPs were located out of the lysosomes after 2 h incubation, and only a few of GNPs were entrapped within the lysosomes


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after 24 h, which was favorable for the miRNA-21 recognition. For the binary system of GNP-1 and GNP-2, the internalized GNPs increased with the incubation time until reached a plateau of approximately 6.5×103 per cell at 6 h. After 24 h, the number of the intracellular nanoparticles did not change, demonstrating that the cells effectively inhibited the exocytosis of miRNA-21 induced GNP aggregates, which we termed as CAIEI (controllable aggregation-induced exocytosis inhibition). Cellular uptake and exocytosis of GNPs. Figure 3a shows the intracellular behavior of single GNP-1 or GNP-2. The MCF-7 cells were treated with GNP-1 or GNP-2 solutions (20 µL GNP-1 or 20 µL GNP-2) respectively. The amount of intracellular GNPs increased with 6 h. However, the nanoparticles began to escape from the cells after 12 h, as shown in the DFM images. In addition, the color of the individual GNPs in DFM images did not show obvious change after the incubation (Figure 3a), indicating no agglomeration. The fluorescence of Cy5 emerged after the incubation, confirming the existence of miRNA-21. Therefore, with only GNP-1 or GNP-2, the nanoparticles could not aggregate, and would be driven out of the cells (Figure 3b). The amount of the GNPs retained inside the cells was determined by ICP-AES, which showed that the internalized GNPs increased to approximately 6.5×103 per cell at 6 h, and since then the number decreased to less than 3.8×103 per cell after 24 h due to the exocytosis (Figure 3c,3d). Compared with the cells treated with the binary system, the number of intracellular GNPs dropped by 42 percent after 24 h incubation (Figure 3e), with subsequent decreased retention time. Evaluation of the Aggregation Efficiency of the Binary System Controlled by MiRNA-21. To investigate the

Figure 4. Different cells treated by the binary system. (a) Microscopic images of MCF-7 control cells (left column) and anti-miRNA-21 (200 nM) treated cells (right column) incubated with the binary system (mixed solution of GNP-1 (10 µL, 0.28 nM) and GNP-2 (10 µL, 0.28 nM)) for 24 h, from up to down: bright-field images, DFM images, Cy5 fluorescence images). (b) Cell images showing the bounds (red dashed lines) and the GNPs (black dots) corresponding to the DFM images in (a). (c) Corresponding color-coded fluorescence intensity of the Cy5 fluorescence images in (a). (d) Amount of intracellular GNPs after the (A) control cells and (B) anti-miRNA-21 treated cells were treated with the binary system for 24 h. The results are presented as mean ± SD (n = 6). **P < 0.01 using a Student’s t test). Scale bar in (a), (b)&(c): 10 µm. Color scale bar in (c): 0-234 from up (black) to down (red).

miRNA-21 controlled intracellular aggregation behavior of the binary system in different cells, the anti-chain of miRNA-21, anti-miRNA-21, was used as an inhibiting agent to down regulate the level of miRNA-21 in MCF-7 cells. As shown in Figure 4a-c, the binary system accumulated efficiently in control MCF-7 cells, as the DFM image contained more orange-red dots and stronger scattering luminescence, indicating the formation of GNP aggregates. The strong fluorescence of Cy5 confirmed the existence of miRNA-21, demonstrating the miRNA-21 induced GNP accumulation. In contrast, in the MCF-7 cells transfected by anti-miRNA-21, there was no obvious Cy5 fluorescence, and the DFM image showed that most of the GNP dots scattered green light with some of them escaping from the cytoplasm. From ICP-AES detection, the intracellular GNPs in miRNA-21 negative cells decreased to less than 3.9×103 per cell compared with 6.5×103 per cell in the control cells (Figure 4d). Therefore, the above results confirmed that the intracellular behavior of the binary system was controlled by the miRNA-21. In the existence of miRNA-21, the GNPs accumulated and retained in the cytoplasm, while in the absence of miRNA-21, the GNPs could not form aggregates and would be exocytosed. Photothermal Therapeutic Application of the Controllable Aggregation-Induced Exocytosis Inhibition (CAIEI). As the above experiments confirmed the efficient CAIEI of the binary system in miRNA-21 positive MCF-7 cells, it is significant to find the clinic applications of the system. In this case, a 680 nm laser was used to perform the NIR irradiation treatment. After incubation with the binary system, the MCF-7 cells were irradiated with the laser at 0.5 W cm−2 at 37 °C for 2 h and then stained by Annexin V-FITC/PI to observe the cell death. As shown in Figure 5a, the miRNA-21 positive cells displayed strong apoptotic fluorescence, and the cell shape was destroyed, demonstrating that the NIR treatment could efficiently kill cancer cells. On the other side, the regular cell shape maintained after the NIR irradiation in miRNA-21 negative cells with no apoptotic fluorescence (Figure 5b).

Figure 5. NIR treatment of the binary system incubated cells. Microscopic images of (a) MCF-7 control cells, (b) anti-miRNA21 (200 nM) treated cells, and (c) MCF-10A cells incubated with the binary system (mixed solution of GNP-1 (10 µL, 0.28 nM) and GNP-2 (10 µL, 0.28 nM)) for 24 h, irradiated under the NIR treatment for 2 h and then stained by Annexin V-FITC/PI (from left to right: bright-field images, PI fluorescence images, Annexin V-FITC fluorescence images, Annexin V-FITC/PI Merged fluorescence images). Scale bar: 10 µm.



The survival rate of the miRNA-21 positive cell was less than 10% compared to more than 90% of the negative cells (Figure S10). A normal breast epithelial cell line, MCF-10A was also cultured with the binary system, irradiated under the laser, and then stained by Annexin V-FITC/PI to visualize the cell death. As shown in Figure 5c, the cells showed negligible fluorescence, indicating good activity. Therefore, the designed CAIEI system could be applied for cancer therapy, with long retain time in the cytoplasm, which was especially favorable for clinical application. In addition, the doubling time of MCF-7 control cells and anti-miRNA-21 treated cells was calculated to be 37.4 and 46.8 h, respectively, indicating a positive correlation between the tumor proliferation and the expression of miRNA-21 (Figure S11).

CONCLUSION In this work, we propose the concept of controllable aggregation-induced exocytosis inhibition (CAIEI), and its potential clinical applications. The controllable exocytosis inhibition of GNPs regulated by miRNA-21 in living cells were studied using the binary system composed by a pair of MBfunctionalized GNPs, GNP-1 and GNP-2, which could form large GNP aggregates in the presence of miRNA-21. Using MCF-7 breast cancer cells as the example, we found that the intracellular retain time of the binary system was greatly improved to 24 h with no obvious exocytosis due to the rapid intracellular accumulation specifically triggered by microRNA-21, which could be used for the following nearinfrared irradiation treatment to killing cancer cells. Using a 680 nm laser, the MCF-7 cells were successfully killed, indicating good potential in the development of more effective drug nanocarriers for the treatment of cancer.

ASSOCIATED CONTENT Supporting Information Characterization of GNPs, discussion of miRNA-21 induced aggregation, stability and cytotoxicity of the binary system, and experimental details. This material is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Phone/fax: 86-21-64252339. *E-mail: [email protected]

ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (21421004, 21327807, 21605048), the Program of Introducing Talents of Discipline to Universities (B16017), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-02-E00023), the Fundamental Research Funds for the Central Universities (222201718001, 222201717003), Chenguang Program (16CG35), and Shanghai Education Development Foundation and Shanghai Municipal Education Commission.

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Figure-1 423x245mm (72 x 72 DPI)



Figure 2 387x746mm (72 x 72 DPI)


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Figure-3 423x313mm (72 x 72 DPI)





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TOC 88x40mm (300 x 300 DPI)


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Controllable Aggregation-Induced Exocytosis Inhibition (CAIEI) of

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