Intracellular Self-Assembly Strategy for ... - ACS Publications

An in Situ Intracellular Self-Assembly Strategy for Quantitatively and Temporally Monitoring Autophagy Yao-Xin Lin,†,‡ Sheng-Lin Qiao,†,‡ Yi Wang,†,‡ Ruo-Xin Zhang,† Hong-Wei An,†,‡ Yang Ma,†,‡ R. P. Yeshan J. Rajapaksha,† Zeng-Ying Qiao,† Lei Wang,*,† and Hao Wang*,†,‡ †

CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: Autophagy plays a crucial role in the metabolic process. So far, conventional methods are incapable of rapid, precise, and real-time monitoring of autophagy in living objects. Herein, we describe an in situ intracellular selfassembly strategy for quantitative and temporal determination of autophagy in living objectives. The intelligent building blocks (DPBP) are composed by a bulky dendrimer as a carrier, a bis(pyrene) derivative (BP) as a signal molecule, and a peptide linker as a responsive unit that can be cleaved by an autophagy-specific enzyme, i.e., ATG4B. DPBP maintains the quenched fluorescence with monomeric BP. However, the responsive peptide is specifically tailored upon activation of autophagy, resulting in self-aggregation of BP residues which emit a 30-fold enhanced fluorescence. By measuring the intensity of fluorescent signal, we are able to quantitatively evaluate the autophagic level. In comparison with traditional techniques, such as TEM, Western blot, and GFP-LC3, the reliability and accuracy of this method are finally validated. We believe this in situ intracellular self-assembly strategy provides a rapid, effective, real-time, and quantitative method for monitoring autophagy in living objects, and it will be a useful tool for autophagy-related fundamental and clinical research. KEYWORDS: autophagy, dendrimer, self-assembly, ATG4B, fluorescence

A

So far, the most reliable and conventional technique is TEM, which is time consuming and cannot be applied on a living specimen for real-time monitoring of studies.17,21 LC3 protein on Western blot is a good method for semiquantitative evaluation of autophagy, but there is an issue with the limited dynamic range of LC3 blots, and it cannot be applied on living cells. GFP-LC3, a fusion of the autophagosome marker LC3 (microtubule-associated protein 1 light chain 3) and GFP (green fluorescent protein), has been widely recognized as a standard for autophagy detection.19,21 However, it is unable to monitor autophagy for a long time, as the GFP fluorescence in autolysosome is attenuated due to the degradation and quenching in the acidic environment.21 Besides, it is not distinguishable between “autophagy active” and “autophagy inactive” states.

utophagy is an essential lysosome-dependent metabolic process to clear unfold proteins and dysfunctional organelles,1 which is closely associated with various mammalian pathologies2,3 such as infections,4 neurodegenerative disorders,5 aging,6 hypoxia,7 and cancer.8−11 Accumulating evidence suggests that autophagy could be a potential therapeutic strategy for some human diseases.3,12,13 However, autophagy plays a dual role in cancer therapy.10,14 On one hand, autophagy can act as a cyto-protective mechanism that maintains cell viability and leads to tumor therapeutic resistance.8,9 On the other hand, excessive or prolonged autophagy enhances the killing effect of an anticancer drug and results in tumor cell death.15,16 Hence, it is important to obtain the data about the autophagic level and function during therapeutic process, which would offer essential evidence for correctly regulating autophagy and ultimately achieving therapeutic potential. To date, several approaches have been utilized to evaluate autophagy, e.g., transmission electron microscopy (TEM),17 Western blot, immunofluorescence of LC3,18,19 and so on.20,21 © 2017 American Chemical Society

Received: November 22, 2016 Accepted: January 23, 2017 Published: January 23, 2017 1826

DOI: 10.1021/acsnano.6b07843 ACS Nano 2017, 11, 1826−1839

Article

www.acsnano.org

Article

ACS Nano Scheme 1. Schematic Illustration of DPBP as a Bioprobe for Autophagy Detection

Scheme 2. Schematic Illustration of the Synthetic Routes of DPBPa

a

Reagents and conditions: (a) 20% piperidine in DMF; (b) Fmoc solid-phase peptide synthesis method: Fmoc protected amino acids, NMM, HBTU, DMF, 2 h; (c) BP, NMM, HBTU, DMF, 4 h; (d) 5% hydrazine in DMF; (e) PAMAM, NMM, HBTU, DMF, 12 h; (f) TFA/TIS/H2O (v/ v/v = 95:2.5:2.5), 3 h.

transfer (FRET) probe44 are reported to detect the ATG4B activity. However, the preparative reproducibility, nonstable fluorescence, and nanomaterial toxicity47,48 somehow limit their application. Recently, our group developed a biological environmentdriven self-assembly strategy for bioimaging49,50 and drug delivery.51−53 Following this strategy, we designed and prepared a series of bis(pyrene) derivatives (BP)54−58 with aggregationinduced emission (AIE) characteristics.59−61 BP exhibited excellent fluorescence properties and biological applications.56,58 Herein, we describe an in situ intracellular self-assembly strategy for quantitative and temporal detection of autophagy. The responsive (DPBP) and control (C-DPBP) building blocks

Environment-responsive bioprobes that are highly sensitive to the polarity,22 pH,23,24 temperature,25 and especially enzyme of the local environment26−30 have been widely used in biological imaging.31−33 Among them, the self-assembly of small molecules under specific physiological or pathological environments to in situ construct nanostructures with particular functions for tumor diagnostics and therapeutics are booming and grabbing a great deal of attention.34−36 ATG4B is one of the most important autophagy-related cysteine proteases37,38 and has been utilized to modulate autophagy for cancer therapy. 39−42 Increasing evidence reveals that ATG4B can act as a potential autophagic biomarker.43−46 Based on these facts, a peptide-conjugated polymeric nanoparticle43 and a fluorescent resonance energy 1827


Article

ACS Nano

Figure 1. The specificity of DPBP to ATG4B in solution. (a) Fluorescence spectra of DPBP (responsive probes) and C-DPBP (control probes) in a Tris buffer. DPBP/C-DPBP (100 μg/mL) were incubated with ATG4B (10 μg/mL) at 37 °C for 2 h. (b) Time-dependent fluorescence spectra of DPBP in the presence of ATG4B. DPBP (100 μg/mL) and ATG4B (10 μg/mL) were mixed at 37 °C. All of the fluorescence spectra were measured in the range from 360 to 650 nm; λex = 350 nm. (c) Fluorescence spectra of DPBP upon incubation with various amounts of ATG4B (0, 1, 2.5, 5, 10, and 20 μg/mL) and the linear relationship between the fluorescence intensities of DPBP and ATG4B. (d) Fluorescence of DPBP incubated with caspase-3 or ATG4B enzyme in the presence of the cysteine protease inhibitor N-ethylmaleimide (NEM, 2 mM) and PMSF (1.5 mM). (e) TEM images of the morphology of aggregates of BP residues from DPBP. DPBP (100 μg/mL) was incubated with ATG4B (10 μg/mL) at 37 °C for 2 h or 24 h. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA for indicated comparison.

RESULTS AND DISCUSSION

composed of a bulky dendrimer as a carrier (Figure S1), a bis(pyrene) derivative (Figure S2) as an AIE signal molecule, and a responsive (DPBP) or nonresponsive (C-DPBP) peptide as a linker were synthesized and characterized comprehensively (Scheme 1). The responsive peptide could be specifically cleaved by the enzyme ATG4B. DPBP maintained the quenched fluorescence due to the monomeric BP in normal cells and could be cleaved by ATG4B upon the activation of autophagy. Subsequently, the BP residue was released and self-assembled into aggregates with significantly enhanced fluorescence (Scheme 1). By measuring the intensity of fluorescent signals, we were able to make a rapid evaluation of the autophagic level in living systems.

Synthesis of DPBP and C-DPBP. DPBP and C-DPBP were prepared by the synthetic routes outlined in Scheme 2. The responsive peptide sequence (GKGSFGFTG)43,45 and the control (GKGSGFFTG) were prepared using a Fmoc protected solid-phase peptide synthesis method.62 BP and fourthgenaration poly(amidoamine) dendrimers (PAMAM) were connected to both ends of the peptide, respectively. The final products were cleaved from the resin, and the responsive probe was named as DPBP and the control as C-DPBP. The modular design of the DPBP significantly compressed the process of synthesis and reduced the purification steps. The PAMAM acted as a bulk carrier and guaranteed monomer state of BP, as it was highly hydrophilic and had spatial hindrance. To obtain the optimal hydrophobic/hydrophilic balance, forming DPBP with 1828


Article

ACS Nano

nanofibers that were about 8.3 ± 0.8 nm in diameter at 24 h (Figure 1e). Measurement of ATG4B by DPBP in Rapa-Treated Cells. The cytotoxicity of DPBP was first studied with MCF-7 cells by CCK-8 assay. MCF-7 cells were treated with DPBP/CDPBP for 24 h, respectively. As shown in Figure S11, the cell viabilities of DPBP/C-DPBP were still close to 100% at the highest dose concentration of 200 μg/mL. These results implied that our DPBP had low cytotoxicity. To obtain insight into the potential application of DPBP in living cells, we studied the cellular location and cell uptake pathway of the DPBP molecular probe by confocal laser scanning microscopy (CLSM). MCF-7 cells were incubated with Cy5-labeled DPBP (DPBP-Cy5), and then time-course intracellular amounts of the fluorescence signal were estimated qualitatively by CLSM. As shown in Figure S12, the fluorescence signals from DPBP-Cy5 inside of the cells increased with time. In order to clarify the cell uptake pathway of DPBP, we detected the fluorescence intensity of the MCF-7 cells incubated with DPBP-Cy5 at different temperatures and in the presence of various endocytosis inhibitors, i.e., amiloride (2 mM), β-CD (5 mM, inhibitors of caveolae-dependent endocytosis), and hypertonic sucrose (450 mM, clathrindependent endocytosis). As shown in Figure 2, incubation at 4

suitable weight and high stability, the molar ratios of BP to PAMAM in the reaction mixtures were adjusted to 1:1, 5:1 and 10:1. We found that 5:1 was optimal, in which the BP units were at a monomer state and could self-aggregate upon treatment with ATG4B (Figures S3−4). As a result, the ratio of BP to PAMAM in feed was optimized at 5:1. Characterization and Cytotoxicity of DPBP. The responsive peptide consisted of a ATG4B-recognition sequence “GTFG,” and hence specifically cleaved by ATG4B.43−45,63 Selective cleavage of this responsive peptide was validated using HPLC (Figure S5). The chemical structure of BP, DPBP, and CDPBP was characterized with 1H NMR (Figures S2, S6−8), respectively. 1H NMR measurement showed the actual molar ratio of BP to PAMAM was ∼2.7. Specificity of DPBP to ATG4B in Solution. Our previous studies concluded that BP was virtually nonfluorescent in good solvents but emitted intensely upon aggregation in poor solvents.54,55 As visible on the fluorescence spectrum as illustrated in Figure S9, BP showed intense fluorescence (525 nm) in DMSO/H2O mixtures. In order to explore potential applications of DPBP on autophagy detection, we measured their responses to ATG4B in solution. Initially, DPBP/C-DPBP (100 μg/mL) was mixed with ATG4B (10 μg/mL) and incubated at 37 °C for 2 h, and the fluorescence spectra were measured in the range of 375−650 nm. As illustrated in Figure 1a, both probes had a similar and very weak fluorescence in the absence of ATG4B. However, when ATG4B was added, a strong fluorescence signal (525 nm) was recorded in the DPBP group, but not in C-DPBP (Figure 1a). The kinetics of enzymecatalyzed reactions were subsequently evaluated by monitoring the changes in fluorescence of DPBP over time. The results revealed that the fluorescence increased and reached to a plateau in 140 min (Figures 1b and S10), which was a ∼30-fold higher intensity in comparison with initial intrinsic emission. Next, the effect of ATG4B concentration on the fluorescence emission of DPBP in solution was tested. DPBP was incubated in a series of ATG4B concentrations ranging from 0 to 20 μg/mL at 37 °C for 2.5 h. The results displayed that the fluorescence intensity gradually raised with the increasing concentration of ATG4B, which also indicated that the fluorescence intensity (525 nm) from DPBP was closely linked to the ATG4B activity (Figure 1c). Additionally, the fluorescence intensities of the DPBP assay increased linearly with the concentrations of ATG4B (Figure 1c). Above all, the ATG4B activity, i.e., hydrolysis kinetics of DPBP catalyzed by ATG4B, could be quantitatively studied on the basis of fluorescence intensity. Moreover, we measured the fluorescence of DPBP upon addition of ATG4B together with the inhibitors, such as NEM and PMSF. The results displayed that there was no detectable fluorescence observed (Figure 1d). In addition, we detected the fluorescence of DPBP in the presence of another cysteine protease (caspase-3). As shown in Figure 1d, in contrast to ATG4B, there was no fluorescence signal. Based on the above results, we concluded that DPBP was specifically recognized and cleaved by ATG4B, and such a cleavage could result in a fluorescence enhancement. Our previous studies also demonstrated that the BP residues in the initial stage could self-assemble into nanoparticles and finally transform into nanofibers.64 Using transmission electron microscopy (TEM), we observed the morphology of BP aggregates released as residues from DPBP. The results illustrated that the size of the formed nanoparticles at 2 h was 33.6 ± 4.6 nm, and the particles further transformed into

Figure 2. Cell uptake pathway detection. CLSM images of MCF-7 cells incubated with DPBP (100 μg/mL) at different temperatures and in the presence of various endocytosis inhibitors, such as amiloride (2 mM), β-CD (5 mM), and hypertonic sucrose (450 mM), respectively. The quantitative analysis of average cellular fluorescence intensity of MCF-7 cells in the lower panel. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA for indicated comparison. 1829


Article

ACS Nano

Figure 3. Measurements of ATG4B activity using DPBP in Rapa treated MCF-7 cells. (a) Western blot of ATG4B expression in MCF-7 cells. MCF-7 cells were incubated with Rapa (0, 0.1, 0.5, and 1.0 μM) at 37 °C for 2 h. (b) Quantitative analysis of Western blot results from (a). Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA for indicated comparison. (c) Normal and autophagic MCF-7 cells were treated with DPBP (responsive probe, 100 μg/mL) or C-DPBP (control probe, 100 μg/mL) for 1 h at 37 °C and then treated with 1.0 μM Rapa or PBS for 2 h. CLSM was used to detect the normal and autophagic MCF-7 cells. 405: λex = 405 nm, λem = 525 ± 50 nm; BF: bright field.

°C significantly reduced intracellular DPBP-Cy5. Meanwhile, treatment of the cells with β-CD and hypertonic sucrose decreased the cell uptake pathway of DPBP-Cy5, implying that DPBP might enter the cell by caveolae-dependent and clathrindependent endocytosis. Endocytosis was a complex process.65 To confirm the intracellular trafficking route of DPBP-Cy5, the MCF-7 cells were further costained with endocytotic vesicle markers (caveolin-1, clathrin) and subcellular organelle markers (early endosome, lysosome, endoplasmic reticulum (ER)). CLSM images spotted that DPBP-Cy5 (red signal) could colocalize with caveolae (anticaveolin-1, green signal) immediately at 0.5 h, but the DPBP-Cy5 signal was significantly separated from the caveolae signal and distributed the cytosol at 2 h (Figure S13). Similarly, DPBP-Cy5 (red signal) could colocalize with clathrin (green signal) immediately at 0.5 h and quickly escaped from the clathrin-coated vesicles at 2 h (Figure S14). Next, we detected the colocalization between DPBP-Cy5 and early endosomes. The CLSM images displayed that a part of the red signals (DPBP-Cy5) could costain with a green signal (early endo-

some), but a large amount of the red signals were dispersed in the cytoplasm (Figure S15). In addition, the experiments of DPBPCy5 colocalized with subcellular organelles (lysosome, ER) were carried out. The results revealed most of the red signals (DPBPCy5) could colocalize with green signals (lysosomes) (Figure S16), and the overlap coefficient of DPBP with lysosomes was 0.62 (0: no colocalization, 1: all pixels colocalized). Meanwhile, we found that most of the red signals (DPBP-Cy5) could colocalize with ER (Figure S17), and the overlap coefficient of DPBP with ER was 0.73. The colocalization experimental results further suggested the caveolae-dependent and clathrin-dependent endocytosis mechanism of cellular uptake of DPBP. Previous studies reported that rapamycin (Rapa) could markedly increase the ATG4B expression, which paralleled the accumulation of LC3 II.45 To confirm the expression of ATG4B was proportional to the increased concentration of Rapa, MCF-7 cells were first treated with different concentrations of Rapa (0, 0.1, 0.5, and 1.0 μM) for 2 h, and then the ATG4B on Western blot experiment was carried out. As shown in Figure 3a,b, the ATG4B expression was observed in the presence of Rapa. 1830


Article

ACS Nano

Figure 4. Analyzing the specificity of DPBP to ATG4B in living cells. (a) Western blot of ATG4B expression in MCF-7 cells. (b) Quantitative analysis of band intensity of ATG4B from (a). (c) ATG4B activity was detected by DPBP in MCF-7 cells. MCF-7 cells were treated with 10 μL of PBS in a regular culture medium (lane 1); 10 μL Rapa (1.0 μM) (lane 2); transfection of a control siRNA (lane 3); transfection of a siRNA against ATG4B (lane 4), followed by DPBP (100 μg/mL) for 2 h. (d) Correlation plot of DPBP and LC3. An overlap coefficient of 0.46 (0: no colocalization, 1: all pixels colocalized) confirmed that the BP spots from DPBP did not colocalize with LC3. Statistical significance: *p < 0.05 and #p < 0.05, one-way ANOVA for indicated comparison. 405: λex = 405 nm, λem = 525 ± 50 nm; BF: bright field.

(autophagy initial marker), LC3 II, and P62 (autophagy substrate marker) expressions did not increase in 100 μg/mL DPBP treated cells (Figure S19). Furthermore, the LC3 II expression of cells pretreated with Rapa (1.0 μM) did not enhance in the presence of DPBP (100 μg/mL), confirming that 100 μg/mL DPBP had no impact on the normal autophagic response. In addition, CLSM was employed to observe the variation of fluorescence signals of the living cells. MCF-7 cells were treated with different concentrations of DPBP for 1 h and then treated with Rapa (1.0 μM) for 2 h. As shown in Figure S21, fluorescence signals were observed in the Rapa treated cells, and the

Quantitative analysis showed that the ATG4B/β-actin ratio increased from 1.38 to 2.71 as the concentration increased from 0.1 μM to 1.0 μM (Figure 3b), confirming the ATG4B could be used as a biomarker for evaluating autophagy in Rapa treated cells. Therefore, the potential of the autophagy cell model with high intracellular ATG4B activity was developed by using MCF-7 cells treated with Rapa. Next, the autophagic effects of DPBP themselves were evaluated by the Western blot method. As shown in Figure S18, there was a minute accumulation of LC3 II (autophagy marker) in 200 μg/mL DPBP treated cells. Meanwhile, BECN1 1831


Article

ACS Nano

Figure 5. Cellular real-time monitoring and quantitatively analyzing autophagy by DPBP. (a) CLSM images of autophagic MCF-7 cells induced by 1.0 μM of Rapa at different time points. MCF-7 cells were first incubated with DPBP (100 μg/mL) for 1 h at 37 °C, then washed with PBS 3 times, and finally treated with 1.0 μM of Rapa. (b) CLSM images of Rapa treated MCF-7 cells and (c) the corresponding quantitative analysis of fluorescence intensity. MCF-7 cells were treated with DPBP (100 μg/mL) for 1 h, followed by Rapa (0, 0.1, 0.5, and 1.0 μM) for 2 h, respectively. The 0 μM Rapa treated group was regarded as 100 (Acontrol), and the fluorescence intensity of sample (Asample) and control (Acontrol) were measured, respectively. Fluorescence intensity (%) was equal to Asample /Acontrol × 100. (d) Western blot of LC3 and corresponding quantitative analysis of band intensity. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA for indicated comparison.

fluorescence was gradually enhanced with the increasing concentration of DPBP. Hence, 100 μg/mL of DPBP was nominated as the optimal concentration for the following cell imaging experiments. With the optimal concentration, we utilized DPBP as specific probes to measure ATG4B activity in living cells. MCF-7 cells were first incubated with DPBP (100 μg/mL) for 1 h at 37 °C and then treated with Rapa (1.0 μM) or the same with PBS (control) for 2 h. The CLSM was used to observe the cells. As shown in Figure 3c, an extremely strong green fluorescence was observed for the DPBP probe in Rapa treated cells, but no fluorescence signals were observed in normal cells (PBS). Meanwhile, in the C-DPBP (control probe) groups, green fluorescence signals could not be observed in the autophagy active or inactive cells. Furthermore, we used Cy5-labeled DPBP and measured the DPBP performance in the absence of Rapa. As shown in Figure S22, DPBP exhibited a quenched fluorescence signal in PBS treated cells, but red signals were highly abundant in DPBP-Cy5 treated cells using the same conditions, indicating

DPBP entered the cells. Once Rapa was added, strong green fluorescence signals were detected, which indicated that the DPBP inside the cells was cleaved and led to BP aggregation. Therefore, we concluded that DPBP was recognized and cleaved by intracellular ATG4B. In order to further determine if the intracellular fluorescence of DPBP was solely due to the increase of ATG4B activity, we carried out the control experiment with Rapa treated cells upon knockdown of ATG4B gene. The Western blot method was utilized to validate the expression of ATG4B. As shown in Figure 4a,b, the ATG4B expression was dramatically decreased in the ATG4B siRNA knockdown cells. Simultaneously, our DPBP assay showed negligible fluorescence in ATG4B siRNA knockdown cells (Figure 4c). These results revealed that intracellular fluorescence signals from DPBP were indeed ascribed to the ATG4B. To justify that our DPBP assay merely reflects the autophagic level, we used the typical immunofluorescence method to detect LC3. As shown in Figure S23, the red immunofluorescence 1832


Article

ACS Nano

Figure 6. The detection of ATG4B activity in other autophagy inducer/inhibitor treated cells. (a) CLSM images of autophagy inducer treated MCF-7 cells. MCF-7 cells were first incubated with DPBP for 1 h and then incubated with PBS, Rapa (1.0 μM), C2-ceramideor (20 μM), and EBSS for 2 h, respectively. (b) CLSM images of autophagy inhibitor treated MCF-7 cells. MCF-7 cells were first incubated with DPBP for 1 h and then treated with 3-MA (5 mM) and Baf (100 nM) for 1 h, respectively. Finally, MCF-7 cells were treated with Rapa (1.0 μM) for 2 h. (c, d) Quantitative analysis of fluorescence intensity for (a) and (b), respectively: PBS treated group was regarded as 100 (Acontrol). The mean fluorescence intensity of sample (Asample) and control (Acontrol) were measured, respectively. Fluorescence intensity (%) was equal to Asample /Acontrol × 100. Statistical significance: n.s.: no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ##p < 0.01, and ###p < 0.01, one-way ANOVA for indicated comparison. 405: λex = 405 nm, λem = 525 ± 50 nm; BF: bright field.

signals from LC3 and green fluorescence from BP were observed in Rapa and DPBP treated cells. However, in the Rapa and CDPBP treated cells, we only observed red immunofluorescence signals. In normal cells, neither red nor green fluorescence signals were observed independent if they were precultured with DPBP or C-DPBP. These findings indicated that DPBP specifically

responded to ATG4B in autophagy-induced cells. We also carried out the colocalization of DPBP with ATG4B by using DPBP-Cy5. The experimental results showed that the overlap coefficient of DPBP-Cy5 with ATG4B is 0.58 (Figure S24), confirming that DPBP-Cy5 was recognized by ATG4B. Furthermore, we investigated the colocalization of BP aggregates 1833


Article

ACS Nano

EBSS for 2 h, respectively. As shown in Figure 6a,c, we observed a strong fluorescence in C2-ceramideor and EBSS treated cells. The quantitative analysis of fluorescence signals displayed that the intensity of fluorescence increased significantly for C2ceramideor (232%) and EBSS (189%), respectively. Based on these results, we concluded that the DPBP array could be used to quantitatively measure the activity of ATG4B in autophagyinduced cells, which was a positive correlation with the autophagic level.45 However, several studies indicated that the ATG4B activity was not parallel to the accumulation of LC3 II in long-time (>3 h) starved cells,45,67 as reactive oxygen species (ROS) from the stimulation could suppress ATG4B activity.67,68 As shown in Figure S28, the fluorescence intensity was decreased from (206%) at 2 h to (108%) in 24 h. To eliminate the sideeffect of ROS, we added NAC (N-acetyl cysteine), which was an antioxidant69 and conditionally rescued the activity of ATG4B.45 As illustrated in Figure S27, there was no fluorescence in cells treated with NAC alone, suggesting that NAC had a negligible effect on the ATG4 activity. Moreover, NAC supplementation did not change the activity of ATG4B on exposure for a short period of time (2 h) in starved cells. However, prolonged starvation, ATG4B activity of cells was significantly enhanced at the 4, 6, 12, and 24 h, respectively (Figure S28), followed by the addition of NAC. These results demonstrated that NAC was capable of conditionally shielding the ATG4B activity in starved cells over a long exposure. Based on above experimental results, we further clarified that our DPBP array could be directly applied to measure the activity of ATG4B in starved cells for a short period of time. Starved cells for a prolonged period of time could also be subjected to our DPBP array along with antioxidant supplementation (e.g., NAC). Subsequently, using DPBP, we measured the activity of ATG4B in MCF-7 cells after incubating with 3-methyladenine70 (3-MA, an inhibitor and suppresses autophagy by inhibiting the PtdIns3K) and Bafilomycin A71,72 (Baf, an inhibitor of the vacuolar-type proton ATPase and blocks autophagic flux by preventing the fusion of autophagosomes and lysosomes). MCF7 cells were first incubated with DPBP for 1 h and treated with 3MA (5 mM) and Baf (100 nM) for 1 h, respectively. As shown in Figure 6, there was no fluorescence signal in the presence of 3MA, and still no fluorescence was observed in 3-MA and Rapa treated cells. Previous studies indicate that 3-MA was an inhibitor of PtdIns3K, which played a crucial role in the early stages of autophagosome formation through an essential complex with BECN1, and suppression of its activity might result in inhibition of ATG4B activity.73 Therefore, we analyzed the ATG4 activity using DPBP in BECN1 knockdown cells. As shown in Figure S29, there was no fluorescence in BECN1 knockdown cells, indicating that BECN1 had a significant effect on ATG4B activity. Interestingly, the Baf treated cells showed a different response in the presence and absence of Rapa. No fluorescence was observed in Baf treated cells. In contrast, the fluorescence remarkably increased in the Baf and Rapa treated cells (Figure 6b). The quantitative analysis displayed that the fluorescence intensity of the Baf and Rapa cotreated group increased to 250% (Figure 6d), but it was lower than that of the Rapa alone group. For a better understanding of the absence of suppressive effect of Baf on Rapa-induced ATG4B activity, we evaluated their autophagy-induced effects through measuring the numbers of autophagosomes and LC3/SQSTM1 (P62) expressions. First, we determined numbers of autophagosomes using LC3 immunofluorescence. As shown in Figure S30a, autophagosome

(DPBP release) with ATG4B. The overlap coefficient of BP aggregates with ATG4B was 0.41, indicating the majority of the BP aggregates did not colocalize with ATG4B (Figure S25). The phenomena could be ascribed to the turn-on mechanism of the DPBP probe. DPBP maintains a suppressing fluorescence in normal cells, but would release BP upon activation of autophagy. Subsequently, self-aggregation of BP residues would emit enhanced fluorescence. Therefore, BP aggregates did not colocalize with ATG4B. Moreover, we investigated the colocalization of BP aggregates (DPBP release) with LC3. The results showed that the overlap coefficient of BP aggregate with LC3 was 0.46, confirming that the greater part of BP aggregates did not colocalize with LC3 (Figure 4d). Cellular Autophagy Real-Time Imaging by DPBP in Rapa Treated Cells. We further investigated whether DPBP could be used as a non-invasive bioprobe for real-time monitoring of autophagy in living cells. First, MCF-7 cells were incubated with DPBP (100 μg/mL) for 1 h at 37 °C. CLSM was used to obtain real-time fluorescence images followed by adding 1.0 μM of Rapa with DMEM. As shown in Figure 5a, fluorescence intensities of our DPBP assay continuously increased with the length of incubation. Moreover, the fluorescence intensity at 20 min was strong enough to evaluate the autophagic level. Finally, the fluorescence intensity reached a maximum at ∼150 min (Figures 5a and S26), which implied that the intracellular DPBP was totally cleaved. Meanwhile, there was no fluorescence in the absence of Rapa (Figure S27). These results demonstrated that DPBP could serve as an effective responsive probe for the real-time monitoring autophagy in living cells. Quantitatively Analyzing the Autophagic Level Using DPBP in Rapa Treated Cells. In order to prove that DPBP could quantitatively evaluate autophagy, we carefully studied the fluorescence intensity of DPBP in the cells treated with different concentrations of Rapa. MCF-7 cells were cocultured with DPBP (100 μg/mL) at 37 °C for 1 h and by a series of Rapa concentrations (0.1, 0.5, 1.0 μM) for 2 h, and CLSM was used to measure the fluorescence signal. As shown in Figure 5b, using DPBP, we observed the variation of fluorescence intensities (indicating variation of ATG4B activity) in the cells treated with different concentrations of Rapa. Quantitative analysis of fluorescence signal displayed that the intensities were observed as 154% at 0.1 μM and 350% at 1.0 μM (Figure 5c), indicating the increase in Rapa concentration favors stronger fluorescence. Hence a stronger fluorescence intensity reflected a higher autophagic level. Meanwhile, Western blot, a semiquantitative method, illustrated that all of the Rapa treatments could induce LC3 II accumulation. Semiquantitative analysis results revealed that the LC3 II/β-actin ratio increased from 2.6 at 0.1 μM to 3.8 at 1.0 μM (Figure 5d). Our previous experiments and other studies45,63 indicated that the ATG4B activity was parallel with the accumulation of LC3 II in Rapa treated cells. Using two different methods, we obtained remarkably similar conclusions, suggesting that DPBP was an effective probe for quantitatively evaluating autophagy in living cells. Assessment of the Autophagic Level in Other Autophagy Inducer/Inhibitor Treated Cells. Taking into account the generality of DPBP as an autophagy detecting probe, other stimuli-induced autophagy models should be considered. Therefore, we detected the ATG4B activity using DPBP under conditions of N-acetyl-D-sphingosine (C2-ceramideor)66 or EBSS (starvation). MCF-7 cells were first treated with DPBP for 1 h and then incubated with C2-ceramideor (20 μM) and 1834


Article

ACS Nano

Figure 7. The detection of ATG4B activity in Zebrafish and other autophagic cells. (a) CLSM images of autophagic cells. (b) Corresponding quantitative analysis of fluorescence intensity from (a). A549, Hela, and U87 cells were cocultured with DPBP (100 μg/mL) at 37 °C for 1 h and then treated with Rapa (1.0 μM) for 2 h. 405: λex = 405 nm, λem = 525 ± 50 nm; BF: bright field. (c) Zebrafish embryos at 2 dpf were treated with PBS or Rapa for 24 h and then stained with DPBP for 4 h before imaging by CLSM. (d) Western blot of ATG4B and LC3 expressions of Zebrafish embryos at 2 dpf treated with PBS or Rapa for 24 h. (e) Quantitative analysis of Western blot results from (d). Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA for indicated comparison.

Rapa (1.0 μM) for 2 h. The intracellular fluorescence signal was measured by CLSM. Strong green fluorescence from DPBP molecular probe was observed in all of the autophagy-activated cells (Figure 7a). The quantitative analysis illustrated that the fluorescence intensity increased to 333% (A549), 282% (Hela), and 347% (U87), respectively (Figure 7b). These experimental results concluded that DPBP was a suitable non-invasive bioprobe for all types of living cells. To further study whether our DPBP assay can be applied in vivo, an autophagic zebrafish model was developed according to a previous report.74 As expected, the Western blot results illustrated that ATG4B and LC3 II expressions increased in Rapa incubated fish (Figure 7d). Semiquantitative analysis results revealed that the ATG4B/β-actin and LC3 II/β-actin ratios increased to 1.9 and 2.2, respectively (Figure 7e). Meanwhile, our DPBP array demonstrated that the fluorescence of DPBP was significantly increased in Rapa incubated fish compared with the control (Figure 7c). The quantitative analysis illustrated that the fluorescence intensity increased to 350%. Based on these results,

accumulation was observed in Baf alone treated cells. Similarly, there was a large number of autophagosomes in Baf and Rapa cotreated cells, and it is higher than that of Baf alone group. Next, we detected degradation of P62 (a substrate is preferentially degraded by autophagy). As shown in Figure S30b, Baf caused P62 and LC3 II accumulation as expected, and adding Rapa increased accumulation of P62 and LC3 II. These results indicated that Baf blocked the autophagic flux, but did not inhibit the initial autophagy activation by Rapa. Previous studies reported that Baf was used to inhibit the fusion between autophagosomes and lysosomes in late autophagy and did not directly suppress the ATG4B activity.71,72 Therefore, there was no suppressive effect of Baf on Rapa-induced ATG4B activity. Cellular Autophagy Monitoring by DPBP in Other Cells and Zebrafish. To validate that the ability of DPBP as a general method to evaluate autophagy, we carefully evaluated the autophagic level by using a DPBP molecular probe in three other types of cells. A549, Hela, and U87 cells were cocultured with DPBP (100 μg/mL) at 37 °C for 1 h and incubated with 1835


Article

ACS Nano we concluded that the DPBP array could be used to quantitatively measure autophagy in living model animals as well.

CONCLUSIONS ATG4B is one of the most important cysteine proteases and is involved in the processing and delipidation of the LC3 (orthologous of yeast ATG8).37,38,63,75,76 In the early stages of autophagosome formation, the LC3-PE, anchored on the autophagosome, is cleaved by ATG4B specifically, and LC3 is released.37 Previous studies demonstrated that ATG4B could be used as a potential biomarker to evaluate autophagy in cancer therapy.39−42 To date, TEM, GFP-LC3, and Western blot on LC3 are widely recognized as standard methods for the detection of autophagy. However, these typical methods have a few disadvantages and cautionary notes with application (Table 1). Table 1. Side-by-Side Comparison of Our Newly Developed Method with the Existing TEM, Western Blot, and GFP-LC3 Methods method TEM GFP-LC3 Western Blot DPBP

timeconsuming

real-time monitoring

in vivo detection

technical requirement

several days ≥24 h

no

no

high

yes

medium

≥12 h

no

yes; zebrafish, worm, mice no

≤3 h

yes

yes/zebrafish

low

medium

As shown in Figure 8 and Table 1, TEM measurement can be used for detection of autophagy by morphological analysis of autophagic structures, but it needs several days for sampling artifacts, and it cannot be applied to real-time monitoring in living specimen studies. GFP-LC3 is widely used as a reliable probe for autophagy detecting, but with limitations such as probe construction is time consuming and large transfected protein molecules may have negative influence on the physiological status of the cells. Moreover, the GFP fluorescence in an acid environment is attenuated. It also does not distinguish between “autophagy active” and “autophagy inactive” states. LC3 protein on Western blot is a good method for semiquantitative evaluation of autophagy, but there is still an issue with the limited dynamic range of the LC3 blots, and it cannot be applied to living cells. Certain assay methods, e.g, a fluorescent resonance energy transfer (FRET) nanoprobe44 and peptide-conjugated polymeric nanoparticles,43 have been developed, but the preparative reproducibility, unstable fluorescence, and nanomaterial toxicity47,48 somehow limit their application. In this study, we successfully developed an intracellular selfassembly approach for autophagy detection by measuring the activity of ATG4B in living objects. In comparison with the traditional methods, i.e., TEM, Western Blot, and GFP-LC3, our DPBP array exhibits a few advantages as follows. First, DPBP is a fluorescence probe which can real-time and in situ monitor the autophagy without tedious sample preparation and expensive reagent consumption. Second, DPBP is a safe and specific responsive molecular probe. On one hand, DPBP shows good biocompatibility and vitally does not induce either autophagy or cell death. On the other hand, DPBP shows satisfactory specificity for ATG4B and can clearly distinguish between “autophagy active” and “autophagy inactive” states in living cells. Finally, the DPBP assay provides stable and reliable turn-on fluorescence due to its AIE characteristics, resulting in the

Figure 8. Autophagy was evaluated by (a) TEM, (b) GFP-LC3, (c) DPBP, and (d) Western blot. For TEM, MCF-7 cells were incubated with PBS or Rapa (1 μM) at 37 °C for 2 h. For GFP-LC3, MCF-7 cells that transfected with GFP-LC3 plasmids were first developed and then treated with PBS, Rapa (1 μM), or 3-MA (5 mM) at 37 °C for 2 h. For DPBP, MCF-7 cells were cocultured with DPBP (100 μg/mL) at 37 °C for 1 h and then treated with PBS, Rapa (1 μM), or 3-MA (5 mM) at 37 °C for 2 h. For Western blot, MCF-7 cells were incubated with PBS or Rapa (1 μM) at 37 °C for 2 h. (e) Quantitative analysis of Western blot results from (d). Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA for indicated comparison.

quantitative autophagic measurement. BP is an AIE fluorescence molecule, and its aggregates exhibit stable and excellent fluorescence properties crucial for prolonged imaging with negligible biological background interference. DPBP maintains quenched fluorescence in normal cells, and upon autophagy activation, the signal molecule BP is released and self-assembled into aggregates which emit dramatically enhanced fluorescence. Overall, our in situ self-assembly strategy offers a highly convenient, non-invasive, real-time, and quantitative detection method for autophagy in living objects. It offers an optional approach for autophagy-related fundamental investigations and drug development.

MATERIALS AND METHODS Materials. A fourth-generation dendrimer was purchased from Sigma-Aldrich (USA), and the ATG4B enzyme was obtained from Abnova (Taiwan). ATG4B siRNA and BECN1 siRNA were purchased from Santa Cruz Biotechnology, Inc. 9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and 2-(1H-benzotriazole-1-yl)-1,1,3,3tetramethy-luronium hexafluorophosphate (HBTU) were purchased from GL Biochem (China). LC3 II, P62, β-tubulin, and β-actin antibody 1836


Article

ACS Nano

analysis of fluorescence intensities: the fluorescence intensity of control group was regarded as 100 (Acontrol), and the fluorescence intensity of sample treated group was regarded as Asample. Fluorescence intensity (%) was equal to Asample /Acontrol × 100. Biotransmission Electron Microscopy. MCF-7 cells were first incubated with PBS or Rapa (1 μM) for 2 h, washed with PBS three times, and isolated by centrifugation. Subsequently, the cells were fixed overnight at 4 °C in 2.5% glutaraldehyde PBS buffer. After the fixed cells were harvested and washed with PBS buffer (0.1 M) for 10 min/time three times, the cells were further fixed with 1% osmium-containing PBS buffer for 2 h at room temperature. Subsequently, the cells were washed with PBS buffer three times and dehydrated with a graded series of acetone (50, 70, 80, 90, 95, 100%) for 15 min for each step. After the cells were infiltrated with a graded of series of mixtures (acetone/EPON 812 resin: 2/1, 1/1, 1/2) at room temperature for 1 h for each step, pure resin was added at 4 °C and incubated overnight. Finally, the cells were placed into gelatin capsules and filled with pure EPON 812 resin at 37, 45, and 60 °C for 24 h, respectively. Ultrathin sections were cut with a diamond knife and picked up with Formvar-coated copper grids (300 mesh). Counter-staining of the sections was performed with osmic acid (1%) for 1 h and uranyl acetate (4%) for 20 min, respectively. Finally, JEOL JEM-1400 electron microscope (JEOL, Tokyo, Japan) was used to observe cells. Detection of ATG4B Activity in Zebrafish. Zebrafish embryos at 2 dpf were treated with PBS or Rapa (1 μM) for 24 h and then stained with DPBP (100 μg/mL) for 4 h before imaging by CLSM. Quantitative Analysis of Fluorescence Intensity. The PBS treated group was regarded as 100% (Acontrol). The mean fluorescence intensity of sample (Asample) and control (Acontrol) were measured, respectively. Fluorescence intensity (%) was equal to Asample /Acontrol × 100. Statistical Analysis. All data were expressed as mean ± SD (standard deviation) from at least three independent experiments. Statistically significant, oneway ANOVA with Bonferroni’s post-test analysis was used; p < 0.05 (*), p < 0.01 (**), or p < 0.001(***) are marked in the figures.

were purchased from Cell Signal Technology. ATG4B, BECN1, caveolin-1, and clathrin antibody were purchased from abcam. The cell counting kit-8 assay (CCK-8) was purchased from Beyotime Institute of Biothechnology, China. MCF-7, Hela, A549, and U87 cell lines were purchased from cell culture center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Zebrafish was purchased from China Zebrafish Resource Center. Other solvents and reagents were used as received. Synthesis of DPBP and C-DPBP. The responsive peptide (GKGSFGFTG) and the control peptide (GKGSGFFTG) were prepared using Fmoc solid-phase peptide synthesis method. In brief, the resin (200 mg, loading ∼0.287 mmol/g) that was used as the solidphase support was first swelled in HPLC-grade DMF for 2 h at room temperature. After washing three times with HPLC-grade DMF and DCM, piperidine/DMF (v/v = 1/4) was used to deprotect the Fmoc group. When a ninhydrin test (ninhydrin, phenol, VC 1:1:1 v/v) confirmed that the Fmoc group had been removed, amino acids (0.57 mM), HBTU (0.35 M), and NMM (0.4 M) were added, and then the mixture was shaken at room temperature for 2 h. The above cycle was repeated until the last amino acid was coupled. Next, BP was regarded as an amino acid and added to the reaction solution with the same synthesis method. Finally, hydrazine/DMF (v/v = 1/19) was used as a deprotection agent to remove the Dde group of K, and then dendrimer (0.011 mM) was added into the reaction solution. After the whole coupling process, the products were cleaved in a mixture of 95% TFA, 2.5% TIS, and 2.5% H2O for 3 h at room temperature. Residual TFA in the final products was removed by vacuum rotary evaporator. Characterizations of DPBP and C-DPBP. The chemical structures of DPBP/C-DPBP were proved by NMR measurements. 1H NMR spectra (400 MHz) of the DPBP/C-DPBP (10 mg/mL) in d6-DMSO were recorded on a Bruker ARX 400 MHz spectrometer. The morphology of aggregates of released BP residues from DPBP was examined by TEM (Tecnai G2 20 S-TWIN). The TEM samples were prepared by contacting the aggregates of BP residues droplets with copper grids for 30 s, removing the excess droplets, and staining with uranyl acetate for 10 s before the TEM studies. Enzymatic Assay in Solution. DPBP/C-DPBP was dissolved in Tris buffer (20 mM Tris·HCl, 50 mM NaCl, 0.5 mM DTT, pH 8.0), and ATG4B enzyme was added to mixed solution. The reaction mixture was incubated at 37 °C and then diluted to a total of 300 μL with deionized water for fluorescence measurement. The solution was excited at 350 nm, and the emission was collected from 375 to 650 nm. Cytotoxicity Assay for MCF-7 Cells. CCK-8 assay was utilized to evaluate the cytotoxicity of DPBP/C-DPBP. First, a density of 6 × 103 cells per well was seeded in the 96-well plates. After 15 h, the medium was replaced by 10 μL of the sample solutions with different concentrations, and the solutions were incubated for an additional 24 h. Finally, 10% CCK-8 solutions was added to each well for another 2 h. The UV−vis absorptions of sample wells (Asample), Ablack, and control wells (Acontrol) were measured using a Microplate reader at a test wavelength of 450 nm and a reference wavelength of 690 nm, respectively. Cell viability (%) was equal to (Asample − Ablank)/(Acontrol − Ablank) × 100. All the experiments were performed in triplicate. Western Blot. The MCF-7 cells were collected and resuspended with 100 μL lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Triton-X 100, and protease inhibitor]. After the samples were estimated by BCA kit (Applygen), the samples (∼60 μg) were subjected to SDS-PAGE and then transferred to a nitrocellulose membrane blots. Next, nitrocellulose membrane blots were blocked with a blocking buffer [5% (wt/v) nonfat milk, 0.1% (v/v) Tween 20 in 0.01 M TBS], then incubated with primary antibodies overnight at 4 °C, and incubated with an secondary antibody for 2 h at room temperature. Finally, Typhoon Trio Variable Mode Imager was used to obtain the images. Band density was calculated by NIH ImageJ software. CLSM Observation. MCF-7 cells were first seeded in confocal plates (Costar, United States) at an intensity of 1 × 105 cells/mL. After 15 h, the medium was replaced by DMEM medium that contained a drug, e.g., Rapa, and then the cells were incubated with 100 μg/mL of DPBP for 2 h. Finally, the cells could be detected by Zeiss LSM710 CLSM with a 63× objective lens. The corresponding quantitative

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b07843. 1 H NMR spectra of BP, DPBP, and C-DPBP; FL spectra of BP and DPBP; HPLC characterization of a responsive peptide; cell viabilities; CLSM images of DPBP signal colocalized with lysosome and ER; cell uptake pathway; Western blot data (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Hao Wang: 0000-0002-1961-0787 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, 2013CB932701), National Natural Science Foundation of China (21374026, 51573031, and 51573032), Science Fund for Creative Research Groups of the National Natural Science Foundation of China (11621505),and Key Project of Chinese Academy of Sciences in Cooperation with Foreign Enterprises (GJHZ1541). 1837


Article

ACS Nano

and Biocompatible Polymer Host for Use in Fluorescent Chemical Sensing and Imaging of Oxygen, pH Values and Temperature. Sens. Actuators, B 2015, 221, 37−44. (26) Yuan, Y.; Sun, H. B.; Ge, S. C.; Wang, M. J.; Zhao, H. X.; Wang, L.; An, L. N.; Zhang, J.; Zhang, H. F.; Hu, B.; Wang, J. F.; Liang, G. L. Controlled Intracellular Self-Assembly and Disassembly of F-19 Nanoparticles for MR Imaging of Caspase 3/7 in Zebrafish. ACS Nano 2015, 9, 761−768. (27) Hou, Y.; Zhou, J.; Gao, Z. Y.; Sun, X. Y.; Liu, C. Y.; Shangguan, D. H.; Yang, W. S.; Gao, M. Y. Protease-Activated Ratio Metric Fluorescent Probe for pH Mapping of Malignant Tumors. ACS Nano 2015, 9, 3199− 3205. (28) Rivera-Gil, P.; De Koker, S.; De Geest, B. G.; Parak, W. J. Intracellular Processing of Proteins Mediated by Biodegradable Polyelectrolyte Capsules. Nano Lett. 2009, 9, 4398−4402. (29) Qiu, L. P.; Zhang, T.; Jiang, J. H.; Wu, C. C.; Zhu, G. Z.; You, M. X.; Chen, X. G.; Zhang, L. Q.; Cui, C.; Yu, R. Q.; Tan, W. H. Cell Membrane-Anchored Biosensors for Real-Time Monitoring of the Cellular Microenvironment. J. Am. Chem. Soc. 2014, 136, 13090−13093. (30) Li, J.; Zhao, Y. Nanotechnology in the Programmed Cell Therapy: Nowhere to Escape of Cancer. Sci. Bull. 2016, 61, 45−47. (31) Krueger, A. T.; Kroll, C.; Sanchez, E.; Griffith, L. G.; Imperiali, B. Tailoring Chimeric Ligands for Studying and Biasing ErBB Receptor Family Interactions. Angew. Chem., Int. Ed. 2014, 53, 2662−2666. (32) Yang, Z.; Cao, J.; He, Y.; Yang, J. H.; Kim, T.; Peng, X.; Kim, J. S. Macro-/Micro-Environment-Sensitive Chemosensing and Biological Imaging. Chem. Soc. Rev. 2014, 43, 4563−4601. (33) Yin, W.; Zhao, L.; Zhou, L.; Gu, Z.; Liu, X.; Tian, G.; Jin, S.; Yan, L.; Ren, W.; Xing, G.; Zhao, Y. Enhanced Red Emission from GdF3:Yb3+,Er3+ Upconversion Nanocrystals by Li+ Doping and Their Application for Bioimaging. Chem. - Eur. J. 2012, 18, 9239−9245. (34) Ye, D. J.; Shuhendler, A. J.; Cui, L. N.; Tong, L.; Tee, S. S.; Tikhomirov, G.; Felsher, D. W.; Rao, J. H. Bioorthogonal CyclizationMediated In Situ Self-Assembly of Small-Molecule Probes for Imaging Caspase Activity in vivo. Nat. Chem. 2014, 6, 519−526. (35) Song, Z. G.; Mao, D.; Sung, S. H. P.; Kwok, R. T. K.; Lam, J. W. Y.; Kong, D. L.; Ding, D.; Tang, B. Z. Activatable Fluorescent Nanoprobe With Aggregation-Induced Emission Characteristics for Selective in vivo Imaging of Elevated Peroxynitrite Generation. Adv. Mater. 2016, 28, 7249−7355. (36) Pires, R. A.; Abul-Haija, Y. M.; Costa, D. S.; Novoa-Carballal, R.; Reis, R. L.; Ulijn, R. V.; Pashkuleva, I. Controlling Cancer Cell Fate Using Localized Biocatalytic Self-Assembly of an Aromatic Carbohydrate Amphiphile. J. Am. Chem. Soc. 2015, 137, 576−579. (37) Yu, Z.-Q.; Ni, T.; Hong, B.; Wang, H.-Y.; Jiang, F.-J.; Zou, S.; Chen, Y.; Zheng, X.-L.; Klionsky, D. J.; Liang, Y.; Xie, Z. Dual Roles of Atg8-PE Deconjugation by Atg4 in Autophagy. Autophagy 2012, 8, 883−892. (38) Satoo, K.; Noda, N. N.; Kumeta, H.; Fujioka, Y.; Mizushima, N.; Ohsumi, Y.; Inagaki, F. The Structure of Atg4B-LC3 Complex Reveals the Mechanism of LC3 Processing and Delipidation During Autophagy. EMBO J. 2009, 28, 1341−1350. (39) Tran, E.; Chow, A.; Goda, T.; Wong, A.; Blakely, K.; Rocha, M.; Taeb, S.; Hoang, V. C.; Liu, S. K.; Emmenegger, U. Context-Dependent Role of ATG4B as Target for Autophagy Inhibition in Prostate Cancer Therapy. Biochem. Biophys. Res. Commun. 2013, 441, 726−731. (40) Akin, D.; Wang, S. K.; Habibzadegah-Tari, P.; Law, B.; Ostrov, D.; Li, M.; Yin, X.-M.; Kim, J.-S.; Horenstein, N.; Dunn, W. A., Jr. A Novel ATG4B Antagonist Inhibits Autophagy and Has a Negative Impact on Osteosarcoma Tumors. Autophagy 2014, 10, 2021−2035. (41) Rothe, K.; Lin, H.; Lin, K. B. L.; Leung, A.; Wang, H. M.; Malekesmaeili, M.; Brinkman, R. R.; Forrest, D. L.; Gorski, S. M.; Jiang, X. The Core Autophagy Protein ATG4B Is a Potential Biomarker and Therapeutic Target in CML Stem/Progenitor Cells. Blood 2014, 123, 3622−3634. (42) Cabrera, S.; Maciel, M.; Herrera, I.; Nava, T.; Vergara, F.; Gaxiola, M.; Lopez-Otin, C.; Selman, M.; Pardo, A. Essential Role for the ATG4B Protease and Autophagy in Bleomycin-Induced Pulmonary Fibrosis. Autophagy 2015, 11, 670−684.

REFERENCES (1) Mizushima, N. Autophagy: Process and Function. Genes Dev. 2007, 21, 2861−2873. (2) Levine, B.; Kroemer, G. Autophagy in the Pathogenesis of Disease. Cell 2008, 132, 27−42. (3) Mizushima, N.; Levine, B.; Cuervo, A. M.; Klionsky, D. J. Autophagy Fights Disease Through Cellular Self-Digestion. Nature 2008, 451, 1069−1075. (4) Deretic, V.; Saitoh, T.; Akira, S. Autophagy in Infection, Inflammation and Immunity. Nat. Rev. Immunol. 2013, 13, 722−737. (5) Nixon, R. A. The Role of Autophagy in Neurodegenerative Disease. Nat. Med. 2013, 19, 983−997. (6) Quarles, E. K.; Dai, D.-F.; Tocchi, A.; Basisty, N.; Gitari, L.; Rabinovitch, P. S. Quality Control Systems in Cardiac Aging. Ageing Res. Rev. 2015, 23, 101−115. (7) Noman, M. Z.; Hasmim, M.; Messai, Y.; Terry, S.; Kieda, C.; Janji, B.; Chouaib, S. Hypoxia: a Key Player in Antitumor Immune Response. a Review in the Theme: Cellular Responses to Hypoxia. Am. J. Physiol. Cell Physiol. 2015, 309, C569−579. (8) Alderton, G. K. Autophagy Surviving Stress in Pancreatic Cancer. Nat. Rev. Cancer 2015, 15, 513−513. (9) Guo, J. Y.; Xia, B.; White, E. Autophagy-Mediated Tumor Promotion. Cell 2013, 155, 1216−1219. (10) Mathew, R.; Karantza-Wadsworth, V.; White, E. Role of Autophagy in Cancer. Nat. Rev. Cancer 2007, 7, 961−967. (11) Starobinets, H.; Debnath, J. Cancer a Suppression Switch. Nature 2013, 504, 225−226. (12) Rubinsztein, D. C.; Codogno, P.; Levine, B. Autophagy Modulation as a Potential Therapeutic Target for Diverse Diseases. Nat. Rev. Drug Discovery 2012, 11, 709−784. (13) Rubinsztein, D. C.; Gestwicki, J. E.; Murphy, L. O.; Klionsky, D. J. Potential Therapeutic Applications of Autophagy. Nat. Rev. Drug Discovery 2007, 6, 304−312. (14) Levine, B. Cell Biology - Autophagy and Cancer. Nature 2007, 446, 745−747. (15) Yang, Z. N. J.; Chee, C. E.; Huang, S. B.; Sinicrope, F. A. Autophagy Modulation for Cancer Therapy. Cancer Biol. Ther. 2011, 11, 169−176. (16) Jiang, P. D.; Mizushima, N. Autophagy and Human Diseases. Cell Res. 2014, 24, 69−79. (17) Swanlund, J. M.; Kregel, K. C.; Oberley, T. D. Investigating Autophagy Quantitative Morphometric Analysis Using Electron Microscopy. Autophagy 2010, 6, 270−277. (18) Yoshioka, A.; Miyata, H.; Doki, Y.; Yamasaki, M.; Sohma, I.; Gotoh, K.; Takiguchi, S.; Fujiwara, Y.; Uchiyama, Y.; Monden, M. LC3, an Autophagosome Marker, Is Highly Expressed in Gastrointestinal Cancers. Int. J. Oncol. 2008, 33, 461−468. (19) Kuma, A.; Matsui, M.; Mizushima, N. LC3, an Autophagosome Marker, Can Be Incorporated into Protein Aggregates Independent of Autophagy. Autophagy 2007, 3, 323−328. (20) Mizushima, N.; Yoshimori, T.; Levine, B. Methods in Mammalian Autophagy Research. Cell 2010, 140, 313−326. (21) Klionsky, D. J.; Abdelmohsen, K.; Abe, A.; Abedin, M. J.; Abeliovich, H.; Acevedo-Arozena, A.; Adachi, H.; Adams, C. M.; Adams, P. D.; Adeli, K.; et al. Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy. Autophagy 2016, 12, 1−222. (22) Pandey, S.; Baker, S. N.; Pandey, S.; Baker, G. A. Fluorescent Probe Studies of Polarity and Solvation within Room Temperature Ionic Liquids. J. Fluoresc. 2012, 22, 1313−1343. (23) Fan, L.; Gao, S.-Q.; Li, Z.-B.; Niu, W.-F.; Zhang, W.-J.; Shuang, S.M.; Dong, C. An Indole-Carbazole-Based Ratiometric Emission pH Fluorescent Probe for Imaging Extreme Acidity. Sens. Actuators, B 2015, 221, 1069−1076. (24) Wang, L.; Yang, L.; Cao, D. Probes Based on Diketopyrrolopyrrole and Anthracenone Conjugates with Aggregation-Induced Emission Characteristics for pH and BSA Sensing. Sens. Actuators, B 2015, 221, 155−166. (25) Wang, X.-D.; Meier, R. J.; Schmittlein, C.; Schreml, S.; Schaeferling, M.; Wolfbeis, O. S. A Water-Sprayable, Thermogelating 1838


Article

ACS Nano (43) Choi, K.-M.; Nam, H. Y.; Na, J. H.; Kim, S. W.; Kim, S. Y.; Kim, K.; Kwon, I. C.; Ahn, H. J. A Monitoring Method for Atg4 Activation in Living Cells Using Peptide-Conjugated Polymeric Nanoparticles. Autophagy 2011, 7, 1052−1062. (44) Li, M.; Chen, X.; Ye, Q.-Z.; Vogt, A.; Yin, X.-M. A HighThroughput FRET-Based Assay for Determination of Atg4 Activity. Autophagy 2012, 8, 401−412. (45) Ni, Z.; Gong, Y.; Dai, X.; Ding, W.; Wang, B.; Gong, H.; Qin, L.; Cheng, P.; Li, S.; Lian, J.; He, F. AU4S: a Novel Synthetic Peptide to Measure the Activity of ATG4 in Living Cells. Autophagy 2015, 11, 403−415. (46) Vezenkov, L.; Honson, N. S.; Kumar, N. S.; Bosc, D.; Kovacic, S.; Nguyen, T. G.; Pfeifer, T. A.; Young, R. N. Development of Fluorescent Peptide Substrates and Assays for the Key Autophagy-Initiating Cysteine Protease Enzyme, ATG4B. Bioorg. Med. Chem. 2015, 23, 3237−3247. (47) Peynshaert, K.; Manshian, B. B.; Joris, F.; Braeckmans, K.; De Smedt, S. C.; Demeester, J.; Soenen, S. J. Exploiting Intrinsic Nanoparticle Toxicity: the Pros and Cons of Nanoparticle-Induced Autophagy in Biomedical Research. Chem. Rev. 2014, 114, 7581−7609. (48) Stern, S. T.; Adiseshaiah, P. P.; Crist, R. M. Autophagy and Lysosomal Dysfunction as Emerging Mechanisms of Nanomaterial Toxicity. Part. Fibre Toxicol. 2012, 9, 20. (49) Li, L.-L.; Ma, H.-L.; Qi, G.-B.; Zhang, D.; Yu, F.; Hu, Z.; Wang, H. Pathological-Condition-Driven Construction of Supramolecular Nanoassemblies for Bacterial Infection Detection. Adv. Mater. 2016, 28, 254− 262. (50) Zhang, D.; Qi, G.-B.; Zhao, Y.-X.; Qiao, S.-L.; Yang, C.; Wang, H. In Situ Formation of Nanofibers From Purpurin18-Peptide Conjugates and the Assembly Induced Retention Effect in Tumor Sites. Adv. Mater. 2015, 27, 6125−6130. (51) Li, L.-L.; Xu, J.-H.; Qi, G.-B.; Zhao, X.; Yu, F.; Wang, H. CoreShell Supramolecular Gelatin Nanoparticles for Adaptive and ″OnDemand″ Antibiotic Delivery. ACS Nano 2014, 8, 4975−4983. (52) Wang, Y.; Lin, Y.-X.; Qiao, Z.-Y.; An, H.-W.; Qiao, S.-L.; Wang, L.; Rajapaksha, R. P. Y. J.; Wang, H. Self-Assembled Autophagy-Inducing Polymeric Nanoparticles for Breast Cancer Interference In-Vivo. Adv. Mater. 2015, 27, 2627−2634. (53) Liu, Y.; Zhang, D.; Qiao, Z.-Y.; Qi, G.-B.; Liang, X.-J.; Chen, X.-G.; Wang, H. A Peptide-Network Weaved Nanoplatform With Tumor Microenvironment Responsiveness and Deep Tissue Penetration Capability for Cancer Therapy. Adv. Mater. 2015, 27, 5034−5037. (54) Li, W.; Wang, L.; Zhang, J.-P.; Wang, H. Bis-Pyrene-Based Supramolecular Aggregates With Reversibly Mechanochromic and Vapochromic Responsiveness. J. Mater. Chem. C 2014, 2, 1887−1892. (55) Li, W.; Yang, P.-P.; Wang, L.; Wang, H. Bis-Pyrene Based Reversibly Multi-Responsive Fluorescence Switches. J. Mater. Chem. C 2015, 3, 3783−3789. (56) Qiao, Z.-Y.; Hou, C.-Y.; Zhao, W.-J.; Zhang, D.; Yang, P.-P.; Wang, L.; Wang, H. Synthesis of Self-Reporting Polymeric Nanoparticles for in situ Monitoring of Endocytic Microenvironmental pH. Chem. Commun. 2015, 51, 12609−12612. (57) Wang, L.; Li, W.; Lu, J.; Zhang, J.-P.; Wang, H. Bis-Pyrene Carbocations for Chromogenic and Fluorogenic Dual-Detection of Fluoride Anion. Tetrahedron 2014, 70, 3172−3177. (58) Wang, L.; Li, W.; Lu, J.; Zhao, Y.-X.; Fan, G.; Zhang, J.-P.; Wang, H. Supramolecular Nano-Aggregates Based on Bis(Pyrene) Derivatives for Lysosome-Targeted Cell Imaging. J. Phys. Chem. C 2013, 117, 26811−26820. (59) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Aggregation-Induced Emission. Chem. Soc. Rev. 2011, 40, 5361−5388. (60) Luo, J. D.; Xie, Z. L.; Lam, J. W. Y.; Cheng, L.; Chen, H. Y.; Qiu, C. F.; Kwok, H. S.; Zhan, X. W.; Liu, Y. Q.; Zhu, D. B.; Tang, B. Z. Aggregation-Induced Emission of 1-Methyl-1,2,3,4,5-Pentaphenylsilole. Chem. Commun. 2001, 1740−1741. (61) Shi, H.; Kwok, R. T. K.; Liu, J.; Xing, B.; Tang, B. Z.; Liu, B. RealTime Monitoring of Cell Apoptosis and Drug Screening Using Fluorescent Light-Up Probe With Aggregation-Induced Emission Characteristics. J. Am. Chem. Soc. 2012, 134, 17972−17981.

(62) Ficht, S.; Payne, R. J.; Guy, R. T.; Wong, C.-H. Solid-Phase Synthesis of Peptide and Glycopeptide Thioesters Through Side-ChainAnchoring Strategies. Chem. - Eur. J. 2008, 14, 3620−3629. (63) Park, E.; Woo, J.; Dinesh-Kumar, S. P. Arabidopsis ATG4 Cysteine Proteases Specificity toward ATG8 Substrates. Autophagy 2014, 10, 926−927. (64) Yang, P. P.; Zhao, X. X.; Xu, A. P.; Wang, L.; Wang, H. Reorganization of Self-Assembled Supramolecular Materials Controlled by Hydrogen Bonding and Hydrophilic-Lipophilic Balance. J. Mater. Chem. B 2016, 4, 2662−2668. (65) Kastl, L.; Sasse, D.; Wulf, V.; Hartmann, R.; Mircheski, J.; Ranke, C.; Carregal-Romero, S.; Antonio Martinez-Lopez, J.; FernandezChacon, R.; Parak, W. J.; et al. Multiple Internalization Pathways of Polyelectrolyte Multilayer Capsules into Mammalian Cells. ACS Nano 2013, 7, 6605−6618. (66) Pattingre, S.; Bauvy, C.; Levade, T.; Levine, B.; Codogno, P. Ceramide-Induced Autophagy to Junk or to Protect Cells? Autophagy 2009, 5, 558−560. (67) Scherz-Shouval, R.; Shvets, E.; Fass, E.; Shorer, H.; Gil, L.; Elazar, Z. Reactive Oxygen Species Are Essential for Autophagy and Specifically Regulate the Activity of Atg4. EMBO J. 2007, 26, 1749−1760. (68) Qiao, S.; Dennis, M.; Song, X.; Vadysirisack, D. D.; Salunke, D.; Nash, Z.; Yang, Z.; Liesa, M.; Yoshioka, J.; Matsuzawa, S.-I.; et al. A REDD1/TXNIP Pro-Oxidant Complex Regulates ATG4B Activity to Control Stress-Induced Autophagy and Sustain Exercise Capacity. Nat. Commun. 2015, 6, 7014. (69) Halasi, M.; Wang, M.; Chavan, T. S.; Gaponenko, V.; Hay, N.; Gartel, A. L. ROS Inhibitor N-Acetyl-L-Cysteine Antagonizes the Activity of Proteasome Inhibitors. Biochem. J. 2013, 454, 201−208. (70) Wu, Y.; Wang, X.; Guo, H.; Zhang, B.; Zhang, X.-B.; Shi, Z.-J.; Yu, L. Synthesis and Screening of 3-MA Derivatives for Autophagy Inhibitors. Autophagy 2013, 9, 595−603. (71) Harada, M.; Sakisaka, S.; Yoshitake, M.; Kin, M.; Ohishi, M.; Shakado, S.; Mimura, Y.; Noguchi, K.; Sata, M.; Tanikawa, K. Bafilomycin A1, a Specific Inhibitor of Vacuolar-Type H+-Atpases, Inhibits the Receptor-Mediated Endocytosis of Asialoglycoproteins in Isolated Rat Hepatocytes. J. Hepatol. 1996, 24, 594−603. (72) Klionsky, D. J.; Elazar, Z.; Seglen, P. O.; Rubinsztein, D. C. Does Bafilomycin A(1) Block the Fusion of Autophagosomes with Lysosomes? Autophagy 2008, 4, 849−850. (73) Petiot, A.; Ogier-Denis, E.; Blommaart, E. F. C.; Meijer, A. J.; Codogno, P. Distinct Classes of Phosphatidylinositol 3 ′-Kinases Are Involved in Signaling Pathways That Control Macroautophagy in HT29 cells. J. Biol. Chem. 2000, 275, 992−998. (74) He, C.; Klionsky, D. J. Analyzing Autophagy in Zebrafish. Autophagy 2010, 6, 642−644. (75) Tanida, I.; Sou, Y. S.; Ezaki, J.; Minematsu-Ikeguchi, N.; Ueno, T.; Kominami, E. HsAtg4B/HsApg4B/Autophagin-1 Cleaves the Carboxyl Termini of Three Human Atg8 Homologues and Delipidates Microtubule-Associated Protein Light Chain 3-and GABA(A) ReceptorAssociated Protein-Phospholipid Conjugates. J. Biol. Chem. 2004, 279, 36268−36276. (76) Thumm, M.; Kadowaki, T. The Loss of Drosophila APG4/AUT2 Function Modifies the Phenotypes of Cut and Notch Signaling Pathway Mutants. Mol. Genet. Genomics 2001, 266, 657−663.

1839


Intracellular Self-Assembly Strategy for ... - ACS Publications

Recommend Documents