A Lysosome‐Targeting AIEgen for Autophagy Visualization

In this work, a morpholine-functionalized aggregation-induced emission luminogen (AIEgen), AIE-LysoY, is reported for lysosomal imaging and autophagy visualization. To attain outstanding imaging contrast, AIE-LysoY is equipped with excited state intramolecular proton transfer (ESIPT) characteristic. AIE-LysoY provides a new platform for lysosome visualization with good biocompatibility, large Stokes shift, superior signal-to-noise ratio, and high photostability.

DOI: 10.1002/adhm.201500674 compartment accumulation containing amyloid protein and β-amyloid precursor. A typical autophagy process is depicted in Scheme 1 . A phagophore is formed at the early stages of autophagy, followed by the formation of autophagosome, which is essential to deliver the damaged pieces within the cytoplasm to the lysosomes. The fusion of an autophagosome and lysosome leads to the formation of autolysosome. The encapsulated materials will then be degraded by lysosomal hydrolases. The lysosomes are reformed after the completion of digestion and able to fuse with next autophagosome. [ 8 ] In the whole autophagy process, lysosome is the determining subcellular organelle in autophagy execution.
Owing to the close association of lysosome with autophagy, visualizing and tracking lysosomal activities will enrich the insight into the autophagic process. A proper lysosome-targeting probe not only facilitates the investigation of autophagy but also broadens the mechanistic vision of corresponding drugs. With such regard, a variety of lysosome-targeting probes are developed for exploring lysosome-involved cellular activities, among which LysoTracker Red DND-99 (LTR) is a good representative. The emission of LTR is turned on due to the removal of photoinduced electron-transfer (PET) effect by protonation of its weak base unit in the lysosome. [ 11 ] Unfortunately, the fl uorescence quenching process by PET is not effi cient outside the lysosome, [ 12 ] thus accurate localization of the lysosome is diffi cult. On the other hand, the working concentration of LTR is normally low to prevent the aggregation-caused quenching (ACQ) effect. As a result, LTR displays low photostability. Furthermore, LTR exhibits small Stokes shift with absorption and fl uorescence maxima at 577 and 590 nm, respectively, thus high-resolution image can only be obtained by choosing appropriate excitation and emission ranges. Besides small organic molecules, lysosome labeling can be achieved by utilizing metal complexes. [ 11,[13][14][15][16][17][18][19] However, they may suffer from high background noise, inadequate specifi city, lengthy incubation time, and low photostability.
In 2001, a series of luminogens with propeller-shaped conformation were discovered to demonstrate faint emission in solution but fl uoresce intensely in their aggregates. We termed the phenomenon as aggregation-induced emission (AIE). Systematic investigations revealed restriction of intramolecular motion (RIM) as the underlying mechanism. AIE luminogens (AIEgens) have been utilized in numerous fl uorescence biosensing systems, including organelle targeting, protein probing, DNA differentiation, and so on. [20][21][22][23][24][25][26][27][28][29][30] To obtain higher signal-to-noise ratio, hydrophilic groups, charged moieties or peptide chains can be incorporated into their structures to enhance their water solubility and weaken their emission in aqueous media. [ 20 ] In Lysosome is known as the leading player of autophagy. [ 1 ] Investigating lysosomal activities is indispensable to in-depth investigation of autophagy, through which cells dissemble unnecessary or dysfunctional cellular components in a regulated manner. Autophagy has become a hot research topic, owing to its close relationship with cancers, aging, longevity, and neurodegenerative diseases, such as Danon disease, Pompe disease, etc. [1][2][3][4][5][6][7][8] Proper autophagic fl ux is necessary for controlling the health of an individual. Upregulated and downregulated autophagy may lead to the clearance and accumulation of protein aggregates, which may reduce and exaggerate the symptoms of several neurodegenerative diseases, respectively. [ 9,10 ] Remarkably, active autophagy is observed in the brain of patients and mouse models of Alzheimer's disease, but autophagy-lysosomal degradation is disrupted, leading to a boost of autophagic COMMUNICATION this contribution, we report the design and synthesis of an AIEactive probe with excited-state intramolecular proton-transfer (ESIPT) characteristics, namely AIE-LysoY, and its utility in visualization of the lysosome and lysosome-involved autophagy process ( Scheme 2 ).
The synthetic route to AIE-LysoY is shown in Scheme 2 . M1 and M2 were prepared by one-step modifi cation of commercially available precursors, which then underwent condensation under mild reaction conditions to yield FAS-Br. AIE-LysoY was obtained by conjugating FAS-Br with morpholine group. The morpholine group with moderate alkalinity is employed for lysosome-targeting. [ 31 ] Once entered lysosome, the morpholine will be protonated due to the high acidity of the lysosome, which endows AIE-LysoY with higher hydrophilicity and lysosomal retention. [ 32 ] In this way, fl uorescence of AIE-LysoY can only be observed in the lysosome area.
AIE-LysoY exhibits an absorption band peaked at 390 nm in THF ( Figure S1, Supporting Information). It emits faintly when it is dissolved in THF with a small emission band centered at 565 nm. With an increase in water fraction of the THF/water mixtures, AIE-LysoY becomes highly emissive ( Figure 1 B), demonstrating a phenomenon of AIE. The AIE effect of AIE-LysoY can be rationalized by the activation of RIM and ESIPT upon formation of nanoaggregates. In pure THF solution, AIE-LysoY can undergo dynamic intramolecular motions, thus excitons nonradiatively decay in these solutions. In solvent mixture with low solvating power, AIE-LysoY form nanoaggregates, and thus its intramolecular motion is prohibited. As a result, the excitons of AIE-LysoY decay through the radiative pathway of fl uorescence and its emission is enhanced.
AIE-LysoY also possesses the characteristics of ESIPT. It demonstrates two kinds of emission (Scheme S1, Supporting Information). In aprotic solvent, intramolecular hydrogen bond exists, thus it exhibits keto emission with longer wavelength, while it displays the enol emission with shorter wavelength in protic solvent due to the breakage of such intramolecular hydrogen bond ( Figure S2, Supporting Information). Besides, addition of organic acid can also break the intramolecular hydrogen bond and lead to decreased keto emission intensity. To demonstrate this, a strong organic acid, trifl uoroacetic acid (TFA), with a p K a of about −0.3, is added into the aggregate suspension (THF/water, 1:99) of AIE-LysoY. The emission of AIE-LysoY at 565 nm decreases dramatically when the TFA fraction is increased from 1% to 5%. When the TFA fraction reaches 10%, AIE-LysoY becomes completely nonemissive ( Figure 2 ) due to the protonation of the central nitrogen and destroy of the ESIPT emission.
Then, AIE-LysoY was applied to imaging of the lysosome and its lysosome-targeting performance was assessed by fl uorescence microscope. HeLa cells were stained with 10 × 10 −6 M of AIE-LysoY for 10 min, followed by washing away the unbounded molecules of AIE-LysoY and incubation with 50 × 10 −9 M of LTR for 30 min. As shown in Figure 3 A, AIE-LysoY selectively accumulates in the punctate lysosomes and endows them with yellow emission, which can be clearly distinguished from the background. Besides, the fl uorescence of The high R r value proves that AIE-LysoY is a lysosome-selective probe. In addition, AIE-LysoY shows a higher contrast than LTR even after shorter incubation time, which can be ascribed to the bright emission and high selectivity of AIE-LysoY. Interestingly, if HeLa cells are stained with LTR in the fi rst place and then AIE-LysoY, the latter still demonstrates higher contrast than the former ( Figure S4, Supporting Information). These results suggest that AIE-LysoY may compete with LTR to bind to lysosome, but AIE-LysoY possesses higher affi nity to lysosome than LTR. Additionally, AIE-LysoY also demonstrates advantageous attribute of variable staining time. Changing the staining time from 10 to 60 min, the specifi city to lysosome and emission contrast is well retained ( Figure S5, Supporting Information).
Compared with LTR, the working concentration of AIE-LysoY is much higher. One may think that LTR is more sensitive than AIE-LysoY and thus more advantageous, but it is not true. As mentioned previously, LTR can only be used at low concentration to avoid the ACQ effect. At such low working concentration, the fl uorescent probes molecularly disperse in the organelle and can be easily photo-oxidized by continuous excitation light irradiation, resulting in the low photostability of these probes. For AIE-LysoY, its emission is brightened by aggregation, thus can be used at high concentrations to image the lysosome with high brightness and photostability. In order to evaluate the photostability of AIE-LysoY, confocal microscope (Zeiss laser scanning confocal microscope LSM7 DUO) was applied to continuously scan cells stained by AIE-LysoY. The photostability of LTR was also measured for comparison. The power of excitation light (405 nm for AIE-LysoY and 561 nm for LTR) were unifi ed by using a power meter. After continuous scanning for 50 times with total irradiation time of ≈6 min, the fl uorescence signal from AIE-LysoY almost remain unchanged, indicating its excellent photo stability ( Figure 4 , red line; video 1, Supporting Information). In contrast, the fl uorescence intensity from LTR drops to lower than 50% of its initial value after only 25 scans ( Figure 4 , blue line; video 2, Supporting Information). The slightly fl uctuation of fl uorescence signal can be ascribed to the dynamic movement of the lysosome in living cells. The excellent photostability of AIE-LysoY may stem from its nanoparticles nature in lysosome imaging, which can protect the chromophore inside the particles from being photo-oxidized. In this way, the fl uorescence of AIE-LysoY can remain after long-time irradiation. The good photostability of AIE-LysoY enables its potential in long-term following the biological processes of lysosome.
To build up a more general strategy for designing lysosome-specifi c bioprobe, molecular engineering approach was employed. The precursor of AIE-LysoY, FAS-Br, and two morpholine-functionalized AIEgens, TPE-2Mor and Nred-mor (Scheme S2-S4, Supporting Information), were prepared and applied to cell imaging. FAS-Br lights up intracellular lipid droplets and the reticulum structures of the mitochondria in HeLa cells ( Figure S6, Supporting Information) rather than the lysosome, which suggests the necessity of morpholine for locating the acidic lysosome. However, bearing morpholine group does not guarantee lysosome selectivity. Both TPE-2Mor Adv. Healthcare Mater. 2016, 5, 427-431 www.advhealthmat.de www.MaterialsViews.com   and Nred-mor contain morpholine group, but none of them can selectively light up the lysosome. TPE-2Mor stains all the hydrophobic region, while Nred-mor selectively accumulates in the mitochondria (Figures S7 and S8, Supporting Information). TPE-2Mor contains a hydrophobic tetraphenylethene (TPE) core and Nred-mor carries a charged pyridinium moiety. The hydrophobic interaction of TPE and the electrostatic interaction of positively charged pyridinium with other cellular organelles may compete with the driving force from morpholine and thus decrease or destroy the lysosome selectivity. Therefore, accurate lysosome targeting should go with the manipulation of hydrophobicity and polarity cautiously.
Mammalian target of rapamycin (mTOR) has been identifi ed as the suppressant of autophagy. Rapamycin, a lipophilic macrolide antibiotic (Scheme S5, Supporting Information), can bind to mTOR and enhance autophagy and is thus widely used for autophagy induction. Treatment with rapamycin has been demonstrated to prolong the survival of prion protein infected mice by activating autophagy and prion protein degradation. [ 9 ] During rapamycin treatment, there is an increase in the number, size, and acidity of the lysosome. By employing AIE-LysoY with high lysosome targetability and excellent photostability as fl uorescent visualizer, the autophagy process can be tracked in long term, which will provide more insight into the cellular activities. To demonstrate this feasibility, HeLa cells were incubated with 10 × 10 −6 M of AIE-LysoY for 10 min and then treated with rapamycin for different periods of time, followed by observation under fl uorescence microscope. As shown in Figure 5 A-E, the lysosome can be clearly visualized under the fl uorescence microscope. During autophagy process, the lysosome will increase in amount and they will fuse with autophagosome to form autolysosome. This is indeed the case. The amount of yellow spots, which corresponds to the number of lysosome, are increased with prolonged rapamycin treatment. If we enlarge the image in Figure 5 E, the lysosome can be visualized with excellent resolution and contrast ( Figure 5 F). Notably, the newly formed lysosomes were lit up even when the excessive AIE-LysoY was removed prior to rapamycin treatment. The result strengthened the occurrence of the fusion between the autophagic compartment and primitive lysosome during autophagy. [ 3 ] In addition, LTR provided similar fl uorescence images and showed good overlapping with AIE-LysoY in rapamycin-treated HeLa cells ( Figures S9 and S10, Supporting Information). However, LTR should be co-incubated with rapamycin, otherwise its emission will be greatly diminished when excessive LTR is washed away beforehand ( Figure S11, Supporting Information). By virtue of its high selectivity towards the lysosome and excellent photostability, AIE-LysoY is an excellent candidate as lysosome selective bioprobe for investigating autophagy process.
In conclusion, an AIE-active lysosome-specifi c probe with ESIPT characteristics, namely AIE-LysoY, was designed and developed. Guided by its morpholine functionality, AIE-LysoY selectively accumulates in and lights up the lysosome of cells. Thanks to the collective effect of AIE and ESIPT properties,