Perylene-Based Fluorescent Nanoprobe for Acid-Enhanced Detection

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Perylene-Based Fluorescent Nanoprobe for AcidEnhanced Detection of Formaldehyde in Lysosome Chendong Ji, Le Ma, Hongtao Chen, Yang Cai, Xujie Zhao, and Meizhen Yin ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00699 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 18, 2018

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ACS Applied Bio Materials

Perylene-Based Fluorescent Nanoprobe for Acid-Enhanced Detection of Formaldehyde in Lysosome

Chendong Ji, Le Ma, Hongtao Chen, Yang Cai, Xujie Zhao and Meizhen Yin*

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

Materials,

Key

Laboratory

of

Biomedical

Materials

of

Natural

Macromolecules, Beijing University of Chemical Technology, 100029 Beijing, China

KEYWORDS Perylene, Fluorescent Nanoprobe, Weak Acidity, Formaldehyde Detection, Lysosome

ACS Paragon Plus Environment

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ABSTRACT

Formaldehyde (FA), as a reactive carbonyl species, is extremely hazardous to human health if its concentration is above normal level. In live cells, lysosome is a main organelle to generate endogenous FA. Thus, the design of facile, stable and sensitive probes for the detection of FA in lysosome is essential. Herein, a self-assembled fluorescent nanoprobe based on homoallylamino substituted perylene (P-FA) has been developed for FA detection in lysosome. P-FA can react with FA along with emission color change from blue to green. P-FA exhibited high sensitivity and selectivity to FA in DMSO solution. In aqueous solution, P-FA self-assembled into uniform sphere-like nanoparticle as a fluorescent nanoprobe. Furthermore, the reaction between the nanoprobe and FA was greatly facilitated at pH 4-5, leading to a lower detection limit (0.96 μM at pH 5) than that in DMSO. In live cells, P-FA nanoprobe achieved long-term tracking of lysosome (over 12 h). The fluorescent nanoprobe was then used for both exogenous and endogenous FA detection. Our work provides a facile and effective strategy for the detection of FA in lysosome.


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INTRODUCTION

Formaldehyde (FA) is a widely used raw material, especially in chemical, pharmaceutical and construction industry.1-3 However, its high toxicity and carcinogenic effects have attracted considerable attention. Over exposure to high level of FA can cause damage to human skin, nose, throat, eye, lung, liver, immune system and DNA.4-6 In addition, FA is also endogenously produced during the demethylation or oxidation processes.7,8 The normal level of FA closely relates to the formation of spatial memory and cognitive ability,9,10 while elevations of FA is implicated in neurodegenerative diseases,11 allergic pneumonia,12 cardiovascular disease,13 and cancer.14 Therefore, the monitoring of FA in various environments is of great significance.

Traditional methods for FA detection have been developed such as high-performance chromatography and electrochemical sensors,15-18 which are expensive and timeconsuming. In order to overcome these drawbacks, fluorescence detection technology using small-molecule based probes has received considerable attention owing to their


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non-destructiveness, simplicity, excellent sensitivity and selectivity.19-21 Recently, fluorescent probes for the detection of FA in solution and especially in live cells have been reported.22-26 For instance, lysosome-targeted probes have shown great potential in monitoring endogenous FA in lysosome.27,28 Most of them are fluorophores modified by FA reacting moiety, targeting moiety or/and acid responsive moiety, which is complex. It is still a challenge to design facile, stable and sensitive probes for the detection of FA in lysosome.

Perylene fluorophore has high fluorescence quantum yield and excellent photo/chemical stability,29-31 which has been considered as a suitable fluorophore for bioimaging and detection fields.32-34 Taking advantage of their rigid planar structures, perylene dyes are self-assembled to construct fluorescent nanoparticle that showed enhanced accumulation in lysosome.35-37 For the design of FA probe, homoallylamino derivative have been reported to react with FA selectively via 2-aza-Cope rearrangement and this reaction can be promoted in acid environment.38,39 Thus, nanoprobe based on


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perylene and 2-aza-Cope rearrangement would be promising for FA detection in lysosome with an acidic environment.

Herein, a novel and facile fluorescent nanoprobe based on homoallylamino substituted perylene (P-FA) for the detection of FA in lysosome is developed (Scheme 1A). P-FA can respond to FA along with emission color change from blue to green. P-FA has good selectivity towards FA with a detection limit of 6.1 μM in DMSO solution. In aqueous solution, P-FA is readily self-assembled into uniform sphere-like nanoparticle as a fluorescent nanoprobe. The 2-aza-Cope rearrangement between P-FA nanoprobe and FA is obviously enhanced at pH 4-5, resulting in a lower detection limit of 0.96 μM. P-FA nanoprobe can accumulate in lysosome site over 12 h after endocytosis (Scheme 1B). Furthermore, the nanoprobe achieves exogenous and endogenous FA detection in live HeLa cells through fluorescence ratio change between red/green channels.


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Scheme 1. (A) Perylene-based fluorescent probe (P-FA) for formaldehyde (FA) detection. (B) Illustration of P-FA nanoprobe detecting FA in lysosome.

EXPERIMENTAL SECTION Materials and Instruments Commercially available solvents and reagents were used without

purification.

Fluorescence

spectra

were

measured

with

fluorescence

spectrophotometer FluoroMax-4 NIR (Horiba Jobin Yvon, USA). UV-visible spectra were measured with UV-2600 (Shimadzu, Japan). 1H NMR spectra were recorded on NMR spectrometer (AVANCE III, Switzerland). DMSO-d6 was used as solvent with tetramethylsilane (TMS) as internal reference. Fourier transform infrared (FT-IR) spectra


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were conducted with FT-IR spectroscopy (Thermo Nicolet Nexus, USA). The mass spectra were recorded with XEVO-G2QTOF (Waters, USA).

Synthesis of Compound CHO-P. A mixture of perylene (504 mg, 2 mmol), anhydrous odichlorobenzene (1 mL) and anhydrous DMF (1 mL, 13 mmol) were added to a stirred three-necked reaction flask and the reaction mixture was heated to 100 °C under nitrogen atmosphere. POCl3 (614 mg, 4 mmol) was added dropwise, stirring at 100 °C for 2 h. Then the mixture was cooled to and poured into H2O (500 mL), neutralized by dilute aqueous sodium acetate and kept at 0 °C for 3 h. The precipitate was filtered off, washed with water (3 × 30 mL), and purified by silica gel column (using dichloromethane as eluent). Compound CHO-P (420 mg, 75%) was yielded as orange powder. 1H NMR (400 MHz, DMSO) δ 10.35 (s, 1H), 9.11 (d, J = 8.4 Hz, 1H), 8.63-8.57 (m, 2H), 8.53 (dd, J = 15.0, 7.4 Hz, 2H), 8.18 (d, J = 7.9 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.81-7.75 (m, 1H), 7.65 (dd, J = 17.5, 7.9 Hz, 2H). MALDI-TOF: m/z calcd for C21H12O, 280.04; found, 281.0472 [M+H]+.


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Synthesis of Compound P-FA. A mixture of compound CHO-P (420.5 mg, 1.5 mmol) was added to 3 mL of a NH3 solution (7 N in methanol, 21 mmol) in 30 mL methanol at -10 °C. The reaction was stirred at -10 °C for 1 h and then warmed. Allylboronic acid pinacol ester (303 mg, 18 mmol) was added at 0 °C and reacted for another 12 h at room temperature. The mixture was purified by silica gel column (using dichloromethane/methanol (v/v, 10:1) as eluent).The final product P-FA (170 mg, 36%) was yeiled as yellow powder. 1H NMR (400 MHz, DMSO) δ 8.43-8.32 (m, 4H), 8.07 (d, J = 8.5 Hz, 1H), 7.79 (t, J = 7.8 Hz, 3H), 7.62-7.52 (m, 3H), 5.91 (ddt, J = 17.1, 10.1, 7.0 Hz, 1H), 5.06 (dd, J = 17.8, 13.7 Hz, 2H), 4.75-4.64 (m, 1H), 2.62-2.52 (s, 3H), 2.43-2.34 (m, 1H). MALDI-TOF: m/z calcd for C24H19N, 321.95; found, 322.919 [M+H]+.

General Procedure for UV-vis and Fluorescence Measurement. All of the UV-vis absorption and fluorescence spectrum of P-FA (10 µM) responding to FA were performed at 25 °C. The FA solutions were prepared by diluting commercial FA aqueous solution (37 wt%). The sample solutions for the measurement were 2 mL. The interference of various analytes was studied, including acetaldehyde, propionaldehyde, acetone


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aldehyde, glyoxal and benzaldehyde in distilled water. For all of the fluorescence measurements, the slit width was 0.8 nm/0.8 nm. The excitation wavelengths were 420 nm in DMSO and 450 nm in aqueous solutions.

Determination of the Detection Limit. The detection limit was calculated by 3σ method. The fluorescence intensity of P-FA was measured as blank for five times. The detection limit of P-FA was calculated using the equation of Detection limit = 3σ/k. k is the slope of fluorescence intensity ratio (F550/F452 or F550/F480) versus FA concentration. σ is the standard deviation of the blank measurement,. Density Functional Theoretical (DFT) Calculation. Quantum chemical calculations were uesd to describe the ground state and singlet excited state of P-FA and CHO-P. DFT calculations were operated with Gaussian 09 package. Becke’s three-parameter hybrid method corrected with the Lee-Yang-Parr function (B3LYP) and the 6-31G (d) basis set was employed.

Preparation of P-FA Nanoparticle. P-FA at the concentration of 1×10-4 M were prepared in DMSO. Then 0.1 mL of P-FA was dropped into 10 mL water or phosphate buffered


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saline (PBS) solution. DMSO was removed by dialysis in water before scanning electron microscopy observation.

Cell Culture and Fluorescence Imaging. Human cervical carcinoma (HeLa) cell was cultured in complete high glucose (DMEM, Hyclone) with 10% FBS (fetal bovine serum) and 1% antibiotic (penicillin-streptomycin). The cell was incubated at 37 °C in a 5% CO2 humidified atmosphere for 24 h. For the co-localization experiment, the cells were rinsed with PBS solution, incubated with 100 μL DMEM containing 10 μM P-FA and 10 μM Lysotracker Red (Beyondtime Cooperation) at 37 °C for 30 min. Subsequently, HeLa cells were washed with PBS twice to remove excess dyes. For the exogenous FA detection, HeLa cells were rinsed with PBS, incubated with 100 μL DMEM containing 10 μM P-FA, then incubated with DMEM containing 0.1 mM of FA at 37 °C for 3 h. For the endogenous FA detection, HeLa cells were rinsed with PBS, and incubated with 100 μL DMEM containing 10 μM P-FA. After incubation with P-FA for 30 min, the medium was replaced with fresh DMEM for the experimental group and with DMEM containing 100 mM NaHSO3 for the negative control group. The cells were incubated at 37 °C for another 12 h. The


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fluorescent images were obtained by fluorescence microscope. Green channel: excitation: 470 nm, emission: 480-545 nm, Red channel: excitation: 531 nm, emission: 553-613 nm. Cytotoxicity Assay. The in vitro cytotoxicity of P-FA was evaluated by the standard CCK-8 assay. 100 mL medium containing HeLa cells and 10% FBS were seeded in the 96-well plates at a density of 5×103 cells at 37 °C with 5% CO2 for 24 h. Then the cluster was washed twice with PBS 100 μL/well. The cells were then cultured in medium containing 10% FBS with 0, 1, 5, 10, 20 and 30 μM P-FA for 24 h. Cells in culture medium without probes were used as the control group. Subsequently, 50 μL CCK-8 was added into each well. The cells were incubated for 2 h at 37 °C under 5% CO2. Finally, the absorbance of the samples was measured at the characteristic peak of 450 nm using the microplate reader (Thermo scientific, MULTISCAN MNK3). The relative cell viability was calculated by the average value of six independent experiments.

RESULTS AND DISCUSSION


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Design and Synthesis of P-FA. The synthesis route of P-FA was outlined in Scheme 2. P-FA was synthesized via two simple steps from perylene. The structure of P-FA and compound CHO-P were further fully characterized with standard 1H NMR spectroscopy, mass spectra and optical spectra (Supporting Information, Figure S1-S5). P-FA consists of a perylene fluorophore and a homoallylic amine reactive moiety. After exposure to FA, the homoallylamino group of P-FA would change into an aldehyde group with strong electron withdrawing ability, resulting in enhanced intramolecular charge transfer (ICT)40 process between perylene and the substituent group. This process could induce a red shift in both absorption and fluorescence spectra of the probe. Therefore, P-FA would achieve colorimetric fluorescence signal response for FA detection.

Scheme 2. Synthesis route of P-FA.


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Optical Response to FA. In order to investigate the response properties of the probe to FA, the absorption and fluorescence titration experiments of P-FA were performed with different concentrations of FA in DMSO solution (Figure 1A and 1B). With the increase of FA concentration, the absorption maximum was gradually red shifted to 472 nm with a color change from colorless to light yellow (Figure 1C). The fluorescence signal enhanced at 550 nm while reduced at 452 nm and 480 nm with the elongation of incubation time. Meanwhile, with higher FA concentration, the fluorescence color changed from blue to green under 365 nm UV lamp (Figure 1C). The ratio of fluorescence intensity at 550 nm and 452 nm (F550/F452) showed an excellent linear relationship when FA concentration ranged from 0 to 0.8 mM (R2 = 0.9904, Figure S6). The detection limit of P-FA was 6.1 μM in DMSO solution (calculated by 3δ/k method41). Therefore, P-FA can be used as a sensitive colorimetric fluorescent probe for FA detection.


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Figure 1. (A) UV-vis absorption and (B) fluorescence change of P-FA (10 μM) incubated with FA at various concentrations (0-4 mM) in DMSO solution after 90 min (FL = Fluorescence). (C) Photographs of P-FA (10 μM) in the presence of FA (0, 0.5, 1, 2, 3, 4 mM) under visible light and ultraviolet (UV) light after 90 min, respectively (λex = 420 nm). Subsequently, the response time of P-FA to FA was investigated. Absorption and fluorescence spectra of P-FA were measured after FA was added into the DMSO solution at different incubation time (Figure S7). After 90 min, the optical spectra of P-FA showed no obvious change, indicating termination of the response process.


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Selectivity of P-FA. The selectivity of P-FA was investigated to evaluate the applicability of fluorescent probe. As shown in Figure S8, only FA triggered an obvious change in the absorption and fluorescence spectra of P-FA. Specifically, the absorption and fluorescence spectra of P-FA did not display changes when other related analytes (acetaldehyde, propionaldehyde, acetone aldehyde, glyoxal and benzaldehyde) were added. We then examined F550/F452 value of P-FA after it was treated with various analytes (Figure 2A). P-FA displayed a 15-fold enhancement in the fluorescence intensity ratio (F550/F452) only in the presence of FA. While, other analytes neither cause apparent optical spectra nor color change of the probe (Figures S8 and 2B). Therefore, P-FA had high selectivity toward FA over other analytes.


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Figure 2. (A) Fluorescence intensity ratio (F550/F452) of P-FA (10 μM), in the presence of various relevant analytes (200 equiv.): (1) blank, (2) formaldehyde, (3) acetaldehyde, (4) propionaldehyde, (5) glyoxal, (6) benzaldehyde, (7) acetone aldehyde. (B) Photographs of P-FA (10 μM) with various relevant analytes in DMSO solution under visible light and UV light, respectively. Sensing Mechanism. Similar to previously proposed response mechanism, during the reaction process of P-FA with FA, P-FA would be condensated with FA initially, then undergo rearrangement and hydrolysis (Scheme S1), resulting in the generation of CHO-


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P. In order to examine this mechanism, the reaction was analyzed with mass spectra, infrared spectra and 1H NMR spectra, respectively. As shown in Figure S9, a new signal peak at 280.02 [m/z] emerged, which is ascribed to the reaction product CHO-P. The peak of aldehyde group was also observed in the IR spectra (Figure S10). Moreover, 1H NMR spectra of P-FA added with FA and P-FA alone were studied. After reaction with FA, the signals of homoallylamino moiety at 2.43-2.34 ppm (H2), 2.62-2.52 (H5, H2), 4.75-4.64 (H3), 5.06 (H4) and 5.91 (H1) of adjacent C-H bond disappeared, while a new proton signal of aldehyde appeared at 10.35 ppm (H1’) correspondingly (Figure 3).


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Figure 3. 1H NMR spectra of (A) P-FA, (B) the isolated product of P-FA + FA and (C) CHO-P in DMSO-d6. In order to further investigate the mechanism of optical spectra change, density functional theory (DFT) calculations for CHO-P and P-FA were carried out. As shown in Figure S11, the optimized structures of P-FA and CHO-P showed that CHO-P has a higher planarity than that of P-FA, resulting in a red shift of absorption and emission wavelength.42,43 Moreover, the energy gap (Eg) of P-FA was found to be 3.028 eV, which was larger than that of CHO-P (2.828 eV). Thus, CHO-P underwent stronger ICT process and exhibited red shifted absorption and emission band.44,45 Thus, the theoretical calculation further proved the proposed sensing mechanism and fluorescence spectra results.

P-FA Nanoprobe for FA Detection. Then, the self-assembling property of P-FA was investigated. Due to the rigid planar structure of perylene, P-FA was readily selfassembled into nanoparticle in aqueous solution as a fluorescent nanoprobe. The nanoparticle was sphere-like with a diameter of 130 nm (Figure 4A) and a hydrated radius


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of 160 nm (Figure 4B). P-FA nanoprobe can respond to FA with the red shift of both absorption and emission spectra (Figures 4A and 4D). The emission color of aqueous PFA nanoprobe changed from blue to green, which is similar to that in DMSO. To quantify the responsive ability of the nanoprobe to FA, the ratio of fluorescence intensity at 550 nm and 480 nm (F550/F480) was plotted against reaction time. The response time of P-FA nanoprobe to FA was 120 min (Figure S12).

To confirm acid can accelerate the detection process, P-FA nanoprobe was incubated with FA in PBS buffer at various pH values. As shown in Figure 4E and S13, the ratio of F550/F480 increased obviously at pH 2-6 within 2 h and the optimized pH for FA detection was 4-5. At pH 5, the fluorescence intensity of the nanoprobe decreased at 480 nm and enhanced at around 550 nm with the addition of FA. The detection limit of aqueous P-FA nanoprobe at pH 5 was calculated to be 0.96 μM (Figure S14). The reaction mechanism was shown in Scheme S1, acid could facilitate the generation and rearrangement of the intermediates, resulting in higher F550/F480 ratio. Therefore, P-FA nanoprobe is suitable for FA detection in acidic environment of lysosome.


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Figure 4. (A) Scanning electron microscopy image of P-FA nanoparticle. (B) Dynamic light scattering measurement and photograph (inset) of P-FA nanoparticle in water. (NP = nanoparticle) (C) Normalized absorption and photograph (inset) of P-FA before and after FA addition. (D) Normalized FL intensity and photograph under UV light (inset) of P-FA before and after FA addition. (E) FL intensity ratio (F550/F480) change of P-FA nanoprobe in the presence of FA at pH 2-8 against time. (F) Fluorescence change of P-FA nanoprobe


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(10 μM) incubated with FA at various concentrations (0-3.5 mM) in aqueous solution after 120 min. Cellular Localization of P-FA Nanoprobe. To gain insight into the cellular localization, PFA nanoprobe was incubated with HeLa cells and lysosome-selective marker Lysotracker Red was used to visually evaluate its cell internalization. After co-staining for 30 min, the green fluorescence of P-FA matched the red fluorescence of lysotracker with the Pearson correlation coefficient of 0.88 ± 0.02, demonstrating that P-FA nanoprobe can localize in lysosome (Figure 5). P-FA nanoprobe also exhibited long-term lysosome tracking ability. The green fluorescence of P-FA nanoprobe remained strong over 12 h. While the signal of Lysotracker Red greatly reduced within 12 h (Figure S15). Nanoparticles with size ~160 nm would enter cell through endocytosis process and be transferred to lysosomes.[46] Due to the relative large size, the nanoprobe P-FA is easily trapped and retained in the lysosome.[47] The amino group in P-FA can be protonated at pH 5, leading to strong interaction between P-FA and lysosome membrane.[48] Thus, P-FA nanoprobe have potential for long-term lysosome-specific FA monitoring.


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Figure 5. Co-localization of Lysotacker Red and P-FA nanoprobe after 30 min incubation. Scale bars are 20 μm. The Pearson correlation coefficient of two channels was 0.88 ± 0.02. Detection of FA in Living Cells. The FA response ability of P-FA nanoprobe was also evaluated in HeLa cell. The HeLa cells were firstly treated with P-FA nanoprobe (10 μM) for 60 min. After FA (0.1 mM) was added and incubated for another 2 hours, the fluorescence intensity of green channel decreased while that of red channel enhanced significantly (Figures 6A-6C). The generated CHO-P with red shifted fluorescence in the presence of FA led to the signal enhancement of the red channel. The ratio of fluorescence intensity (Fred/Fgreen) increased by 5 folds (Figure 6D).


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Figure 6. Detection of exogenous FA in HeLa cell. Fluorescent images of HeLa cells incubated with (A) P-FA nanoprobe (10 μM ) alone and (B) P-FA nanoprobe (10 μM) in the presence of FA (100 μM). Scale bars are 30 μm. (C) Relative FL intensity of each channel in A and B. (D) Ratio of relative FL intensity. The nanoprobe was further applied to track endogenous FA in living cells (Figure S16). The fluorescence of P-FA nanoprobe in both green and red channels can be observed, indicating the generation of endogenous FA. For comparison, NaHSO3 was


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used as an inhibitors of cellular generated FA.27,28 P-FA nanoprobe only showed fluorescence in green channel when incubated with HeLa cells pre-treated with NaHSO3. Finally, the cytotoxicity of P-FA nanoprobe was evaluated by CCK-8 assay. The cell viability was above 90% when the concentration of P-FA was below 30 μM after 24 h (Figure S17), indicating that P-FA had low toxicity. Therefore, P-FA nanoprobe is effective for monitoring both exogenous and endogenous FA in living cells.

CONCLUSIONS

In summary, a fluorescent nanoprobe P-FA based on the perylene chromophore was designed and prepared successfully for FA detection in solution and lysosome. P-FA can react with FA via 2-aza-Cope rearrangement, resulting in CHO-P with red shifted absorption/emission bands. P-FA exhibited high selectivity and sensitivity to FA. Furthermore, P-FA was self-assembled into sphere-like nanoparticle with uniform size in aqueous solution and used as a fluorescent nanoprobe. The reaction between P-FA nanoprobe and FA was significantly facilitated at pH 4-5, leading to a lower detection limit (0.96 μM at pH 5) than that in DMSO (6.1 μM). After endocytosis by live HeLa cells, P-FA


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nanoprobe localized in lysosome and exhibited bright green fluorescence for over 12 h. Then, the nanoprobe achieved effective detection of both exogenous and endogenous FA. Our work provides a facile and effective strategy to construct fluorescent nanoprobes for detecting FA in lysosome.

ASSOCIATED CONTENT

Supporting Information. Additional NMR, MS, IR, UV-vis absorption/fluorescence emission spectra, and cell experiments of P-FA.

AUTHOR INFORMATION

Corresponding Author Meizhen Yin: 0000-0001-8519-8578 Tel.: +86-10-64443801. E-mail: [email protected].

ACKNOWLEDGMENT


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This work was financially supported by the National Natural Science Foundation of China (grant numbers 21774007 and 21574009), the Fundamental Research Funds for the Central Universities (PT1811) and Beihuazhongri United Fund (PYBZ1822).

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