Photocontrolled Fluorescence “Double-Check” Bioimaging Enabled

Communication Cite This: J. Am. Chem. Soc. 2018, 140, 8671−8674

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Photocontrolled Fluorescence “Double-Check” Bioimaging Enabled by a Glycoprobe−Protein Hybrid Youxin Fu,†,§ Hai-Hao Han,†,§ Junji Zhang,*,† Xiao-Peng He,*,† Ben L. Feringa,†,‡ and He Tian*,† †

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Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China ‡ Centre for Systems Chemistry, Stratingh Institute for Chemistry and Zernike Institute for Advanced Materials, Faculty of Mathematics and Natural Sciences, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands S Supporting Information *

low fluorescence quantum yields and molecular aggregation in aqueous solutions and cells.10,11 Here we developed a simple hybridization strategy to easily overcome these drawbacks through the host−guest assembly between a fluorophore/ photochromophore conjugate and a serum protein, thereby interrupting dye aggregation and, simultaneously, activating the photochromophore emission. The solution-based protein/ dye complex was characterized by small-angle X-ray scattering (SAXS). The simple host−guest assembly also enables a unique duplexed fluorescence switching between the fluorophore (“on−off” green-channel fluorescence) and the photochromophore (“off-on” red-channel fluorescence) upon alternate UV/vis light irradiation. Remarkably, this duplexed switching can be realized both in solution and, in particular, inside living cells, suggesting the good biocompatibility of the host−guest hybrid system developed. Human serum albumin (HSA) is used to host the carbohydrate-modified (Gal: galactose) naphthalimide (Naph)-spiropyran (SP) dyad (Gal-NSp), whose structure can be reversibly switched to the merocyanine isomer (GalNMr) upon light irradiation (Figure 1a). Owing to the presence of diverse hydrophobic pockets that accommodate hydrophobic molecules, 12 HSA has been used as a biocompatible carrier for drug delivery.13 The “host−guest” inclusion of the glycoprobe to the hydrophobic cavity of HSA significantly enhances the fluorescence emission of merocyanine (MR) in aqueous solution, which leads to a reversible, duplexed fluorescence relay between Naph and MR upon UV/ vis irradiation (Figure 1b). Guided by the carbohydrate group tethered to the photochromic probe, the photocontrolled fluorescence “double-check” mechanism has been made available toward the targeted imaging of living cells (Figure 1c). The hybrid was constructed through the “host−guest” inclusion of our previously synthesized Gal-NSp probe14 (Scheme S1) to HSA. Fluorescence spectroscopy, isothermal titration calorimetry (ITC) and SAXS were used to elaborate the glycoprobe−protein hybridization. Fluorescence titration of the probe with increasing HSA (Figure S1a,b) resulted in a dose-dependent enhancement of Naph emission with good linearity, suggesting the inclusion of Gal-NSp to the protein.

ABSTRACT: Despite the rapid development of imaging techniques, precise probe localization and modulation in living cells is still a challenging task. Here we show that the simple hybridization between a photochromic fluorescent glycoprobe and human serum albumin (HSA) enables a unique fluorescence “double-check” mechanism for precisely localizing and manipulating probe molecules in living cells. Docking of a carbohydrate-modified naphthalimide (Naph)-spiropyran (SP) dyad to a hydrophobic pocket of HSA produces the glycoprobe-protein hybrid, causing the protein conformation to fold as determined by small-angle X-ray scattering. We show that the Naph and merocyanine (the photoisomer of SP) fluorescence of the resulting hybrid can be reversibly switched by light in buffer solution and in target cells overexpressing the carbohydrate receptor.

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patiotemporal sensing of crucial biomarkers relevant to physiologically and pathologically important cellular pathways using exquisitely designed imaging agents,1 e.g., smallmolecular fluorescent probes2 and luminescent materials,3 can facilitate the development of chemical biology and disease diagnosis.4 Despite the elegant achievements made in this area, several issues still remain. For instance, the conventional probes developed are passively internalized by cells, limiting their specificity for a target cell/tissue. On a second front, the majority of the known fluorescent probes only emit a single fluorescence, which can be interfered by the biological autofluorescence, producing false-positive signals. Therefore, the development of smart probes to enhance the precision of bioimaging remains a challenging task. Equipment of probe molecules with a targeting agent (e.g., peptides, aptamers and sugars),5,6 the use of near-infrared fluorophores,7 and the development of multiplexed bioimaging materials8 are among the popular strategies to tackle these issues. Photochromism offers an alternative opportunity to enhance the precision of bioimaging because photochromophores can be light-controlled to reversibly emit fluorescence. This enables the probe’s signal to be differentiated from autobiofluorescence. Although fluorescent diarylethene derivatives have been employed in high-resolution bioimaging,9 the majority of current photochromophores are nonemissive because of the © 2018 American Chemical Society

Received: May 23, 2018 Published: June 25, 2018 8671

DOI: 10.1021/jacs.8b05425 J. Am. Chem. Soc. 2018, 140, 8671−8674

Communication

Journal of the American Chemical Society

Figure 2. (a) X-ray scattering pattern of HSA. (b) Fitting atomic model of HSA superimposed with Gal-NSp/HSA hybrid (1:1 complex; NMA simulation; crystal structure used to fit the data: PDB id 1n5u). (c) X-ray scattering pattern of Gal-NSp/HSA hybrid (1:1 complex). (d) Interatomic distance distribution function, P(r), of the X-ray scattering patterns of HSA and the Gal-NSp/HSA hybrid (1:1 complex).

Figure 1. (a) Light-controlled structural transformation of the photochromic glycoprobe. (b) (I) singlet fluorescence switching and (II) duplexed fluorescence “double-check” switching enabled by host−guest hybridization upon alternate UV/vis light irradiation. (c) Targeted “double-check” fluorescence cell imaging upon alternate UV/vis light irradiation.

inclusion. The SAXS data corroborates the hybridization between HSA and Gal-NSp, leading to protein folding. Next, we tested the photochromic response of Gal-NSp/ HSA alone and the hybrid in a 99.5% phosphate buffer saline (PBS) solution. While the fluorescence “on−off” switching of Naph (green channel) was achieved by alternate UV/vis irradiation (Figure S3a−c), no obvious fluorescence emission from merocyanine was observed (Figure S3a−c, red channel). Though the Naph fluorescence (green channel) could be gradually enhanced in PBS after hybridization with HSA (Figure S1a), we similarly determined that the photocontrolled green-channel fluorescence switching between Gal-NSp/HSA and Gal-NMr/HSA was amplified (Figure 3a−c), which could be ascribed to the enhanced photochromic activity of SP/MR encapsulated in the hydrophobic cavity of HSA (Figures S4 and S5). The photochromic quantum yields of the Gal-NSp/ HSA hybrid were determined to be Φsp‑mr = 7.25% and Φmr‑sp = 17.21% (Table S2), which are higher than those of Gal-NSp alone in an aqueous solution.14 Furthermore, we observed that the spiropyran fluorescence was activated upon inclusion into HSA, leading to an additional “off−on” red-fluorescence switching in the aqueous solution (Figure 3a−c, red channel). Moreover, the Gal-NSp/HSA hybrid exhibited good stability over a pH range of 4−9 (Figure S6). We further tested whether the duplexed fluorescence modulation of the Gal-NSp/HSA hybrid could be achieved in living cells. We used a hepatoma cell line (Hep-G2) that overly expresses the asialoglycoprotein receptor (ASGPr, galactose-selective) for fluorescence imaging. A clear duplexed fluorescence switching in Hep-G2 cells (Figure 4a,b) was observed. Upon UV irradiation, while the green-channel fluorescence was suppressed, the red-channel fluorescence was enhanced. In contrast, the subsequent vis light irradiation recovered the green-channel fluorescence but quenched the red-channel fluorescence. This duplexed fluorescence “blink-

This is in agreement with previous observations that association of a fluorophore with HSA resulted in an enhanced fluorescence emission.15 The glycoprobe−HSA binding ratio was determined to be 1:1 according to the fluorescence Job’s plot (Figure S1c). In addition, the emission of the probe largely decreased with the addition of trypsin, suggesting the disassociation between Gal-NSp and HSA because of protein degradation (Figure S1d). The ITC results gave positive ΔH and ΔS values (Table S1), suggesting a hydrophobic interaction between the probe and protein (K = 3.69 × 103 M−1).16 To determine the association position of the glycoprobe, a replacement assay was carried out using albumin-binding drugs, ibuprofen and phenylbutazone that are known to bind the IIIA and IIA domain of HSA, respectively.17 The selective replacement of Gal-NSp by ibuprofen suggests the preferential localization of the probe at around IIIA domain of HSA (Figure S2). To better analyze the glycoprobe-protein hybridization, SAXS was used (Figure 2).18 We obtained the ab initio lowresolution models based on the single merged X-ray scattering pattern of the homogeneous solution of HSA (Figure 2a) and a 1:1 Gal-NSp/HSA hybrid (Figure 2c). The 3D SAXS model of probe−HSA hybrid (red, Figure 2b) was then superimposed with that of HSA (light gray, Figure 2b). We observed that, around the IIIA region, the hybrid was conformationally more altered than that of HSA alone. This again suggests that the glycoprobe might dock to the IIIA domain of HSA. A comparison of the interatomic distance distribution function (P(r)) between the two models (Figure 2d) showed a slightly decreased maximum diameter (Dmax) of HSA after probe 8672

DOI: 10.1021/jacs.8b05425 J. Am. Chem. Soc. 2018, 140, 8671−8674

Communication

Journal of the American Chemical Society

targeting agent; Scheme S1) to prove the receptor-targeting ability of Gal-NSp/HSA hybrid. The hybrids were incubated with Hep-G2 and two control cell lines, HeLa (human cervical cancer) and A549 (human lung cancer) with minimal ASGPr expression.19 We determined that while the fluorescence blinking was only shown in Hep-G2 cells for Gal-NSp/HSA (Figure S8), all the cells tested exhibited the duplexed fluorescence switching with PEG-NSp/HSA that lacks the targeting agent (Figure S9). We also determined that pretreatment of Hep-G2 with increasing free D-galactose led to a gradually suppressed fluorescence of the hybrid (Figure S10), and that lowering the cell culture temperature to 4 °C inhibited the endocytosis of the hybrid (Figure S11).20 The reduction of ASGPr expression in Hep-G2 also suppressed the fluorescence of the hybrid (Figure S12), suggesting that the fluorescence imaging is receptor dependent. To test the stability of Gal-NSp/HSA on receptor binding, we examined the fluorescence change of the hybrid in the presence of peanut agglutinin (PNA, a lectin that selectively binds galactose).21 We observed that the addition of PNA did not suppress the fluorescence of the hybrid (Figure S13a), implying that the hybrid was not dissociated when bound to a carbohydrate receptor. Furthermore, the fluorescence enhancement of the glycoprobe bound to increasing HSA was determined to be slightly changed in a cell lysate of Hep-G2 (Figure S13b,c). A cell viability assay showed that the hybrids were not toxic to Hep-G2 cells, and that the UV/vis irradiations did not cause phototoxicity (Figures S14 and S15). To better analyze the intracellular localization of the GalNSp/HSA hybrid, confocal laser-scanning microscopy (CLSM) was used. We obtained a resolution-enhanced photoswitchable duplexed fluorescence imaging with CLSM upon alternate UV/vis irradiation (Figure 4c), clearly demonstrating the optically controlled, reversible “on−off” and “off-on” fluorescence blinking of Naph (green) and MR (red) inside the target cell, respectively. Because ASGPr is responsible for trafficking endocytosed species to the lysosome, colocalization of Gal-NSp/HSA with a lysosomal tracker (lysotracker) was carried out (Figure S16)22 using a mitochondrial tracker (mito-tracker) as control. The good colocalization of Gal-NSp/HSA fluorescence with that of lyso-tracker (Pearson’s correlation coefficient: 0.892) rather than mito-tracker (Pearson’s correlation coefficient: 0.529) suggests the predominant localization of the hybrids in the lysosome. To conclude, we have developed a glycoprobe−protein hybrid based on the simple host−guest assembly between a photochromic glycoprobe and a serum protein. The assembly activated the aqueous fluorescence of the photochromophore, thus enabling a unique duplexed fluorescence blinking manner for precision bioimaging because the dual fluorescence channels can be reversibly tuned by light intracellularly. We note that, to the best of our knowledge, the use of host−guest assembly for inclusion of a ligand-modified photochromic probe into a serum protein, leading to light-controllable duplexed bioimaging, is unprecedented. We postulate that this strategy can also be extended toward the simple construction of other light-contolled functional imaging tools through the assembly between other photochromic compouds and naturally existing proteins.

Figure 3. (a) Fluorescence intensity of Gal-NSp/HSA (5/100 μM) upon UV light irradiation with time (0−120 s; interval: 20 s) excited by 440 nm (green) or 550 nm (red). (b) Fluorescence intensity of Gal-NMr/HSA (5/100 μM) upon vis light irradiation with time (0− 300 s; interval: 60 s) excited by 440 nm (green) or 550 nm (red). (c) Fluorescence intensity cycling between Gal-NSp/HSA (I530; excitation: 440 nm; UV irradiation time: 120 s) and Gal-NMr/HSA (I628; excitation: 550 nm; vis irradiation time: 300 s).

Figure 4. (a) Fluorescent imaging with Gal-NSp/HSA (10 μM/100 μM) of Hep-G2 cells upon alternate UV/vis irradiation (green/red channel excitation: 460−490/520−550 nm; emission: 500−550/ 580−650 nm; scale bar = 100 μm). The cell nuclei were stained by Hoechst 33342. Error bars represent S. D. (n = 3). Intracellular fluorescence intensity switching between (b) Gal-NSp/HSA and GalNMr/HSA upon alternate UV/vis irradiation. (c) UV/vis cycling of the two imaging channels of Gal-NSp/HSA and Gal-NMr/HSA visualized by confocal laser-scanning microscopy (green/red channel excitation: 440/579 nm; emission: 535/603 nm; scale bar = 25 μm).

ing” was repeatedly achieved in Hep-G2 cells through alternate UV/vis irradiation (Figure 4b). In contrast, when cells were incubated solely with Gal-NSp, only the green channel was detectable (Figure S7). The absence of red channel is probably due to the low quantum yield of merocyanine intracellularly Next, we used a control probe (PEG-NSp/HSA hybrid)14 (polyethylene glycol [PEG] is used to replace the galactose 8673

DOI: 10.1021/jacs.8b05425 J. Am. Chem. Soc. 2018, 140, 8671−8674

Communication

Journal of the American Chemical Society

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b05425. Materials and methods, control experiments in buffer solution and additional cell-based assays (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] *[email protected] ORCID

Junji Zhang: 0000-0003-2823-4637 Xiao-Peng He: 0000-0002-8736-3511 Ben L. Feringa: 0000-0003-0588-8435 He Tian: 0000-0003-3547-7485 Author Contributions §

Equal contribution.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the NSFC (Nos. 21788102, 21722801, 21572058, 21402050 and 21420102004), the Programme of Introducing Talents of Discipline to Universities (B16017) and the Shanghai Rising-Star Program (16QA1401400 to X.-P.H.) for generous financial support. We are also grateful to Dr. Na Li of the National Center for Protein Sciences Shanghai and the staff from BL19U2 beamline of the Shanghai Synchrotron Radiation Facility for data collection and processing of SAXS.

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DOI: 10.1021/jacs.8b05425 J. Am. Chem. Soc. 2018, 140, 8671−8674