Development of a Silicon-Rhodamine Based Near-Infrared Emissive

Letter pubs.acs.org/ac

Development of a Silicon-Rhodamine Based Near-Infrared Emissive Two-Photon Fluorescent Probe for Nitric Oxide Zhiqiang Mao, Hong Jiang, Xinjian Song, Wei Hu, and Zhihong Liu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei 430072, China S Supporting Information *

ABSTRACT: Two-photon (TP) fluorescent probes are potential candidates for near-infrared (NIR) imaging which holds great promise in biological research. However, currently, most TP probes emit at wavelength 650 nm at the NIR region) TP fluorophores have been reported, which still suffer from deficiencies such as poor water solubility, low fluorescence quantum yield, or limited two-photon action cross section (Φδ).15−19 Therefore, it is still a great challenge and highly desired to develop TP probes with both NIR emission and satisfaction of comprehensive properties, which is a bottleneck to achieve “NIR-to-NIR” imaging. To address the above challenge, we herein propose a new “NIR-to-NIR” TP fluorescent probe, which for the first time uses a Si-rhodamine dye (SiR)20−23 as the scaffold and exhibits so far the best comprehensive performance. The probe was designed for nitric oxide (NO), a critical signal molecule in biological systems that has triggered tremendous interest in

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© 2017 American Chemical Society

chemistry and biology communities.24,25 Although there has been a considerable number of probes reported for NO,26−29 the tracking of subtle variation of this signal molecule in physiological and pathological events in situ, such as in tumor development, is still difficult and rarely achieved. This situation also highlights the pressing needs to develop new probes with higher capabilities in bioimaging. The probe SiRNO was constructed following a photoinduced electron transfer (PeT) principle. A Si-rhodamine derivate was used as the two-photon excitable fluorophore, and 4-methoxy-N-methylaniline moiety was selected as an NO recognition site based on the N-nitrosation reaction under aerobic condition.18,30 The two moieties were covalently linked by a short carbon−carbon single bond (Scheme 1), which facilitates the occurrence of the PeT process to quench the fluorescence of probe.31−36 Upon the N-nitrosation of the methylamino group by NO, the fluorescence of SiRNO would be lightened up due to the suppression of the PeT process. SiRNO was synthesized and characterized by 1H NMR, 13C NMR, and HRMS (Scheme S1 and Figures S9−S18). In order to explore the two-photon fluorescence properties of SiR based fluorophore, we first synthesized another dye SiROMe (Scheme S1). SiR-OMe has a similar structure to SiRNO but is highly fluorescent due to the absence of PeT. The photophysical properties of SiR-OMe were investigated in 10 Received: July 11, 2017 Accepted: August 28, 2017 Published: August 28, 2017 9620

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Letter

Analytical Chemistry Scheme 1. Design of a Silicon-Rhodamine Based “NIR-toNIR” Two-Photon Fluorescent Probe SiRNO for the Detection of NO in Cells and Mouse

only 90 s with 81-fold emission enhancement, indicating a quite fast response to the target. The probe shows a maximal absorption at 651 (ε = 1.17 × 105 M−1cm−1) and 653 nm (ε = 1.17 × 105 M−1cm−1), respectively, before and after reacting with excessive NO (Figure 1b). As expected, the probe itself exhibited very weak fluorescence emission at 672 nm (Φ = 0.002), as a consequence of the PeT process which was confirmed by the DFT calculation results (Figure S3). After the reaction with varying amounts of NO, a gradually increasing fluorescence enhancement at 672 nm (with a maximal Φ = 0.23) was observed (Figure 1c). The fluorescence intensity of the reaction product also exhibited a linear relationship with NO concentration in the range of 0.4−20 μM (R2 = 0.98), with a limit of detection (LOD) value of 14 nM according to the 3Sb/m criterion, where m is the slope for the range of linearity and Sb is the standard deviation of the blank (n = 11) (Figure 1d). Such a LOD represents a high sensitivity among the reported small-molecule probes for NO. Thereafter, we tested the response of the probe under two-photon excitation mode. The probe itself showed a maximal Φδ value of 0.14 GM, while the product of the probe-to-NO reaction showed a maximal Φδ value of 62 GM (Table S1). The remarkable enhancement of Φδ value (ca. 440-fold) suggests that SiRNO can be a sensitive TP probe for NO detection and imaging under two-photon microscopy. The specificity of SiRNO toward NO was examined by detecting its fluorescence response to a variety of biomolecules and other reactive species, including ascorbic acid (AA), dehydroascorbic acid (DHA), methylglyoxal (MGO), GSH, Cys, Hcy, H2O2, ClO−, ·OH, O2−, NO2−, and ONOO−. As shown in Figure S4, none of these interfering species caused obvious fluorescence enhancement. The result revealed pronounced selectivity of SiRNO toward NO, which is in agreement with our recent study using the N-nitrosation strategy for NO recognition.18 It was also verified that both SiRNO and its N-nitrosation product are pH insensitive in the range of 5.5−8.0 (Figure S5), showing that SiRNO is suitable for the detection of NO in the physiological pH range. We next looked into the reaction mechanism between SiRNO and NO. As an initial step, we made a model compound 4-methoxy-Nmethylaniline containing the recognition site of NO and studied its reaction with NO in PBS/CH3CN (2:1) solution. The results revealed that 4-methoxy-N-methylaniline can be easily converted into its N-nitrosation product with a high yield of 95%, which was confirmed by 1H NMR, 13C NMR, and MS analysis (Scheme S2 and Figures S19−S21). Then, the probe was allowed to react with NO in the PBS buffer, and the resulting solution was analyzed by LC-MS. The LC-MS analysis also clearly indicated that the major product was the Nnitrosation product (Figure S6), which rationalized our probe design. The cytotoxicity of SiRNO was evaluated by the tetrazolium based colorimetric (MTT) assay. As illustrated in Figure S7, SiRNO exhibits a low cytotoxicity to HeLa cells (cell viability >90%) at a concentration up to 15 μM. We thus attempted to utilize the probe to detect intracellular NO. To clarify the distribution of SiRNO in live cells, RAW 264.7 cells loaded with SiRNO were coincubated with MitoTracker Green (MTG, a commercial mitochondria marker) and LysoTracker Green (LTG, a commercial lysosome marker), respectively. As shown in Figure 2, there is a large fluorescence overlay between SiRNO and LTG with overlay coefficient of 0.98, while the overlay coefficient for SiRNO and MTG is 0.60. Unlike a

mM PBS solution (pH = 7.4, 0.5% DMSO). To our delight, SiR-OMe showed a NIR emission band centering at 670 nm, with a maximal active cross section (Φδ value, Φ is the fluorescence quantum yield and δ is the TP absorption cross section) of 75 GM under 820 nm excitation (Figures 1a and

Figure 1. (a) Two-photon active cross section spectra of SiR-OMe, SiRNO, and the reaction product of SiRNO and NO. (b) UV−vis absorption spectra of 5.0 μM SiRNO before and after reacting with 25 μM NO. (c) Fluorescence spectra of SiRNO with various NO concentrations (0−30 μM). (d) Plot of fluorescence intensity versus NO concentration (0−30 μM). Inset: the linear relationship between fluorescence intensity and NO concentration in the range of 0.4−20 μM. All data were measured in 10 mM PBS buffer (pH = 7.4, 0.5% DMSO). Reaction time: 30 min.

S1). This Φδ value is comparable to that of most current TP fluorophores, standing for a high enough brightness in imaging experiments. Besides, the dye SiR-OMe also has good water solubility, which is always a concern of long-wavelength emissive dyes. It suggests that the Si-rhodamine scaffold might be an ideal platform to develop “NIR-to-NIR” probes. We then examined the response of the probe SiRNO to NO. To start, the fluorescence change of SiRNO upon reacting with excessive NO as a function of time was checked. As shown in Figure S2, the fluorescence intensity reached the plateau within 9621

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Analytical Chemistry

Figure 4. TP images of the NO production in RAW 264.7 cells. (a) Cells were incubated with SiRNO for 30 min. (b) Cells were pretreated with NO stimulants (0.5 mg/mL L-Arg, 20 μg/mL LPS, and 150 U/mL IFN-γ) for 6 h and subsequently incubated with SiRNO for 30 min. (c) Cells were pretreated with NO stimulants and L-NNA, followed by incubation with SiRNO for 30 min. (d) Cells were pretreated with NO stimulants for 6 h, followed by incubation with 0.5 mM Carboxy-PTIO for 30 min, and then incubated with SiRNO. (e) Relative fluorescence intensity of images a−d. The fluorescence emission was collected at 650−750 nm upon the excitation of 820 nm. Scale bar: 20 μm.

Figure 2. Colocalization images of SiRNO with MitoTracker Green (a−c) and LysoTracker Green (d−f) in RAW 264.7 cells. The SiRNOloaded cells were coincubated with 200 nM MitoTracker Green and 100 nM Lysotracker Green for 1 h and imaged, respectively. The excitation wavelengths were 633 nm (SiRNO) and 488 nm (MTG and LTG), and the corresponding emission was collected at 650−750 and 500−600 nm. Scale bar, 10 μm.

previously reported Si-rhodamine dye that targeted mitochondria due to the lipophilic cationic dye structure,11 our results implied that SiRNO is mainly localized in lysosomes. This may be attributed to the inclusion of the alkaline secondary aniline moiety (ArNHMe) in the molecule, which tends to accumulate in lysosomes.37,38 The ability of SiRNO to detect NO in live cells was first verified through capturing exogenously supplied NO, where the SiRNO stained HeLa cells were incubated with various concentrations (0, 20, 50 μM) of NO solution. As shown in Figure 3a, the SiRNO loaded cells with no external addition of

fluorescence was observed due to the recognition of the generated NO by SiRNO (Figure 4b). To gain more information, two more groups were further tested, in which the generation of intracellular NO was down regulated by different means. In one group, the cells were pretreated with the NO stimulants together with 20 μM L-NG-nitroarginine (LNNA, a well-known inducible NO synthase (iNOS) inhibitor) for 6 h before loading the probe and imaging. In this case, the intracellular fluorescence signal (Figure 4c) was suppressed, because of the inhibited generation of NO. In the other group, the cells were pretreated with the NO stimulants and 0.5 mM Carboxyl-PTIO42 (a commercial NO scavenger). As expected, the intracellular fluorescence (Figure 4d) was also largely decreased (compared with Figure 4b), as a result of the scavenging of the as-generated NO. All these results have vividly proven that SiRNO is sensitive enough to monitor NO at low concentrations in live cells. The success of detecting the target molecule in live cells inspired us to further use our probe in animal tissues. Using a mouse liver tissue as an example, we first examined the ability of SiRNO to stain tissue samples. After incubating the liver tissue with SiRNO for 1 h, followed by incubation with external NO solution for 0.5 h, the sample was observed using the zscan mode of the two-photon microscope, and the fluorescence intensities at different depths were recorded. As shown in Figure S8, the 3D reconstructed TP images revealed that the probe can well stain the tissue sample and the imaging depth was up to 200 μm, approving the advantage of NIR imaging. It was documented that the induction of NO in tumor cells may result in some protective effects by mediating cell proliferation, survival, and resistance, and the NO can be produced by the highly expressed iNOS and activated macrophages during inflammatory response.43,44 In this context, it is meaningful to monitor the generation of NO in the development of tumor, which has been seldom achieved before. To this end, we employed the probe in three groups of mice, i.e., one group of a normal mouse as the control and two groups of tumor-bearing mice. Each group of mice was subcutaneously injected with 150

Figure 3. TP images of 5.0 μM SiRNO-stained HeLa cells incubated with various concentrations of NO for 30 min. (a) 0 μM NO; (b) 10 μM NO; (c) 20 μM NO. (d) Relative fluorescence intensity of images a−c. The fluorescence emission was collected at 650−750 nm upon the excitation at 820 nm. Scale bar, 20 μm.

NO showed rather faint NIR fluorescence. Upon supplying NO to the SiRNO loaded cells, the intracellular fluorescence intensity was dramatically elevated, and the intensity is obviously dependent on the NO concentration (Figure 3b−d). We further demonstrated the capability of SiRNO to track the subtle change of endogenously generated NO in live cells upon stimulation, which is more challenging since the signal molecule is always at very low concentration levels (nM to pM).39 RAW 264.7 cells were chosen for the test as they generate NO when stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ).40,41 Similar to the above case of HeLa cells, the RAW 264.7 cells loaded only with the probe showed weak fluorescence (Figure 4a). In contrast, when the SiRNO stained cells were pretreated with the NO stimulants (0.5 mg/ mL L-arginine, the substrate for nitric oxide synthase, 20 μg/mL LPS, and 150 U/mL IFN-γ), much brighter intracellular 9622

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μL of 1.0 mM probe solution 1.0 h before the tissues were harvested and imaged. As shown in Figure 5a, the tissue of the

Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b02697. Materials and apparatus, experimental detail, synthesis and characterization, spectroscopic properties of SiRNO, cytotoxicity assay, 3D reconstructed TP images of mouse liver tissue, and NMR and MS spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 86-27-8721-7886. Fax: 86-27-6875-4067. ORCID

Zhihong Liu: 0000-0003-1500-9342 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21625503, 21535005).

Figure 5. TP images of the NO production in situ in the tumor tissues of a xenograft mouse model. A normal mouse and tumor-bearing mice were injected with 150 μL of 1.0 mM SiRNO 1.0 h before the tissues were harvested and imaged under a two-photon microscope. (a) Tissue imaging of the normal mouse. (b) Tissue imaging of xenograft tumor for 2 weeks. (c) Tissue imaging of xenograft tumor for 4 weeks. (d) The tumor-bearing mouse was injected with 150 μL of 1.0 mM SiRNO and 1.0 mM Carboxyl-PTIO, and then, the tissues were harvested and imaged. (e) The relative fluorescence intensities of images a−d. The fluorescence emission was collected at 650−750 nm upon the excitation at 820 nm. Scale bar, 100 μm.

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control exhibited weak NIR fluorescence (similar to the cell experiments), while the tissues of tumor-bearing mice showed much brighter fluorescence (Figure 5b,c). After the xenograft mice were grown for 2 and 4 weeks, the fluorescence intensity of the tumor tissues was increased by 2.5- and 4.4-fold, respectively. To further verify that the increased fluorescence signals were induced by NO, another tumor-bearing mouse was tested as the control, which was injected with probe and Carboxyl-PTIO (NO scavenger). Similar to the NO detection in live cells, the fluorescence signal of the tumor tissue was sharply decreased (Figure 5d,e). The results evidently prove that the fluorescence enhancement was a result of the reaction of the probe with NO. Although this was just a simple proof-ofconcept test, the results have unambiguously confirmed that the probe SiRNO can be a practical tool for NO detection in biological and biomedical applications. In conclusion, we have developed a Si-rhodamine based TP fluorescent probe SiRNO for NO detection. The “NIR-to-NIR” probe possesses favorable two-photon properties and exhibits high sensitivity and selectivity for NO. The probe shows a limit of detection of 14 nM for NO in a solution assay. It is able to recognize NO in live cells, thus tracking the subtle variation of endogenously generated NO upon various stimulations. The probe can be used to monitor NO in the development of tumor in a xenograft mouse model. To the best of our knowledge, SiRNO is the first “NIR-to-NIR” probe using a Si-rhodamine scaffold as the fluorophore. The work not only affords a practical tool for NO imaging but also proposes a new platform for the design of NIR emissive TP probes. 9623

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