Alkaline Phosphatase-Initiated Sensitive Responsiveness of Activatable Probes to Hydrogen Sulfide for Accurate Cancer Imaging and Differentiation

Open AccessCCS ChemistryCOMMUNICATION7 Dec 2022Alkaline Phosphatase-Initiated Sensitive Responsiveness of Activatable Probes to Hydrogen Sulfide for Accurate Cancer Imaging and Differentiation Rongchen Wang, Kai Yin, Muye Ma, Tianli Zhu, Jinzhu Gao, Jie Sun, Xuemei Dong, Chengjun Dong, Xianfeng Gu, He Tian and Chunchang Zhao Rongchen Wang Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237 , Kai Yin Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203 , Muye Ma Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203 , Tianli Zhu Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237 , Jinzhu Gao Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237 , Jie Sun Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237 , Xuemei Dong Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237 , Chengjun Dong Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237 , Xianfeng Gu *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203 , He Tian Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237 and Chunchang Zhao *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237 https://doi.org/10.31635/ccschem.022.202201971 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Optical imaging with molecular probes is becoming an essential tool for advancing biological research and clinical applications. However, most currently available molecular probes show limited sensitivity, specificity, and accuracy due to their typical responsiveness to a single stimulation for biomarker-based imaging. In this study, we develop a novel molecular probe that shows alkaline phosphatase (ALP)-instructed sensitive responsiveness to hydrogen sulfide for accurate cancer imaging and differentiation. This designed probe in an aggregated state under physiological conditions bears negatively charged surfaces, giving poor optical response to H2S. The ALP-mediated dephosphorylation reaction yields an assembled product with a positively charged surface, affording significantly aggregation-enhanced responsiveness to H2S with light-up NIR fluorescence at 755 nm. Such charge reversal of assembled probe from negative to positive plays a vital role in allowing precise visualization and differentiation of cancers based on differences in ALP upregulation and H2S content. We envisage that our charge-reversal strategy for multiple-parameter-activated molecule probes will facilitate boosting the specificity and precision of cancer imaging. Download figure Download PowerPoint Introduction Optical imaging with molecular probes is becoming an essential tool for advancing biological research and clinical applications.1–3 To date, various activatable molecular imaging probes have been developed to detect biological targets of interest in vitro and in vivo.4–7 These reported probes are generally designed to be tumor microenvironment (TME)-responsive molecules that emit various signals upon interaction with cancer-related biomarkers including cancer-associated enzymes, hypoxia, acidic pH, high levels of reactive species, and so on, thus facilitating targeted cancer imaging.8–17 However, most currently available molecular probes typically respond to a single stimulation for biomarker-based imaging,18–20 showing limited sensitivity, specificity, and accuracy. Such unsatisfactory outcomes largely restrict the practical applications for bioimaging.21 Recently, to improve the precision of cancer imaging, molecular probes that can directly target multiple cancer-associated biomarkers have been constructed.22–31 The activation by the presence of multiple cancer-associated biomarkers is capable of boosting the specificity and precision of cancer imaging. Although multiple-parameter-activated molecule probes are now available, pH/enzymes or pH/redox have typically been exploited to activate these probes.32,33 Considering the complicated TME, it is highly desirable to develop versatile molecular probes responsive to various multifactor stimulations for accurate tumor imaging in vivo. Because alkaline phosphatase (ALP) is highly expressed on the cancer cell membrane while increased H2S production leads to tumorigenesis in many cancers,34–39 molecular probes that successively respond to ALP and H2S should unambiguously shed light on precise cancer imaging. Although numerous studies have individually explored how to monitor phosphatase activity and H2S in vitro and in vivo,40–47 reports about ALP/H2S-activatable probes developed for evaluating the correlation of phosphatase activity and H2S are rare. More recently, a single fluorescent probe has been designed to append two different reactive sites responsive to phosphatase and H2S simultaneously, showing different spectral signals.48 Unfortunately, no in vivo imaging was explored, presumably due to the requirement of the short excitation wavelength that limits the bioimaging applications. In vivo visualization and differentiation of cancers based on differences in ALP upregulation and H2S content represent an unmet need. Herein, we report a molecular probe BOD-Py-PA that shows ALP-instructed sensitive responsiveness to hydrogen sulfide for accurate cancer imaging and differentiation. In our design, a new charge-reversal strategy was employed to get a multiple-parameter-activated molecule probe. As depicted in Scheme 1, the rationally designed probe consists of (1) monochlorinated boron-dipyrromethene (BODIPY) as the H2S-responsive unit49 and (2) l-Phenylalanine methyl ester linker capped with phosphate group (–PO3H), the ALP recognition, appending to BOD-Py via an amide bond. The amphiphilic structure enabled BOD-Py-PA to self-assemble into a nanoprobe in buffer solution. Interestingly, the negatively charged surface of assembled BOD-Py-PA initially prevented anion HS− (main forms of H2S in physiological pH) from accessing the probe, giving poor optical response to H2S. In sharp contrast, an efficient dephosphorylation reaction was initiated upon introduction of ALP, yielding the assembled product BOD-Py-Phe with a positively charged surface. As a result, the charge reversal process enriched the local concentrations of HS− close to the H2S-responsive unit, thus showing significant aggregation-enhanced responsiveness (AER) to H2S. Such ALP-instructed sensitive responsiveness to hydrogen sulfide gave rise to bright near-infrared (NIR) emissions lighting up, allowing precise visualization and differentiation of cancers based on differences in ALP upregulation and H2S content. We envisage that such a charge-reversal strategy for multiple-parameter-activated molecular probes will facilitate the boosting of the specificity and precision of cancer imaging. Scheme 1 | Schematic illustration of BOD-Py-PA with ALP-initiated sensitive responsiveness to H2S for cancer imaging. Download figure Download PowerPoint Results and Discussion The dual-parameter-activated probe BOD-Py-PA was synthesized according to the procedure as depicted in Supporting Information Scheme S1. And the dephosphorylated product BOD-Py-Phe, as a control probe, was also prepared to validate the vital role of the phosphate group in such a multiple-parameter-activated design strategy ( Supporting Information Scheme S2). All the chemical structures were identified by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS) spectrometry. Since BOD-Py-PA contains the phosphate group (–PO3H) and the H2S-responsive unit, we first investigated the optical response of probe to ALP and H2S in the CH3CN/Tris mixtures with fw = 90% (volume fraction of water), respectively. Upon incubation with 100 U/L ALP, the fluorescence intensity at 578 nm displayed a time-dependent quenching in 30 min ( Supporting Information Figure S1a). Moreover, it was found that the degree of such fluorescence quenching depended on the ALP concentration (0–100 U/L) ( Supporting Information Figure S1b). These results can be ascribed to the ALP-mediated dephosphorylation, which transformed BOD-Py-PA into dephosphorylated product BOD-Py-Phe and subsequently enhanced the intermolecular aggregation, thus leading to the intermolecular-aggregation-caused quenching (ACQ). The ALP-activated dephosphorylation was confirmed by HRMS analysis ( Supporting Information Figure S2). In addition, at fw = 90%, it was further identified that the fluorescence intensity at 578 nm of BOD-Py-PA was stronger than that of chemically synthesized BOD-Py-Phe ( Supporting Information Figure S3), revealing that the introduction of phosphate group suppressed the intermolecular ACQ effect. Notably, there was no obvious fluorescence quenching when BOD-Py-PA was incubated with other representative enzymes ( Supporting Information Figure S4), demonstrating high selectivity of BOD-Py-PA for ALP. Interestingly, when BOD-Py-PA was treated with 100 μM NaHS, minimal optical changes were observed (Figure 1a,b and Supporting Information Figure S5), indicating that BOD-Py-PA cannot be a suitable tool for direct detection of H2S. Figure 1 | (a) and (b) Time-dependent absorption and fluorescence responses of BOD-Py-PA (10 μM) to NaHS (100 μM) at fw = 90%, λex = 640 nm. (c) and (d) Time-dependent absorption and NIR fluorescence responses of BOD-Py-PA (10 μM) to NaHS (100 μM) after being pretreated with ALP (100 U/L) for 30 min. (e) and (f) Normalized absorption and fluorescence responses of BOD-Py-PA (10 μM) to NaHS (100 μM) after being pretreated with different ALP concentration (0, 1, 5, 50, and 100 U/L) for 30 min, λex = 640 nm. Data were recorded 120 min after addition of NaHS. Download figure Download PowerPoint Since ALP initiated the production of BOD-Py-Phe that also retained the H2S-responsive unit, we then interrogated the capability of ALP-mediated dephosphorylation for instructing sensitive responsiveness to H2S. Accordingly, we evaluated the optical changes of BOD-Py-PA when successively treated with ALP and H2S. First, BOD-Py-PA (10 μM) was incubated with different concentrations of ALP for 30 min at fw = 90%, and then 100 μM NaHS was introduced to the buffer solutions. Surprisingly, we found that BOD-Py-PA exhibited significantly enhanced responsiveness to H2S after incubation with ALP. As shown in Figure 1c–1f and Supporting Information Figure S6, with the increase of ALP concentration from 0 to 100 U/L, the absorption band around 656 nm enhanced sharply, accompanied by the decrease of original absorption band at 528 nm in the presence of NaHS (100 μM). When the excitation wavelength was 640 nm, a new NIR emission at 755 nm was activated. More importantly, compared with the NIR fluorescence activated by single H2S, successive treatment of BOD-Py-PA with 100 U/L ALP and 100 μM NaHS could produce about 4.3-fold fluorescence enhancement, indicating that probe BOD-Py-PA might be an ideal candidate for in vivo imaging due to efficient reduction of nonspecific activation caused by a single stimulation. Such ALP-instructed sensitive responsiveness to H2S could be attributed to the conversion of BOD-Py-PA to BOD-Py-Phe via the ALP-mediated dephosphorylation, followed by the improved thiol-halogen nucleophilic aromatic substitution reaction (SNAr) to afford thiol-substituted product BOD-Py-Phe-SH, proven by HRMS analysis ( Supporting Information Figure S7). To acquire insight into the mechanism of ALP-initiated sensitive responsiveness, the optical changes of the control probe BOD-Py-Phe to H2S were next explored. Interestingly, BOD-Py-Phe showed enhanced responsiveness to H2S as the volume fraction of water (fw) increased from 50% to 90% (Figure 2a and Supporting Information Figure S8). At fw = 90%, H2S activated the best optical changes, consistent to the ALP-instructed responsiveness of BOD-Py-PA to H2S. However, negligible optical changes were observed at fw = 50%. It is also worth noting that minimal optical enhancement was initiated when BOD-Py-PA was successively treated with ALP and NaHS at fw = 50% (Figure 2b). ALP-instructed optical response of BOD-Py-PA to H2S was consistent with the features of previous monochlorinated BODIPY probes upon single-stimulation, which showed unprecedented AER to H2S.43 In order to verify the AER, the molecular states of BOD-Py-PA and BOD-Py-Phe were confirmed by dynamic light scattering (DLS) characterization. At fw = 50%, no DLS signals were detectable, indicating that both probes were in the molecularly dissolved state. At fw = 90%, BOD-Py-PA and BOD-Py-Phe were found to form aggregates with average diameters of 56 ± 15 nm, 107 ± 20 nm, respectively (Figure 2c). Of note, assembled BOD-Py-PA showed good stability in buffer solutions for a long time ( Supporting Information Figure S9). The intermolecular aggregation by BOD-Py-PA formed relatively small particles, which proved the introduction of anion phosphate group suppressed the intermolecular interaction. Interestingly, an enhanced aggregation was observed upon the treatment of BOD-Py-PA with ALP, which was caused by the ALP-mediated dephosphorylation. Obviously, BOD-Py-Phe showed AER to H2S. In contrast, BOD-Py-PA showed negligible H2S-activated optical changes both at fw = 50% and 90% without ALP-pretreatment ( Supporting Information Figure S10), indicating the important role of phosphate group (–PO3H) in modulating the responsive capability of BOD-Py-PA to H2S. Given the deprotonation feature of phosphate group (pH ≧ 3),50 the assembled BOD-Py-PA presumably showed negatively charged surface in buffers, which prevented anion HS− from access to the probe and thus gave poor optical response to H2S. On the contrary, the dephosphorylated BOD-Py-Phe formed aggregates with a positively charged surface that enriched the local concentrations of HS− close to the reaction site and thus facilitated the SNAr reaction, showing significantly AER to H2S. As expected, the mean zeta potential values characterization confirmed such an inference (Figure 2d). Undoubtedly, the assembled BOD-Py-PA with negatively charged surface in buffer solution afforded ALP-initiated dephosphorylation to form assembled BOD-Py-Phe with a positively charged surface. Such a charge reversal process led to ALP-instructed sensitive responsiveness to H2S (Figure 2e), making probe BOD-Py-PA a good candidate for precise visualization of cancers based on differences in ALP upregulation and H2S content. Figure 2 | Normalized absorption responses of (a) BOD-Py-Phe (10 μM) and (b) BOD-Py-PA (10 μM, pretreated by 100 U/L ALP) to NaHS (100 μM) in CH3CN/Tris mixtures with various fw. Data were recorded 120 min after addition of NaHS. (c) DLS characterization of BOD-Py-PA (10 μM, pretreated with or without ALP) and BOD-Py-Phe (10 μM) at fw = 90%. (d) Mean zeta potential of BOD-Py-PA and BOD-Py-Phe at fw = 90%. (e) Proposed mechanism of ALP-initiated sensitive responsiveness of BOD-Py-PA to H2S. Download figure Download PowerPoint To confirm that the enhanced responsiveness to H2S was indeed initiated by ALP, H2S-induced optical changes were then examined after the pretreatment of BOD-Py-PA with other enzymes. As expected, only ALP but not other interfering species could obviously augment H2S-activated NIR fluorescence under identical conditions ( Supporting Information Figure S11a). Of note, such fluorescence enhancement was greatly suppressed upon the addition of Na3VO4 (an ALP inhibitor) ( Supporting Information Figure S11b). Importantly, BOD-Py-PA still maintained a good responsiveness to H2S after incubation with ALP in 100% Tris buffer ( Supporting Information Figure S12), indicating its good performance in living systems. Furthermore, the control probe BOD-Py-Phe exhibited a good selectivity toward H2S, and minimal fluorescence enhancement was triggered by other interfering species ( Supporting Information Figure S13). The NIR fluorescence intensity at 755 nm showed a good linear correlation within the H2S concentration range from 0 to 50 μM ( Supporting Information Figure S14), and the detection limit was determined to be 60 nM, indicative of a good sensitivity for H2S. BOD-Py-PA was also found to afford good optical changes upon successive addition of ALP and H2S at different physiological pH (8.5–5.5) ( Supporting Information Figure S15). Such dramatic responsiveness was maintained when BOD-Py-PA was incubated with ALP and H2S at the same time ( Supporting Information Figure S16). Encouraged by the aforementioned promising results, we examined the capability of BOD-Py-PA for precise fluorescence visualization in living cells. Due to the expression of ALP and H2S, HeLa cells were chosen as the model.51,52 After demonstrating the low cytotoxicity of BOD-Py-PA ( Supporting Information Figure S17), HeLa cells were subsequently incubated with BOD-Py-PA for 2 h. Fortunately, bright NIR fluorescence signals were collected from living cells (Figure 3a). By contrast, when HeLa cells were pretreated with Na3VO4 for 1 h to inhibit cellular ALP activity, followed by the incubation with BOD-Py-PA for another 2 h, the fluorescence intensity decreased significantly (Figure 3b). Similarly, weak fluorescence in the cells was obtained (Figure 3c) by the pretreatment with aminooxyacetic acid (AOAA, a cystathionine-β-synthase (CBS) inhibitor), owing to the inhibition of the cellular H2S production. These results suggested that BOD-Py-PA was synergistically activated by ALP and H2S in living cells, affording light-up NIR fluorescence signals. Additionally, H2S-deficient human umbilical vein endothelial cells (HUVECs) were chosen as a control, to confirm the capability of BOD-Py-PA for differentiation between cancer cells and normal cells. As a result, negligible fluorescence signals could be obtained after treatment of HUVEC cells with BOD-Py-PA (Figure 3d). Notably, the activated NIR fluorescence intensity of BOD-Py-PA in HeLa cells was almost eightfold more than that in normal HUVEC cells. For comparison, we further evaluated the capability of dephosphorylated probe BOD-Py-Phe for imaging. As shown in Figure 3e, distinct NIR emission can be observed after incubation of HeLa cells with BOD-Py-Phe. Notably, due to the lack of the ALP recognition group, bright fluorescence signals were still found in Na3VO4-treated HeLa cells (Figure 3f). However, the fluorescence intensity was significantly attenuated by the addition of AOAA (Figure 3g), showing that BOD-Py-Phe was activated by single parameter H2S in living cells. Importantly, we found that the NIR fluorescence intensity of BOD-Py-Phe in HeLa cells was only 1.3-fold more than that in normal HUVEC cells, which was much lower than that of BOD-Py-PA (Figure 3h–3j). Collectively, these imaging experiments indicate that dual-parameter-activated BOD-Py-PA can efficiently improve the accuracy of imaging in living cells, reducing false positive or negative signals from nontargeted cells. Figure 3 | Confocal microscopy images of probes in living cells. (a) HeLa cells incubated with BOD-Py-PA (10 μM) for 2 h. HeLa cells pretreated with (b) 5 mM Na3VO4 or (c) AOAA (1 mM) for 1 h, followed by incubation with BOD-Py-PA for 2 h. (d) HUVEC cells incubated with BOD-Py-PA (10 μM) for 2 h. (e) HeLa cells incubated with BOD-Py-Phe (10 μM) for 2 h. HeLa cells pretreated with (f) 5 mM Na3VO4 or (g) AOAA (1 mM) for 1 h, followed by incubation with BOD-Py-Phe for 2 h. (h) HUVEC cells incubated with BOD-Py-Phe (10 μM) for 2 h. Scale bar: 10 μm. (i) and (j) Relative fluorescence intensities quantified from images in each group. Download figure Download PowerPoint Then we explored the ability of our probes for in vivo ALP-instructed sensitive responsiveness to H2S (Figure 4a,b and Supporting Information Figure S18). After subcutaneous injection of BOD-Py-PA into HeLa tumor-bearing mouse models, the tumor region displayed gradually enhancing NIR emission over time, which leveled off at 3 h. However, this fluorescence intensity was remarkably attenuated after the HeLa tumor was pretreated by Na3VO4. It was found that the NIR fluorescence signal in the HeLa tumor region was almost fourfold more than in Na3VO4-pretreated tumors. In contrast, when BOD-Py-Phe was injected into the tumor with or without Na3VO4 pretreatment, the light-up fluorescence signals had no significant difference. Next, in vivo biodistribution of BOD-Py-PA was further investigated by intravenous injection into tumor-bearing mice ( Supporting Information Figure S19). Detectable fluorescent signals could be observed at the tumor site at 1 h post injection. The ex vivo fluorescence imaging of tumor and major organs also evidenced an efficient accumulation of probe in tumor. These imaging results demonstrate that the activity of ALP determines the ability of BOD-Py-PA to detect endogenous H2S in vivo. Thus probe BOD-Py-PA is a promising tool to visualize and distinguish cancers based on differences in ALP upregulation and H2S content. Figure 4 | NIR fluorescence images of HeLa tumor-bearing mice at different time post-subcutaneous injection of (a) BOD-Py-PA and (b) BOD-Py-Phe into tumor-bearing mouse models with or without pretreatment of Na3VO4. Download figure Download PowerPoint Conclusion We have developed a dual-parameter-activated probe BOD-Py-PA for precise cancer imaging through successive activation by ALP and H2S. This designed probe was constructed by combining the AER mechanism with the charge reversal strategy. In the absence of ALP, the negatively charged surface of assembled BOD-Py-PA inhibited the response to H2S. Upon the ALP-mediated dephosphorylation process, assembled BOD-Py-PA with a negatively charged surface was efficiently converted into assembled BOD-Py-Phe with a positively charged surface. Such a process activated AER to H2S and generated increased light-up NIR fluorescence for imaging. Importantly, our designed probe with the capacity of ALP-initiated sensitive responsiveness to H2S showed high accuracy and great promise for in vitro and in vivo imaging and differentiation based on differences in ALP upregulation and H2S content. We expect that this dual-parameter-activated design approach can promote the development of optical probes for precisely targeted cancer imaging. Supporting Information Supporting Information is available and includes synthesis procedures, structure characterization of compounds, and supplementary figures. Conflict of Interest There is no conflict of interest to report. Funding Information This research was made possible as a result of generous grants from the National Natural Science Foundation of China (grant nos. 21874043, 22077030, and 21977018), the Shanghai Municipal Science and Technology Major Project (grant no. 2018SHZDZX03), and the China Postdoctoral Science Foundation (grant no. 2021M701196). Disclosures All animal experiments were carried out in compliance with the relevant laws and institutional guidelines for the Care and Use of Research Animals established by Fudan University which approved the experimental protocols and procedures. References 1. Li J.; Pu K.Development of Organic Semiconducting Materials for Deep-Tissue Optical Imaging, Phototherapy and Photoactivation.Chem. Soc. Rev.2019, 48, 38–71. Google Scholar 2. Niu L.-Y.; Chen Y.-Z.; Zheng H.-R.; Wu L.-Z.; Tung C.-H.; Yang Q.-Z.Design Strategies of Fluorescent Probes for Selective Detection Among Biothiols.Chem. 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