Engineering of Electrochromic Materials as Activatable Probes for

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Engineering of electrochromic materials as activatable probes for molecular imaging and photodynamic therapy Luyan Wu, Yidan Sun, Keisuke Sugimoto, Zhiliang Luo, Yusuke Ishigaki, Kanyi Pu, Takanori Suzuki, Hong-Yuan Chen, and Deju Ye J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10176 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 2, 2018

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Journal of the American Chemical Society

Engineering of electrochromic materials as activatable probes for molecular imaging and photodynamic therapy Luyan Wu†, Yidan Sun†, Keisuke Sugimoto‡, Zhiliang Luo†, Yusuke Ishigaki‡, Kanyi Pu§, Takanori Suzuki ‡,*, Hong-Yuan Chen†, and Deju Ye†,‖,* †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China ‡

Department of Chemistry, Faculty of Science, Hokkaido University, N10 W8, North-ward, Sapporo 060-0810, Japan § School ‖

of Chemical and Biomedical Engineering Nanyang Technological University, 637457, Singapore

Research Center for Environmental Nanotechnology (ReCent), Nanjing University, Nanjing, 210023, China

KEYWORDS: Electrochromic materials, activatable probe, H2S, molecular imaging, photodynamic therapy

ABSTRACT: Electrochromic materials (EMs) are widely used color-switchable materials, but their applications as stimuliresponsive biomaterials to monitor and control biological processes remains unexplored. This study reports the engineering of an organic π -electron structure-based EM (dicationic 1,1,4,4-tetraarylbutadiene, 12+) as a unique H2S-responsive chromophore amenable to build H2S-activatable fluorescent probes (12+-SPNs) for in vivo H2S detection. We demonstrate that EM 12+ with a strong absorption (500–850 nm) efficiently quenches different fluorescence (580, 700, or 830 nm) of fluorophores within 12+-SPNs, while the selective conversion into colorless diene 2 via H2S-mediated two-electron reduction significantly recovers fluorescence, allowing for non-invasive imaging of hepatic and tumor H2S in mice in real time. Strikingly, EM 12+ is further applied to design a near-infrared photosensitizer (12+-PSs-FA) with tumor-targeting and H2Sactivatable ability for effective photodynamic therapy (PDT) of H2S-related tumors in mice. This study demonstrates promise for applying EMs to build activatable probes for molecular imaging of H2S and selective PDT of tumors, which may arise the development of new EMs capable of detecting and regulating essential biological processes in vivo.

■ INTRODUCTION Smart activatable fluorescent probes that demonstrate enhanced fluorescence in response to a molecular target can offer high signal-to-noise ratios and real-time information, greatly improving sensitivity and specificity for molecular imaging.1-4 A number of activatable fluorescent probes capable of detecting diverse molecular targets at pathological conditions have shown promising results in early diagnosis of diseases.5-8 In particular, hydrogen sulfide (H2S)-activatable fluorescent probes have attracted considerable attention due to the essential roles of H2S in biology.9,10 H2S is identified as the third gasotransmitter in biology, which can elicit diverse physiological and pathological functions.11-13 Abnormal H2S levels are closely involved in various conditions, including liver cirrhosis, Alzheimer ’ s disease, hypertension, inflammation and cancers;14-16 accurate detection of H2S levels is thus invaluable for the study of H2S-related biological processes and disease diagnosis. To date, many

H2S-activatable fluorescent probes have been developed using H2S ’s unique chemical reactivity to trigger selective nucleophilic additions,17 azido18-23 and nitro group reduction,24,25 copper sulfide precipitation,26 or thiolysis reactions.27-29 These probes provide a useful tool for detecting H2S because of sensitive, non-invasive, nonradioactive and real-time capacities of fluorescence imaging. Nevertheless, most of them still suffer from limited tissue penetration, insufficient reaction kinetics and poor in vivo targeting ability, restricting the detection accuracy for H2S in living animals. In addition to fluorescence imaging, activatable photosensitizers (PSs) with enhanced photodynamic therapy (PDT) efficacy against cancers are another important research subject.30 Different to traditional “ always-on ” PSs that cause phototoxicity to noncancerous tissues, the activation of PSs for controllable reactive oxygen species (ROS) generation by a specific cancerassociated biomarker allows to specifically destroy

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tumor cells upon irradiation, largely reducing phototoxicity to normal tissues.31-34 In the past few years, activatable PSs being responsive to different biological signals such as pH,35 biothiols,36-40 ATP41 and proteases42-45 in cancers have received substantial attention, as the cancer-specific PDT can greatly improve treatment accuracy. Considering that H2S level is significantly increased in many cancers (e.g., colon, breast and ovarian cancers),46-48 the use of H2S-activatable PSs to control ROS release in cancer cells can offer an efficient means for precise cancer treatment. To date, PSs that are selectively activated by H2S are still quite few; there is only one H2Sactivatable PS consisting of zinc porphyrin-bridged metal organic framework nanoparticles (MOF NPs) has been reported.11 However, MOF NPs are lacking near-infrared (NIR) absorption and cancer targeting ability,

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compromising PDT efficacy in vivo. Therefore, it is necessary to develop H2S-activatable PSs that allow selective accumulation in cancers and remarkable ROS production under NIR light irradiation to elicit effective cancer PDT in living animals. Electrochromic materials (EMs) that display distinct color changes during an electrochemistry-induced electron transfer (redox) process have been widely used as optical memories and electrochromic devices, including autodimming mirrors, smart windows and electronic displays.49-51 Recent applications integrating EMs for redox flow battery and solar cells have prompted people to develop many prominent EMs such as transition metal oxides,52,53 viologens,54 conducting polymers55 and Prussian Blue systems.56 In addition, there is a growing interest of using EMs as optical sensors to track electron

Figure 1. Schematic illustration shows the engineering of electrochromic materials (EM 12+) as H2S-activatable fluorescent probes (12+-SNPs) for molecular imaging. (a) Chemical structure of dication EM 12+ and proposed H2S-mediated two-electron reduction into diene 2. (b) Absorption spectra and photographs (inset) of 12+ and 2 (PBS buffer, 1×, pH 7.4). (c) Preparation of 12+-SNPs and proposed mechanism of 12+-SNPs for H2S detection in vivo. 12+-SNPs were prepared via amphiphilic DSPE-PEG-assisted encapsulation of hydrophobic EM 12+ and semiconducting polymers. 12+-SNPs show low fluorescence (OFF) owing to the fluorescence quenching effect of EM 12+ via a FRET process. The H2S-triggered reduction of 12+ into colorless 2 within 12+-SNPs can eliminate the FRET process and recover fluorescence (ON), allowing for non-invasive imaging of hepatic or tumor H2S in living mice. (d) Chemical structures of three different semiconducting polymers, including PCPDTBT, PFDPP and MEH-PPV for the preparation of 12+-SNPs.


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transfer process.54 Thus far, however, these EMs with distinguished spectroscopic properties have not yet been explored for molecular imaging. We have previously reported a member of organic π-electron structures like dicationic 1,1,4,4-tetraarylbutadiene (EM 12+) that exhibit remarkable electrochromic behavior (Figure 1a).57 EM 12+ displays a violet color with strong absorption between 500 and 850 nm owing to two units of Michler ’s Hydrol Blue, which can be converted into nearly colorless diene (2) upon electrochemical two-electron reduction (Figure 1b). Given the reduction potential for converting 12+ into 2 (Ered = +0.18 V) is more positive than the oxidation potential of H2S (Eox = -0.476 V),58 we posit here that H2S may serve as a strong reductant capable of fast donating two electrons to reduce EM 12+ into 2. The resultant distinct decline in absorption among 500–850 nm can offer a novel means of constructing smart H2S-activatable fluorescent probes and PSs for non-invasive imaging of H2S and selective tumor PDT in live mice. In this work, we report the engineering of EM 12+ as a H2S-responsive chromophore, and demonstrate its ability to develop a class of H2S-activatable fluorescent probes (12+-SPNs) by doping into semiconducting polymer nanoparticles (SPNs) (Figure 1c). Within 12+-SPNs, the strong absorption of EM 12+ can serve as an effective quencher to turn off the SPNs fluorescence via a fluorescence resonance energy transfer (FRET) process. The subsequent reduction of 12+ into colorless 2 by H2S can eliminate the FRET process and recover fluorescence. Thus, 12+-SPNs show significant fluorescence enhancement toward H2S, enabling to rapidly detect H2S with high sensitivity and specificity. Using 12+-SPN830 that emits NIR fluorescence at 830 nm upon H2S activation, non-invasive and reliable monitoring of hepatic H2S levels in mice was realized in real time, revealing the upregulated expression of cystathionine γlyase (CSE) and enhanced production of H2S in lipopolysaccharide (LPS)-induced liver inflammation. In addition, a tumor-targeting and H2S-activatable fluorescent probe (12+-SNP830-FA) was developed for H2S-related tumors imaging upon systemic intravenous (i.v.) administration. Based on imaging results, EM 12+ was further applied to develop tumor-targeting and H2Sactivatable PSs (12+-PSs-FA) by replacing semiconducting polymers with organic PSs. 12+-PSs-FA showed preferable tumor accumulation and H2S-specific activation to produce ROS under an 808 nm laser irradiation, which was promising for effective PDT of tumors with negligible phototoxicity to normal tissues in mice. Notably, because EM 12+ possesses a broad and strong absorption that can theoretically quench multiple fluorescence within 500– 850 nm, H2S-activatable probes emitting diverse fluorescence can be created using different fluorophores. These smart probes can serve as promising tools to assess biological functions of H2S in vivo.

■ RESULTS

Characterization of the reaction between EM 12+ and H2S. We first investigated whether EM 12+ could be selectively reduced by H2S to form colorless diene 2 under physiological conditions. UV-Vis absorption and HPLC analysis showed that 12+ was reduced not only by NaHS (H2S donor) upon two-electron reduction, but also by other biologically relevant reducing agents (e.g., homocysteine (Hcy), L-cysteine (L-Cys), glutathione (GSH) and vitamin C) (Figure S1). However, dynamic measurement of the absorption in the course of reaction revealed a much faster reduction process (second-order reaction rate: k2 = 304 ± 8 M-1 s-1) for NaHS than other reductants, suggesting that H2S is a more efficient reductant for 12+ (Figure S1d). To improve the selectivity of 12+ toward H2S over other biological reductants, we employed an amphiphilic phospholipid polymer (DSPEPEG2000) to engineer 12+ into mono-disperse micellar nanoparticles (12+-NPs) (Figure S2a). Owing to the hydrophobic π-electron structure, EM 12+ could be easily encapsulated by the DSPE-PEG2000, affording 12+-NPs with high loading efficiency (Table S1). We postulated that the phospholipid layer on 12+-NPs would only allow small H2S (but not other biothiols) to diffuse into and react with 12+ inside nanoparticles, thus achieving high selectivity for H2S. Indeed, we found that only NaHS triggered the reduction of 12+ into 2 within 12+-NPs, inducing a fast and dose-dependent decline of absorption between 500 and 850 nm (Figure S2e-h). Moreover, both 12+ and 12+-NPs were quite stable in aqueous solution, with little change in absorption upon incubation (PBS, pH 7.4) for 14 days (Figure S3). We also noticed a high photostability of 12+-NPs (Figure S3c). These results demonstrated that EM 12+ within 12+-NPs could serve as an efficient H2S-responsive chromophore. Preparation of 12+-SNPs as H2S-activatable fluorescent probes. By leveraging the strong absorption and distinct color change in response to H2S, we then employed EM 12+ to prepare H2S-activatable fluorescent probes (12+-SNPs) by doping it into SPNs (Figure 1c). SPNs are newly emerging biocompatible fluorophores, which can offer reliable fluorescence for molecular imaging due to their excellent in vivo stability and high photostability.59-62 Three different semiconducting polymers (PCPDTBT, PFDPP, and MEH-PPV) with respective fluorescence at 830, 700, and 580 nm were chosen to prepare 12+-SNP830, 12+-SNP700, and 12+SNP580, respectively (Figure 1d and Table S1). The optimal ratio between the semiconducting polymers and 12+(BF4-)2 in 12+-SNPs was evaluated as 0.58 by weight (Figure S4), with a good reproducibility for the synthesis of 12+-SNPs (Table S2). As expected, the fluorescence of 12+-SNPs was remarkably quenched by 12+ via the FRET process (~15-fold in 12+-SNP830, ~60-fold in 12+-SNP700 and ~25-fold in 12+-SNP580) (Figure S5). The different quenching efficacy aligned well with the distinct spectral overlaps between SPN emissions and 12+ absorption (Figure 2a). Dynamic light scattering (DLS) analysis of 12+-SNPs demonstrated a good



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Figure 2. Characterization of 12+-SNP830. (a) UV−Vis absorption of EM 12+ (black) and fluorescence emission of MEH-PPV (blue), PFDPP (green) and PCPDTBT (red). (b) DLS result, (c) TEM (left) and AFM (right) images of 12+-SNP830. (d) Normalized absorption and (e) fluorescence spectra of 12+-SNP830 (28/48 µg/mL PCPDTBT/12+(BF4-)2) upon incubation with NaHS (350 µM) at r.t. for 0–10 min. Inset: The dark blue solution turned light green (PCPDTBT’s color) after incubation for 10 min. (f) Fluorescence spectra of 12+-SNP830 upon incubation with NaHS (0,10, 30, 55, 90, 125, 160, 190, 220, 240, 260, 350, 500 µM) at 37 ºC for 10 min. (g) Fluorescence spectra of 12+-SNP830 upon incubation with different reductants or ROS at 37 ºC for 10 min. 1: NaHS (350 µM); 2: LCys (1.25 mM); 3: GSH (10 mM); 4: Hcy (1 mM); 5: VC (1.25 mM); 6: DTT (1.25 mM); 7: BSA (10 µg/mL); 8: H2O2 (1 mM); 9: ClO− (1 mM); 10: O2.- (100 µM xanthine + 22 mU XO); 11: 1O2 (1 mM H2O2 + 1 mM ClO−); 12: ONOO− (1 mM NaNO2 +1 mM H2O2); 13: 300 µM t-BuOOH; 14: 300 µM CuOOH; 15: NADPH (100 μM); 16 Fe2+ (100 μM); 17: Cu+ (100 μM); 18: NO (Diathylamine NONOate, 100 μM); 19: CO (CORM-3, 100 μM). (h) Fluorescence spectra of human plasma, 12+-SNP830 or 12+-SNP830 incubating in human plasma with the addition of 0, 5, 10, 20, 30, 40 and 50 µM NaHS at 37 ºC for 10 min. (i) Plot of the fluorescence intensity and HS- concentration (0, x, x + 5, x + 10, x + 20, x + 30, x + 40, and x + 50) to determine the H2S concentration in human plasma. The 0 point was obtained by adding ZnCl2 into plasma to trap sulfide. λex = 650 nm.Values denote mean ± standard deviation (SD, n = 3).

mono-dispersity in aqueous solution with a mean hydrodynamic diameter of ~50 nm (Figure 2b and Table S1); transmission electron microscopy (TEM) and atomic force microscopy (AFM) analysis confirmed this finding(Figure 2c and S6). Notably, both DLS and fluorescence spectroscopic analysis revealed that 12+-SPNs were very stable in PBS buffer, with little leakage of EM 12+ observed after incubation in PBS buffer for 7 days (Figure S7). The response of 12+-SNPs to H2S was next investigated by incubating 12+-SNPs with 100 µM NaHS in PBS buffer. As presented in Figure 2d, absorption of 12+-SNP830 at 555 and 700 nm rapidly declined. The initially quenched

fluorescence of 12+-SNP830 at 830 nm increased concomitantly, with a maximum ~15-fold turn-on ratio observed after 10 min (Figure 2e). We then determined the apparent reaction rate (k2) of ~91.6 M-1 s-1 between 12+SNP830 and H2S (Figure S8), which was much faster compared to most reported H2S-activatable NIR fluorescent probes (Table S3). The subsequent incubation of 12+-SNP830 with varying NaHS concentrations for 10 min revealed a concentration-dependent fluorescence enhancement at 830 nm (Figure 2f), with a good linearity from 1.0–90 µM. The detection limit of 12+-SNP830 for H2S was identified as ~0.7 µM (blank + 3σ) (Figure S9).20 We also found that 12+-SNP830 exerted a similar activation ratio


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at pH ranging from 4.0–9.0 (Figure S10). Moreover, fast kinetics and high sensitivity toward H2S were also observed for 12+-SNP700 and 12+- SNP580, with detection limits of ~79 nM and ~0.14 µM, respectively (Figure S11 and S12). The subsequent examination of selectivity toward H2S revealed that only NaHS could activate 12+-SNP830 and induce substantially enhanced fluorescence at 830 nm (Figure 2g and S13), demonstrating that 12+-SNP830 was highly specific for H2S detection. The high specificity coupled with fast reaction kinetics and high sensitivity could allow 12+SNP830 to rapidly and accurately measure H2S concentration in human plasma. As shown in Figure 2h, blank human plasma showed very low autofluorescence at 830 nm, which had little effect on the quantitative detection of H2S. Taking NaHS as an internal standard, the H2S concentration in human plasma was found to be 12.9 ± 3.3 μ M (Figure 2i), which is within the range reported in healthy human plasma (10.5–22.5 μM).63 Visualization of H2S in live cells. To verify the capacity of 12+-SNPs to recognize H2S in living cells, we first revealed that 12+-SNP830 was capable of selective activation by H2S under biologically relevant environment (Figure S14). The MTT assay showed that 12+-SNPs had little toxicity to RAW264.7 macrophages, suggesting a high biocompatibility for cell studies (Figure S15). We subsequently applied 12+-SNP830 to assess H2S in

RAW264.7 macrophages. As illustrated in Figure 3a, cells treated with 12+-SNP830 for 3 h exhibited increased intracellular fluorescence, which could be inhibited by the H2S scavenger, ZnCl2. The addition of extraneous NaHS (1 mM, 1 h) could significantly augment intracellular fluorescence (~7-fold, Figure 3b). Similarly increased fluorescence was also found in RAW264.7 cells after being incubated with 12+-SNP580 (Figure S16). The subsequent colocalization studies with a lysosome staining dye (Lysotracker red) demonstrated that the intracellular fluorescence matched well with that of Lyso-tracker, indicating that 12+-SNPs localized primarily in lysosomes (Figure 3c). These findings demonstrated that 12+-SNPs could be effectively internalized by RAW264.7 macrophages and become fluorescence upon activation by H2S. We then applied 12+-SNP830 to visualize endogenously generated H2S in RAW264.7 cells stimulated with L-Cys, the main substrate of CSE responsible for H2S biosynthesis. RAW264.7 cells treated with 12+-SNP830 displayed an L-Cys’ dose- and incubation time-dependent increment of intracellular fluorescence, indicating more H2S production in cells supplement with L-Cys (Figure S17). The fluorescence intensity in L-Cys-treated cells (200 µM, 1 h) was remarkably ~3.7-fold higher than in control cells, which was significantly suppressed by

Figure 3. Imaging of H2S in RAW264.7 macrophages. (a) Fluorescence images and (b) average fluorescence intensity of macrophages incubated with 12+-SNP830 and indicated reagents. Cells were untreated (control) or pretreated with ZnCl2 (300 µM, 10 min), NaHS (1 mM,1 h), L-Cys (200 μM, 1 h), PAG (50 mg/L, 0.5 h) + L-Cys (200 μM, 1 h), LPS (1 μg/mL, 6 h) + L-Cys (200 μM, 1 h), or PAG(50 mg/L, 0.5 h) + LPS (1 μg/mL, 6 h) + L-Cys (200 μM, 1 h), and incubated with 12+-SNP830 (28/48 µg/mL PCPDTBT/12+(BF4-)2) for 3 h. The fluorescence regions in cells were chosen for the region-of-interest (ROI) measurement to quantify cellular fluorescence. Values represent mean ± SD (n = 3, * P < 0.05, *** P < 0.001). (c) Colocalization study of macrophages incubating with 12+-SNP580 (green) and Lyso-tracker (red). Macrophages were pretreated with 12+-SNP580 (28/48 µg/mL MEH-PPV/12+(BF4-)2, 3 h) and co-stained with Lyso-tracker red (1 µM, 20 min).



DL-pargylglycine (PAG, 50 µg/mL), a potent CSE inhibitor (Figure 3a). As H2S is an important gasotransmitter participating in inflammation diseases,13,64 the H2S levels in LPS-stimulated RAW264.7 cells was investigated using 12+SNP830. Figure 3b showed that the fluorescence of cells increased obviously after being treated with both LPS and L-Cys. In contrast, pretreatment of LPS-stimulated cells with PAG (50 µg/mL) lowered fluorescence significantly. This finding clearly demonstrated the positive role of LPS on upregulating CSE expression and H2S production in inflammatory RAW264.7 cells, which was consistent with elevated CSE miRNA and CSE enzyme expression (Figure S18). Moreover, the different endogenous levels of H2S were also validated by the methylene blue (MB) method65,66 and a previously reported fluorescent probe for H2S67 (Figure S19). Thus, 12+-SNP830 permitted efficient

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assessment of endogenous H2S fluctuation within cells through fluorescence imaging. Fluorescence imaging of hepatic H2S in vivo. Considering that the longer NIR fluorescence at 830 nm could facilitate deep tissue penetration and high sensitivity for in vivo imaging, we first applied 12+-SNP830 to monitor exogenous H2S in living mice. Mice receiving an intraperitoneal (i.p.) injection of 12+-SNP830 and NaHS (1 mM) showed a substantially greater signal-to-background ratio (SBR) compared to that of 12+-SNP830 and saline in the i.p. cavity (Figure S20). A maximum ~4-fold turn-on fluorescence in mice was observed after 20 min, indicating that 12+-SNP830 could provide reliable NIR fluorescence for real-time detection of H2S in live mice. We then used 12+-SNP830 to non-invasively detect endogenous H2S in mice. Figure 4a showed that

Figure 4. Non-invasive imaging of hepatic H2S in vivo. (a) Longitudial fluorescence images and (b) SBR of saline-, L-Cys- or PAGtreated mice following i.v. injection of 12+-SNP830 (78.4/134 µg PCPDTBT/12+(BF4-)2, 100 µL). (c) Schematic for fluorescence imaging of hepatic H2S in live mice treated with LPS. (d) Fluorescence images and (e) SBR of mice following i.p. injection of varying doses of LPS (0, 5, 10, 20 mg/kg, or 5 mg/kg PAG + 10 mg/kg LPS) and L-Cys (5 nmol/kg), and then i.v. injection of 12+-SNP830 (78.4/134 µg PCPDTBT/12+(BF4-)2, 100 µL) at 6 h. (f) Western blot analysis showed different CSE expression in liver homogenates resected from mice treated with different dose of LPS for 6 h (n = 3). Fluorescence images were acquired with Ex/Em = 780/845 nm. Red arrows show the locations of fluoresence signals in livers. Black circles show the background locationsin mice. Values denote mean ± SD (* P < 0.05, ** P < 0.01, *** P

Engineering of Electrochromic Materials as Activatable Probes for

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