Unimolecular near-infrared chemiluminescent reporter for cascaded multiplex imaging of ischemia-reperfusion injury in the liver-kidney axis

Abstract

Acute kidney injury (AKI) frequently arises as a complication of hepatic ischemia-reperfusion injury (HIRI), yet simultaneous optical imaging of both remains challenging due to the lack of unimolecular dual-responsive probes. Herein, we report a library of hemicyanine-based chemiluminophores (HCLs) with tunable emission to second near-infrared window (725 − 1025 nm) achieved by integrating bicyclic dioxetane onto hemicyanine skeletons to develop multiple-responsive chemiluminescent probes. HCL1 and HCL5 respectively emitting at 725 nm and 1025 nm are selected to construct a cascaded activatable reporter CAR for crosstalk-free duplex chemiluminescence imaging of interlinked biomarkers. Following systemic injection to male mice, CAR preferentially accumulates in the liver and reports HIRI-associated superoxide anion (O₂^•−), which initiates self-fragmentation and liberates the secondary reporter KIR into kidneys to report AKI-associated N-acetyl-β-D-glucosaminidase (NAG). Such mechanism allows CAR to serve as a reservoir for gradual release of AKI reporters, providing a significantly prolonged imaging window compared to co-administering separate probes. CAR further permits remote detection of HIRI-induced AKI via urinalysis. This study not only offers a powerful tool for simultaneous detection of HIRI and HIRI-induced AKI, but also highlights a unimolecular probe design for ultrasensitive detection of deeply-seated intercorrelated diseases.

Introduction

Hepatic ischemia reperfusion injury (HIRI), which occurs when the liver experiences inadequate oxygen and nutrient supply, followed by the restoration of blood flow, often leads to oxidative stress and inflammation^1,2,3. It is a common complication in various clinical scenarios such as hemorrhagic shock, trauma, liver resection, and transplantation, and stands as a leading cause of acute liver failure (ALF) during the perioperative period^4,5. Patients with ALF frequently develop acute kidney injury (AKI) (Fig. 1a), with the incidence varying between 40% and 85%, depending on the etiology^6,7,8. Notably, the occurrence of AKI in orthotopic liver transplantation can reach up to ~95%, significantly increasing mortality^6,7,8. Timely therapeutic intervention has been shown to improve patient outcomes, highlighting the need for early diagnostic methods that can simultaneously detect HIRI and HIRI-induced AKI, as well as advancing the understanding of elusive pathophysiological mechanisms linking HIRI with AKI^9,10.

Fig. 1: Design and mechanisms of CAR for multiplex imaging and urinalysis of IRI in the liver-kidney axis.

a The effects of liver ischemia duration on the induction of AKI. The severity of HIRI-induced AKI is highly associated with the duration of liver ischemia. b Schematic illustration of CAR for imaging of O₂^•− and NAG and urinalysis. After systemic administration of CAR into living mice, it efficiently accumulates into the liver and specifically liberates NIR-II CL signal to report O₂^•− levels, followed by inducing self-elimination to defragment the backbone of CAR and effectively releasing the NIR-I CL reporter (KIR) to the kidneys for sensing NAG levels, finally excreting out for fluorescently urinalysis. c Chemical structures of CAR and its fragments in response to O₂^•− and NAG, respectively. Source data are provided as a Source Data file.

Current clinical diagnostic techniques primarily rely on assessing serological biomarkers such as serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and serum creatinine (sCr)/blood urea nitrogen (BUN) for liver and kidney dysfunction, respectively. However, these biomarkers are late-stage indicators and lack the sensitivity to detect HIRI and AKI at an early stage¹¹. Biopsy is another routine method for diagnosing organ damage; whereas it is invasive, carries a risk of secondary injury and provides only a limited and static view of the pathological state¹². Although non-invasive imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) have been used to reveal anatomic and functional changes in both liver and kidneys, these methods are insensitive to detecting early biomarkers at the molecular level during HIRI-induced injury¹³.

Chemiluminescence (CL) imaging known for its ultrahigh sensitivity and high signal-to-background ratio (SBR) has become one of the most reliable bioimaging techniques¹⁴. In particular, the chemiluminophore Schaap’s dioxane with excellent chemical amenability has been constructed into versatile activatable probes that inspire chemiluminescence imaging of a diverse array of pathological biomarkers including reactive species (superoxide anion, hydrogen peroxide, singlet oxygen, peroxynitrite, etc.) and enzymes (β-galactosidase, alkaline phosphatase, γ-glutamyl transpeptidase, cathepsin B, granzyme B, etc)^{15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30}. However, most of these probes have visible emission with limited tissue penetration depth, and the red shift of emission wavelength remains challenging^{31,32,33,34,35,36,37,38,39,40}. In addition, existing probes mostly respond to merely one biomarker, failing to simultaneously diagnose the primary and the related complication diseases, which requires detecting two interlinked biomarkers. While co-administration of two separate probes may seem like a feasible approach, it demands precise alignment of imaging windows for both probes, which can be challenging due to their differing pharmacokinetic profiles^{41,42,43,44,45}. Unimolecular dual-locked probes might overcome these challenges by using a single probe that sequentially responds to two biomarkers at primary and complication disease sites, with the initial response triggering the liberation of a secondary reporter^46,47,48,49. Such sequential logic response also favors the pharmacokinetic conversion of the probe for multiorgan imaging, providing opportunities to investigate the pathophysiological mechanisms between the primary disease and its remote complication^{50,51,52,53,54,55}. However, unimolecular dual-responsive reporters are rarely explored, especially for detecting interlinked diseases such as HIRI and HIRI-induced remote AKI.

Here, we develop a unimolecular cascaded activatable reporter (CAR) with dual responses and pharmacokinetic conversion for non-invasive multiplex chemiluminescence imaging and fluorescent urinalysis of HIRI and associated AKI (Fig. 1b, c). We first synthesize a library of hemicyanine-based chemiluminophores (HCLs) with tunable emission from 725 to 1025 nm and structural flexibility for constructing activatable probes. Unlike conventional chemiluminescence resonance energy transfer (CRET) approaches, HCLs are constructed by respectively integrating a benz[e]indolium, benz[c,d]indolium, naphtho[c,d]indolium and thiophen-benz[c,d]indolium moiety onto xanthonium- based bicyclic dioxetane to expand linear π-conjugation, affording record-long-wavelength emission among reported bicyclic dioxetane chemiluminophores (Fig. 2e and Supplementary Table 1). From this library, HCL1 emitting in the first near-infrared window (NIR-I) and HCL5 in the second NIR window (NIR-II) are selected to construct CAR because of their minimal spectral overlap. HCL1 and HCL5 are respectively caged with cleavable moieties responsive to N-acetyl-β-D-glucosaminidase (NAG), a biomarker of renal tubular damage in AKI, and superoxide anion (O₂^•−), a biomarker of oxidative stresses in HIRI, and are linked with a renal-clearable unit polyethylene glycol (PEG) (Fig. 1c)^28,29. After systemic administration to living mice (Fig. 1b), CAR efficiently accumulates in the liver, where it specifically reports O₂^•− level via NIR-II CL. This response further triggers its self-elimination to expose and release the NIR-I CL reporter (KIR), which travels to the kidneys and detects NAG, finally getting excreted out and enabling in vitro urinalysis³⁰. Such cascaded activation thus enables crosstalk-free and simultaneous monitoring of two intercorrelated pathological events in the liver-kidney axis during the onset and progression of HIR.

Fig. 2: NIR-II Hemicyanine chemiluminophores.

a, b Schematic illustration of the redshift of chemiluminescence via energy-transfer-based indirect chemiluminescence emission pathway and direct chemiluminescence emission pathway. c Activation and chemiexcitation pathway of adamantyl-dioxetanes and bicyclic dioxetanes. d Evaluation of the thermal stability of BD-OTf and AD-OTf. Comparison of the reserved CL intensities of BD-OTf and AD-OTf when stored at 25 °C for different duration time. (n = 3, mean ± s.d.). e Structures of adamantyl-dioxetanes scaffold^15,17,23,56 and hemicyanine-based bicyclic dioxetanes and their CL emission wavelengths are indicated by arrows. Their maxima CL emissions are given underneath the corresponding structures. f Absorption, (g) fluorescence spectra and (h) Chemiluminescence spectra of HCL_S (10 μM) in dimethyl sulfoxide/H₂O (v/v = 1/1). (a.u. means arbitrary units). i Molecular orbitals of HCL_S. The HOMO and LUMO energy levels are indicated. j Calculated HOMO/LUMO energy gaps (ΔE) of HCL_S. Source data are provided as a Source Data file.

Results

Design, synthesis, and characterization of NIR-II chemiluminophores

To shift the chemiluminescence towards a more biologically transparent NIR window, dioxetane substrates are often conjugated onto fluorophores, forming CRET systems (Fig. 2a). However, this indirect approach significantly reduces chemiluminescent brightness due to low energy transfer efficiencies and additional energy relay processes. A more effective strategy is to directly modify dioxetane onto NIR fluorophores (Fig. 2b). However, due to the challenges in chemical synthesis, dioxetane-based chemiluminophores with the NIR-I emission (660–780 nm) are scarcely studied, and those reaching beyond 900 nm are rarely reported^16,56,57,58. Moreover, improved thermal stability is essential for chemiluminophores. To address this, we synthesized the bicyclic dioxetane (BD-1) and adamantyl dioxetane (AD-1) (Fig. 2c) and determined their reserved chemiluminescence after placing on the bench at room temperature for 6 h. As shown in Fig. 2d and Supplementary Fig. 1, BD-OTf demonstrated superior thermal stability, as evidenced by a negligible decrease on the reserved chemiluminescence intensity compared to AD-OTf. Thereafter, we tailored the bicyclic dioxetane scaffold by conjugating xanthonium and indolium derivatives, which feature an extended D-π-A motif and render rigid conjugated structures (Fig. 2e). Such conjugation extension effectively tuned and redshifted the chemiluminescence emission of the HCL family, ranging from 725 nm to a maximum of 1025 nm (Fig. 2e), which was ~ 225 nm longer than the previously reported bicyclic dioxetane probe (~ 800 nm)^59,60. The detailed synthetic routes of HCLs were shown in Fig. 3. Briefly, the key precursor 5 was synthesized from commercial 3,5-dimethoxybenzyl bromide through substitution of the benzyl bromide and oxidation reaction to yield compound 1. In the presence of lithium diisopropylamide, compound 1 was converted to compound 2 through a cyclization reaction, which was further converted to aldehyde compound 3 via AgOTf-aided direct formyl transfer. Compound 3 was directly deprotected by BBr₃ to afford ortho-hydroxy-formyl 4, followed by cascaded oxa-Michael addition and dehydration to yield aldehyde compound 5. It was then condensed with indoles 11, 12, 14, 16 and 19 and subsequently oxidation via [2 + 2] cycloaddition with singlet oxygen (¹O₂) using methylene blue as the photosensitizer to yield HCL1-5. The chemical structures of the intermediates and final HCLs were confirmed through ¹H NMR, ¹³C NMR and mass spectrometry. The photophysical properties of HCL1-5 were measured and shown in Fig. 2f–h and Supplementary Fig. 2. The absorption maxima in dimethyl sulfoxide/H₂O were at 652, 695, 755, 798, and 822 nm for HCL1-HCL5, respectively. Furthermore, HCL1-5 exhibited strong chemiluminescence across NIR-I and NIR-II regions with maximum emission peaks at 725, 735, 850, 920, and 1025 nm, respectively, which corresponded well with their fluorescence spectra (Fig. 2g and Supplementary Table 1) and implied their abilities for both NIR chemiluminescence and fluorescence imaging. These findings also confirmed that integrating different π-extended indolium derivatives onto the xanthonium moiety successfully induced bathochromic shift in their emission wavelengths. To understand the mechanism of this spectral shift, time-dependent density functional theory (TD-DFT) calculations and electron-hole analysis were performed. Overall, HCL1-HCL5 displayed gradually decreased LUMO levels and increased HOMO levels (Fig. 2i), showing reduced energy gaps from 2.19, 2.08, 1.93, 1.92 to 1.77 eV, respectively (Fig. 2j). Decreased HOMO-LUMO energy gaps and the excitation energy were observed, which in accordance with the red shift of the emission wavelengths from HCL1 to HCL5 (Supplementary Fig. 3). This finding aligned well with the bathochromic shifts in emission and provided the theoretical validation for the strategy of expanding π-conjugation on indolium moieties. Among HCLs, HCL1 and HCL5 respectively emitting at 725 and 1025 nm demonstrate negligible spectral overlap and high potential for crosstalk-free duplex chemiluminescence imaging. We herein selected HCL1 and HCL5 for the construction of unimolecular dual-responsive probes in the following studies.

Fig. 3: Synthetic Routes for chemiluminophores and probes.

a Synthetic routes of HCLs. Reagent and conditions: (1) Compound 21, sodium hydride, THF, rt, 6 h. (2) Pyridinium Chlorochromate, DCM, rt, 12 h. (3) Lithium diisopropylamide, THF, − 78 °C, 6 h. (4) Silver trifluoromethanesulfonate, Dichloromethyl methyl ether, DCM, − 78 °C, 8 h. (5) Boron tribromide, DCM, − 78 °C, 3 h. (6) Compound 25, DMF, Cs₂CO₃, rt, 12 h. (7) AcONa, EtOH, 50 °C, 6 h. (8) Methylene blue, O₂, hv, 1 h. b Synthetic routes of probe CAR. (1) Compound 22, Cs₂CO₃, K₂CO₃, DCM, rt, 2 h. (2) NaOMe, DCM, rt, 1 h. (3) SH-PEG₂₀₀₀-alkyne, sodium ascorbate, CuSO₄, THF, H₂O, rt, 2 h. (4) Compound 23, 24, triphosgene, 4-di-methylaminopyridine, 0 °C, 2 h. (5) Triethylamine, THF, rt, 3 h. (6) Methylene blue (MB), MeOH, O₂, hv, 2 h. c Synthetic routes of probe LIR. (1) Trifluorosulfonic anhydride, Pyridine, DCM, rt, 2 h. (2) Methylene blue (MB), O₂, hv, 1 h. d Synthetic routes of probe KIR. (1) Compound 24, THF, trimethylamine, 3 h. (2) Methylene blue (MB), MeOH, O₂, hv, 1 h. Source data are provided as a Source Data file.

Synthesis and in vitro characterization of CAR

Unimolecular dual-responsive chemiluminescence probe CAR was constructed through three main steps (Fig. 3b): (i) Synthesis of the N-acetyl-β-D-glucosamine-caged NIR-I chemiluminescent substrate 8, (ii) synthesis of the trifluoromethanesulfonate-caged NIR-II chemiluminescent substrate 10, and (iii) conjugation of NIRF-I and NIR-II chemiluminescent substrates (8&10) followed by oxidation to afford CAR. Briefly, compound 6 was synthesized similarly to HCL1, then reacted with a bromo-glucose derivative to afford compound 7 after hydrolysis. The azide group on 7 was conjugated with the alkyne-functionalized PEG to produce substrate 8. The trifluoromethanesulfonate-caged self-immolative substrate 23 was synthesized as described in previous studies (Supplementary Fig. 4), enabling conjugation with the precursor 9 and maleimide linker 24 to afford NIR-II chemiluminescent substrate 10. The thiol group on the terminal of substrate 8 was conjugated with the maleimide group of substrate 10 via a click reaction, followed by [2 + 2] cycloaddition with ¹O₂ using methylene blue to yield CAR. ¹H NMR, ¹³C NMR and mass spectra confirmed the chemical structures of the intermediates and CAR. Two control probes liver injury reporter (LIR) and kidney injury reporter (KIR) were also synthesized and characterized (Fig. 3b, c). Synthesis routes of 11, 12, 14, 16 and 19 were depicted in Supplementary Fig. 4. ¹H NMR and mass spectra confirmed their chemical structures.

The amphiphilicity of CAR allowed it to spontaneous assemble into spherical nanoparticles, as shown by transmission electron microscopy (TEM) (Fig. 4l). The average hydrodynamic size of CAR was measured to be ~180 nm (Fig. 4j). No precipitation and obvious change in fluorescence were detected for CAR during the storage in phosphate buffer solution (PBS) or fetal bovine serum (FBS) for 5 days (Supplementary Fig. 5a, b) and in different pH buffer solution (Supplementary Fig. 6). Zeta potential measurement revealed that CAR was negatively charged (Supplementary Fig. 5c). CAR had two absorption peaks at ~650 nm and ~800 nm, corresponding to the absorbance of HCL1 and HCL5 moieties, respectively (Supplementary Fig. 5d). CAR by itself was non-chemiluminescent and non-fluorescent because both HCL1 and HCL5 moieties were in ‘caged’ states that the electron-donating abilities of the aromatic hydroxyl groups were diminished. Upon exposure to KO₂ and NAG, CAR demonstrated red-shifted absorption peaks at ~ 700 nm and ~ 820 nm (Supplementary Fig. 5d). In the presence of NAG, NIR-I (~ 720 nm) chemiluminescence and fluorescence from CAR increased by ~ 8500-fold and ~8-fold, respectively (Fig. 4a, b). Similarly, incubation with KO₂ increased NIR-II (~ 1025 nm) chemiluminescence and fluorescence increased by ~6500-fold and ~ 7.5-fold, respectively. Such dramatic emission enhancement were ascribed to the cleavage of the glycosidic bond of N-acetyl-β-D-glucosamine moiety by NAG and trifluoromethanesulfonate group by O₂^•−, which triggered 1,4-elimination and led to the fragmentation into uncaged probes FL1R and FL2R (Fig. 1c), both with a strong electron-donating phenolate group on the luminophore.

Fig. 4: In vitro sensing evaluation of CAR.

a, b NIR-I chemiluminescence and fluorescence spectra of CAR (20 μM) in the absence or presence of NAG (40 mU) in PBS (10 mM, pH 7.4). Inset: The corresponding fluorescence and chemiluminescence images using the IVIS spectrum imaging system. c Michaelis-Menten saturation curve for NAG (10 mU) toward CAR (10, 20, 30, 60 and 90 μM) in PBS (10 mM, pH 7.4). (n = 3, mean ± s.d.). d NIR-I chemiluminescence and fluorescence changes of CAR (10 μM) after incubation with ROS (40 μM), metal ions (40 μM), glutathione (GSH), β-galactosidase (β-Gal), gamma-glutamyl transferase (GGT), and NAG in PBS (10 mM, pH 7.4). (n = 3, mean ± s.d.). e, f NIR-II chemiluminescence and fluorescence spectra of CAR (20 μM) in the absence or presence of KO₂ (80 μM) in PBS (10 mM, pH 7.4). g NIR-II chemiluminescence kinetic profiles of CAR in the presence of KO₂ in PBS (10 mM, pH 7.4). h NIR-II chemiluminescence and fluorescence changes of CAR (10 μM) after incubation with ROS (40 μM), metal ions (40 μM), GSH, β-Gal, GGT, and KO₂ (80 μM) in PBS (10 mM, pH 7.4). (n = 3, mean ± s.d.). i NIR-II chemiluminescence intensities of CAR as a function of the concentration of KO₂ (0–50 nM). (n = 3, mean ± s.d.; a.u. means arbitrary units). j, k Dynamic light scattering of CAR before and after incubation with KO₂ in buffer solution. l Representative TEM images of CAR before and after incubation with KO₂ in buffer solution. Source data are provided as a Source Data file.

This fragmentation was validated by TEM and dynamic light scattering (DLS), which indicated that the size of CAR decreased to ~ 30 nm and ~ 4 nm after exposure to KO₂ (Fig. 4j, k). CAR showed minimal change in chemiluminescence and fluorescence in the presence of interfering analytes, including other reactive oxygen species, metal ions, and other enzymes, confirming their excellent stability and specificity (Fig. 4d, h). In addition, the catalytic efficiency (K_cat/K_m) of NAG towards CAR was calculated to be 0.85 μM⁻¹S⁻¹ (Fig. 4c). A good linear relationship was observed between the NIR-II chemiluminescent signal and KO₂ concentration, with an estimated limit of detection (LOD) of approximately 12 nM (Fig. 4i). The chemiluminescence half-life of CAR was ~ 12 min (Fig. 4g), which is suitable for in vivo imaging. In addition, optical changes of control probes LIR and KIR in response to KO₂ and NAG were characterized and shown in Supplementary Fig. 5e–g.

Tissue penetration and clearance pathways

We then compared the penetration depth and imaging sensitivity of chemiluminescence and fluorescence channels from HCL1 and HCL5 (Fig. 5a). To do so, three trifluoromethanesulfonate caged-luminophores, including BD-OTf, HCL1-OTf and HCL5-OTf were synthesized (Fig. 5b). In the presence of KO₂, the trifluoromethanesulfonate group was cleaved and resulted in maximum chemiluminescence emission at approximately 450, 725 and 1025 nm for these three probes, respectively (Fig. 5c). The triggered chemiluminescence as well as fluorescence signals were recorded under different depths of chicken breast tissue. Chemiluminescence signals from BD-OTf and HCL1-OTf decreased to the background levels at tissue thickness of 0.5 cm and 2 cm, respectively; whereas that from HCL5-OTf remained discernible even at 3 cm (Fig. 5d). In contrast, fluorescence signals from BD-OTf, HCL1-OTf and HCL5-OTf declined to background levels at tissue thickness of 0.5 cm, 1 cm and 1.5 cm, respectively (Fig. 5d). In addition, the SBRs of chemiluminescence for HCL5-OTf (591 and 124) were 5.8 and 10.3-fold higher than that of BD-OTf (101 and 12) and 1.6 and 8.2-fold higher than that of HCL1-OTf (302 and 15) at chicken tissue thicknesses of 0.5 and 3.0 cm, respectively (Fig. 5e). Although fluorescence SBRs improved with increasing emission wavelengths, they were much lower than the corresponding chemiluminescence SBRs (Fig. 5f). These findings verified that chemiluminescence provides enhanced imaging sensitivities and deeper tissue penetration than fluorescence, highlighting its superiority for in vivo imaging.

Fig. 5: Tissue penetration and clearance pathways.

a Schematic illustration of tissue-penetration study. b, c Chemical structures and chemiluminescence spectra of BD-OTf, HCL1-OTf and HCL5-OTf. d Representative chemiluminescence and fluorescence images of BD-OTf, HCL1-OTf and HCL5-OTf (40 µM) solutions upon overlaying chicken tissues with the desired thickness on top of the samples. e, f SBRs for chemiluminescence and fluorescence imaging of BD-OTf, HCL1-OTf and HCL5-OTf as a function of chicken tissue depth in panel d. (n = 3, mean ± s.d.). g Schematic illustration of renal and hepatobiliary clearance pathways in living mice. h Blood concentration (% ID g⁻¹) decay of CAR, KIR and LIR after i.v. injection into living mice. (n = 3, mean ± s.d.). i The total renal and fecal clearance efficiency of CAR, KIR and LIR after 7 days of injection. (n = 3, mean ± s.d.). j Fluorescence images of the abdominal cavity of mice after 1 h injection of CAR, LIR, FL2R, PBS, KIR and FL1R (2.5 μmol kg⁻¹ body weight). Bladder (Bl), kidneys (Ki), liver (Li), muscle (Mu), spleen (Sp). k NIR-II fluorescence intensity of liver from mice after 1 h injection of CAR, LIR and FL2R. (n = 3, mean ± s.d.; a.u. means arbitrary units) l NIR-I fluorescence intensity of kidneys after 1 h injection of PBS, KIR and FL1R. (n = 3, mean ± s.d.). Source data are provided as a Source Data file.

To investigate the pharmacokinetics of CAR and the control probes (LIR and KIR), their concentrations in the blood were analyzed by high-performance liquid chromatography (HPLC) after intravenous (i.v.) injection into living mice (Fig. 5g). Blood concentration curves showed that LIR and KIR rapidly decreased to 0% injected doses (ID) g⁻¹ at 2 h post-injection (Fig. 5h), while CAR persisted for up to 24 h. And CAR had a much longer elimination half-life (t_1/2β) (~ 5 h) compared to LIR (~ 32 min) and KIR (~ 25 min). Moreover, the renal clearance efficiency of KIR was ~ 92% ± 2% ID at 24 h post-injection in living mice, in sharp contrast to ~ 5% ± 1.3% ID for LIR and ~ 3% ± 1.2% ID for CAR. On the contrary, the fecal clearance efficiency of LIR and CAR were determined to be ~ 70% ± 3% ID and ~ 58% ± 2.2%, respectively, much higher than 2.1% ± 0.5% ID for KIR (Fig. 5i). Furthermore, the biodistributions of CAR, LIR, KIR and their activation forms (FL2R and FL1R) were investigated by NIR-II and NIR-I fluorescence imaging after i.v. injection. NIR-II fluorescence of FL2R mainly located in the liver with minimal signal in other organs; while no obvious NIR-II fluorescence signal of CAR and LIR was found, as they are intrinsically non-fluorescent (Fig. 5j). Similarly, a bright NIR-I fluorescence signal of FL1R was observed in the kidneys (Fig. 5j), whereas minimal signal from KIR was observed. In addition, the optical and chemical profiles of CAR, KIR and their fragments excreted renally from mice were measured. The absorption and HPLC of KIR recovered from urine (CAR recovered from feces) and their as-synthesized forms were almost identical (Supplementary Fig. 7), confirming their structural stability and minimal in vivo metabolism in mice. Moreover, no increment in serological levels of sCr, BUN, ALT and AST were observed between PBS groups and CAR groups in healthy mice (Supplementary Fig. 8). Histological staining revealed that no signs of cell death or necrosis observed in the major organs, implying CAR and its fragments had high biocompatibility (Supplementary Fig. 9). Collectively, these findings indicated that CAR and LIR showed liver-oriented accumulation and were mainly excreted through the hepatobiliary clearance pathway, which should be attributed to the large particle size (~ 180 nm) of CAR that far exceeded the kidney filtration threshold (~ 6 nm) and the hydrophobicity of LIR. In contrast, KIR and FL1R were mainly eliminated through the renal clearance pathway because of its high hydrophilicity (distribution coefficient Log D = − 9.58) and low molecular weight (~ 2.5 kDa) relative to the glomerular filtration molecular weight cutoff (~ 50 kDa).

Cascaded multiplex imaging and urinalysis of HIRI and HIRI-induced AKI using a unimolecular dual-responsive probe CAR

The performance of CAR for real-time non-invasive imaging of HIRI and HIRI-induced AKI was evaluated in living mice. A partial hepatic warm IR model was established according to previous reports²⁸. Briefly, the abdomen was opened to dissect the interlobular ligaments when male mice were anesthetized. A microvascular clamp was then used to interrupt the arterial and portal venous blood supply to the left lateral and median liver lobes for 15, 30, 45, or 60 min (Fig. 6a). Afterwards, the clamp was removed and the abdominal cavity was closed. Such an operation procedure resulted in a segmental (~ 70%) liver ischemia. A sham-operation group was also established using the same procedure, involving only abdominal trauma without vascular occlusion. Healthy mice or mice treated with an antioxidative drug (N-acetylcysteine (NAC)) prior to the operation were used as control groups.

Fig. 6: Real-time cascaded multiplex imaging and urinalysis of HIRI and HIRI-induced AKI using CAR.

a Schematic illustration of the development of the hepatic ischemia-reperfusion mouse model with different ischemia durations and chemiluminescence imaging after injection of CAR (10 μmol kg⁻¹ body weight). b, c The dynamic NIR-II chemiluminescence intensities of liver and the dynamic NIR-I chemiluminescence intensities of kidney in living mice after injection of CAR. (n = 3, mean ± s.d.). Two-tailed Student’s t test. d, e Representative NIR-II and NIR-I chemiluminescence images of living mice subjected to liver ischemia with different durations (15, 30, 45 and 60 min) after injection of CAR (10 μmol kg⁻¹ body weight). NIR-II chemiluminescence images were acquired with an acquisition time of 30 s. NIR-I chemiluminescence images were acquired under bioluminescence mode of the IVIS spectrum imaging system with the acquisition time of 60 s. f Online urinalysis: representative fluorescence images of excreted FL1R in the urine from living mice subjected to liver ischemia with different durations; Offline urinalysis: representative fluorescence images of CAR after incubation with the urine samples collected from living mice subjected to liver ischemia with different duration. g, i Change fold of NIRF intensity in urinalysis, ALT, AST, sCr, BUN, TNF-α and IL-6. h The cascaded injury scores and the complication index from living mice subjected to liver ischemia after injection of CAR. (n = 3, mean ± s.d.). Two-tailed Student’s t test. Healthy group versus control and experimental groups (**p < 0.01, ***p < 0.001, ****p < 0.0001). N.S.: no statistically significant differences. Source data are provided as a Source Data file.

Longitudinal chemiluminescence imaging was conducted after injection of CAR at 4 h post-reperfusion. For mice subjected to 15 min and 30 min of liver ischemia, the injection of CAR led to a gradual increase of NIR-II chemiluminescence signals in the liver (Fig. 6d), peaking at 30 min of post-injection (Fig. 6b). Such hepatic activation of CAR indicated the upregulation of O₂^•− during the progression of HIRI. At this time point, NIR-II chemiluminescence signals were 15.2-fold and 17.3-fold higher than that of the control mice, respectively. However, no obvious NIR-I chemiluminescence signal was observed in kidneys from mice subjected to 15 min and 30 min of liver ischemia (Fig. 6d), suggesting that the kidneys were unaffected under these conditions. For mice subjected to 45 min and 60 min of liver ischemia, similar trends in NIR-II chemiluminescence signal changes were observed in the liver, showing a 26.5-fold and 41.2-fold higher than that of the control group at 30 min post-injection of CAR, respectively (Fig. 6b). Notably, bright NIR-I chemiluminescence signals were observed in the kidneys (Fig. 6d), reflecting upregulated NAG detected by liberated KIR, which indicated the occurrence of HIRI-associated AKI. Importantly, the signals in the kidneys remained detectable even at 60 min post-injection of CAR. At 30 min post-injection of CAR, kidney signals from mice subjected to 45 min and 60 min of liver ischemia were 32.4-fold and 42.6-fold higher than that of the control group (Fig. 6c). By contrast, NIR-II chemiluminescence signals in the liver and NIR-I signals in the kidneys decreased by 4.8-fold and 1.2-fold when mice were pre-treated with NAC to protect the liver against IRI insult (Fig. 6b–d). Although O₂^•− is associated with lesion formation in physical trauma, sham-operated mice with only abdominal trauma showed negligible chemiluminescence increase in the liver and kidneys, showing the high specificity of CAR to report HIRI instead of other injuries. This should be ascribed to the liver-oriented accumulation of CAR, resulting in minimal probe accumulation in other traumatic tissues. Such observation was further confirmed by in vivo fluorescence imaging and ex vivo biodistribution studies (Supplementary Fig. 10–12). In addition, chemiluminescence imaging by CAR showed ~ 4.5 and ~ 4.2-fold higher SBRs at 45- and 60 min post-injection, respectively, compared to fluorescence imaging (Supplementary Fig. 13), due to the absence of tissue autofluorescence.

Moreover, HIRI-induced AKI was detected remotely through urinalysis using two CAR-based methods (Supplementary Fig. 14a): (i) online urinalysis, which involved direct NIR-I fluorescence analysis of urinary FL1R from mice subjected to liver ischemia after CAR injection; and (ii) offline urinalysis, which involved collecting urine samples from mice subjected to liver ischemia, followed by in vitro CAR incubation and fluorescence readout. In the online urinalysis (Fig. 6f, g and Supplementary Fig. 14b, c), no significant fluorescence enhancement was observed in mice subjected to 15 min and 30 min liver ischemia. However, the fluorescence increased by 10.8- and 14.3-fold when liver ischemia duration reached 45 min and 60 min, respectively. These fluorescence signal changes in urinary FL1R correlated well with the abovementioned chemiluminescence imaging results, as the excreted FL1R was activated by renal tubular NAG during the onset and progression of HIRI-induced AKI. The HPLC and mass spectra further confirmed the production of FL1R in urine (Supplementary Fig. 15). A similar trend was found in the offline urinalysis (Fig. 6f, g). CAR had no statistically significant enhancement after incubation with urine from mice subjected to 15- and 30 min liver ischemia, but the fluorescence increased by 4.3- and 6.5-fold after 45 and 60 min of liver ischemia. This occurs because NAG is normally secreted in very low concentrations from renal tubules into urine, but its secretion rises dramatically during the onset and progression of AKI⁶¹. In addition, urinary signals were reduced after pre-treatment with NAC or in sham-operation mice, which were consistent with the real-time in vivo imaging results.

To assess the correlation between the severity of HIRI and associated AKI, the kidney-to-liver ratio of signals (defined as cascaded injury score) and urinary-to-liver ratio of signals (termed as complication index) were calculated as a function of liver ischemia duration (Fig. 6h). The cascaded injury score was close to 0 when mice were subjected to 15 and 30 min liver ischemia but sharply increased to 42 and 47 when liver ischemia duration reached 45 min and 60 min, respectively (Fig. 6h). A similar trend was observed for complication index when increasing ischemia duration. These findings indicate that mice subjected to more than 45 min of liver ischemia develop severe AKI, and the severity of kidney damage correlates with the extent of liver injury (Fig. 7a). The diagnosis of HIRI induced-AKI was further confirmed by serum creatinine and BUN results (Fig. 6iand Supplementary Fig. 16).

Fig. 7: The correlation of the severity between HIRI and associated AKI.

a Schematic illustration of the correlation between liver ischemia duration and the chemiluminescence signals in the liver and kidneys and fluorescence signals in urine. b Schematic illustration for the pathological mechanisms of HIRI-induced AKI. c Mean fluorescence intensity of anti-caspase-3 in the liver. (n = 3, mean ± s.d.). d Mean fluorescence intensity of FL1R in the kidney after injection of CAR. (n = 3, mean ± s.d.; a.u. means arbitrary units). e Representative photomicroscope images of H&E staining in paraffin-embedded liver and kidney sections and confocal fluorescence microscopy images of liver and kidney from mice subjected to liver ischemia with different duration, followed by 4 h reperfusion. The blue and green signals are from 4’,6-diamidino-2-phenylindole (DAPI) and caspase-3 antibody, respectively. The red signal is from FL1R. Two-tailed Student’s t test. Healthy group versus control and experimental groups (**p < 0.01, ***p < 0.001, ****p < 0.0001). NS: no statistically significant differences. Source data are provided as a Source Data file.

Clinical and preclinical assays were used to test the liver function (ALT and AST) and the level of proinflammatory cytokines, including tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in sera of mice (Fig. 6i and Supplementary Fig. 17, 18). The ALT and AST had a negligible increase in mice subjected to 15 and 30 min liver ischemia relative to the control mice, but they significantly increased by 9.5- and 5.2-fold, 13.2- and 9.5-fold in mice subjected to 45 and 60 min liver ischemia. Elevated levels of serum pro-inflammatory cytokines TNF-α (8.5-, 10.5-, 26.5- and 35.9-fold) and IL-6 (3.5-, 5.6-, 18.6- and 22.5-fold) were observed in mice subjected to 15, 30, 45, and 60 min liver ischemia, implying a heightened inflammatory response. Although these cytokine levels were upregulated in mice subjected to liver ischemia, they also increased in sham-operated mice, suggesting that cytokine measurements have low specificity to diagnose HIRI. In comparison, CAR-based imaging is more sensitive than these assays and achieved the earliest prediction of HIRI. Another conventional assessment by histological analysis revealed noticeable loss of normal liver architecture and severe hepatocyte necrosis in mice subjected to 45 and 60 min liver ischemia followed by 4 h reperfusion (Fig. 7e); while no significant lesion was observed in mice subjected to 15 and 30 min liver ischemia, healthy control mice, NAC-pretreated mice, or sham-operation mice. Hepatocyte necrosis was observed when the reperfusion period was extended to 8 h for mice subjected to 15 and 30 min liver ischemia (Supplementary Fig. 19), while kidneys remained unaffected. Histological abnormalities in the kidneys were only seen in mice subjected to 45 and 60 min liver ischemia followed by 4 h reperfusion, showing marked vascular stasis in the interstitial capillary lumina and focal tubular dilatation with intraluminal shedding of cytoplasmic debris and formation of granular eosinophilic casts (Fig. 7e). While no abnormalities were found in other groups. No obvious histological changes in other major organs, including heart, spleen and lung, were found (Supplementary Fig. 20), consistent with previous studies that the kidneys are the most clinically affected organs in HIR, followed by lung, spleen and heart⁶². The proposed mechanisms behind this include the initiation of oxidative stress, the recruitment of leukocytes, intrarenal vasoconstriction, induced intrarenal ischemia, as well as renal tubular necrosis (Fig. 7b)^1,2,3,4,62. Immunostaining of liver sections from mice subjected to 45 and 60 min ischemia displayed strong green fluorescence for caspase-3, signifying the apoptosis of hepatocytes after liver ischemia (Fig. 7e). The intensities were ~ 4.8-, and 6.6-fold higher than that of control mice (Fig. 7c). NIR-I fluorescence signals of FL1R in the kidney sections from mice subjected to 45 and 60 min liver ischemia were ~ 6.5-fold and 8.8-fold higher than that of control mice (Fig. 7d) while no or slightly enhancement was found in other groups, consistent with the real-time in vivo imaging results.

Multiplex imaging of HIRI and HIRI-induced AKI using two single-channel probes LIR and KIR

We then compared the performance of the unimolecular CAR with the co-injection of two monochromatic probes, LIR and KIR. The latter were simultaneously coadministered into mice subjected to liver ischemia at 4 h post-reperfusion (Fig. 8a), followed by longitudinal chemiluminescence imaging. In mice subjected to 30 min liver ischemia, co-injection of LIR and KIR led to a gradual NIR-II chemiluminescence signals increase in the liver, reaching their peaks at 30 min after LIR injection and decreasing at later time points (Fig. 8b, c). The liver signal was 21.8-fold higher than that in healthy controls at 30 min post-LIR injection (Fig. 8c), indicating O₂^•− upregulation during HIRI progression. A similar trend was observed in the liver of mice subjected to 45 and 60 min liver ischemia, while the liver signals increased by 29.6- and 41.3-fold relative to that of control mice (Fig. 8c). Ex vivo biodistribution studies indicated consistent results (Supplementary Fig. 21, 22).

Fig. 8: Real-time in vivo imaging of HIRI and HIRI induced-AKI using LIR and KIR.

a Schematic illustration of the development of the hepatic ischemia-reperfusion mouse model with different ischemia durations and chemiluminescence imaging after simultaneously injection of LIR and KIR (10 μmol kg⁻¹ body weight). b Representative NIR-II and NIR-I chemiluminescence images of living mice subjected to different ischemia durations (30, 45 and 60 min) after injection of LIR and KIR (10 μmol kg⁻¹ body weight). NIR-II chemiluminescence images were acquired with an acquisition time of 30 s. NIR-I chemiluminescence images were acquired under bioluminescence mode of the IVIS spectrum imaging system with the acquisition time of 60 s. c The dynamic NIR-II chemiluminescence intensities of liver (n = 3, mean ± s.d.). d the dynamic NIR-I chemiluminescence intensities of the kidney in living mice after injection of LIR and KIR (10 μmol kg⁻¹ body weight). (n = 3, mean ± s.d.). Two-tailed Student’s t test. Healthy group versus control and experimental groups (**p < 0.01, ***p < 0.001, ****p < 0.0001). N.S: no statistically significant differences. e A scheme diagram of cascade intrahepatic and intrarenal activation of CAR from blood circulation and following excretion. Injected CAR can firstly react with O₂^•− to undergo in situ fragmentation and release FL2R and KIR. The resulted FL2R would excreted through the hepatobiliary elimination pathway (liver→bile→intestine→feces); released KIR can accumulate in the kidneys and activated by upregulated NAG, liberating FL1R for non-invasive urinalysis (kidney→bladder→urine). f A scheme diagram of simultaneous injection of LIR and KIR and their independent intrahepatic and intrarenal activation from blood circulation and following excretion. Injected LIR can react with O₂^•− and excrete through the hepatobiliary elimination pathway; injected KIR can accumulate in the kidneys and activated by upregulated NAG, releasing FL1R for non-invasive urinalysis. g Comparison of the entire imaging window period of CAR and co-injection of LIR and KIR. Source data are provided as a Source Data file.

In mice subjected to 30 min liver ischemia, NIR-I chemiluminescence signals in the kidneys were almost undetectable, but they increased dramatically when liver ischemia duration increased to 45 and 60 min (Fig. 8b, d). However, the maximum kidney signals appeared at 15 min post-injection of KIR and rapidly decreased to background levels at 30 min post-injection, which differs from the performance of CAR that the maximum kidney signals were observed at 30 min post-injection and remained detectable even at 60 min (Fig. 6c). The short kidney imaging window for KIR is likely attributed to its short plasma elimination half-life (t_1/2β) of ~ 25 min after a bolus injection (Figs. 5h and 8f). In contrast, CAR, with its cascade activation mechanism, acted as a reservoir to release AKI reporters (KIR) upon reaction with O₂^•−, which circumvented its rapid clearance issue (Fig. 8e) and resulted in an extended imaging window that aligned with O₂^•− detection (Fig. 8g). Therefore, CAR outperforms coadministration of LIR and KIR for real-time and simultaneous imaging of HIRI and AKI in the liver-kidney axis.

Discussion

AKI is a common complication in the setting of HIRI; however, in vivo simultaneous optical imaging of HIRI and HIRI-induced AKI remains challenging because of the lack of unimolecular dual-responsive probes. Despite substantial progress made recently in developing dual-locked probes, their use for simultaneous detection of two intercorrelated diseases progress remains underexplored. To address this, unimolecular dual-responsive probes with spontaneous pharmacokinetic conversion for probing in multiple organs provide a promising strategy. To this end, we design a library of HCLs with tunable, long-wavelength chemiluminescence emission by incorporating benz[e]indolium, benz[c,d]indolium, naphtho[c,d]indolium and thiophen-benz[c,d]indolium moieties onto xanthonium-based bicyclic dioxetane to enlarged π conjugation (Supplementary Table 1). The representative chemiluminophores HCL1 and HCL5 with crosstalk-free emissions at 725 nm and 1025 nm are then respectively caged with NAG- and O₂^•− -responsive moieties and hooked together through a renal clearance unit to obtain a unimolecular cascaded activatable reporter CAR. In mouse models, CAR demonstrates liver-oriented accumulation and enables NIR-II CL imaging specific to HIRI-associated O₂^•−, which in turn initiates the in situ fragmentation of CAR and liberates KIR to enter the kidneys to image AKI-associated NAG via NIR-I CL. Notably, this cascaded activation allows CAR to act as a reservoir to slowly release KIR, which extends the imaging window for AKI detection and enables a synchronized window for both HIRI and associated AKI detections. Such alignment provides a more accurate diagnosis for the onset of complications, which is not possible with the coadministration of two separate probes, LIR and KIR. Furthermore, CAR permits remote detection of HIRI-induced AKI through either directly measuring excreted FL1R or incubating CAR with urine, serving as a ‘urine fingerprint’ to sensitively reflect the severity of HIRI. Thereby, CAR offers a powerful tool for diagnosing HIRI and HIRI-induced AKI in clinics, either through imaging inspections or point-of-care testing.

In summary, this study highlights a unimolecular dual-responsive chemiluminescent probe with crosstalk-free record-long emissions and switchable pharmacokinetics for the simultaneous diagnosis of two intercorrelated diseases. The flexible molecular design can be feasibly tailored to detect a diverse range of biomarkers, not only expanding the probe-box available for detection of intercorrelated diseases beyond the liver-kidney axis (for example, chemotherapy and its associated AKI, or interlinked diseases in the gut-liver axis) but also contributing to more accurate and early diagnosis of these diseases in clinical settings.

Methods

General Information

All chemicals were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai Bide Pharmatech Ltd, Energy Chemical Co., Ltd., Shanghai Macklin Biochemical Co., Ltd., unless otherwise stated. ALT activity assay kit, AST activity assay kit, IL-6 activity assay kit, TNF-α activity assay kit, creatinine assay kit and urea assay kit were purchased from Sigma-Aldrich. Silica gel (200–300 mesh) was used for column chromatography. Cleaved-caspase-3 antibody was purchased from Cell Signaling Technology. TLC was conducted on silica gel HSGF254 glass plates. UV-Vis absorption and fluorescence spectra were measure on a Shimadzu UV2600 spectrometer and a HORIBA Fluoromax-4 fluorescence spectrophotometer, respectively. Chemiluminescence was recorded on SpectraMax i3x (Molecular Devices, USA). HPLC was performed on a Shimadzu LC-16P system equipped with an Ascentis C18 HPLC Column (25 cm × 21.2 mm, 5 μm) using gradient elution. Proton-nuclear magnetic resonance (¹H NMR) was conducted with a Bruker 400 MHz NMR instrument. Chemical shifts are reported in ppm relative to residual protic solvent resonances. Mestre Nova LITE v12.0.1.20560 software (Mestre lab Research S.L.) was used to analyze the NMR spectra. All the NMR spectra and Mass spectra are provided in Supplementary Figs. 23–109. Electrospray ionization-mass spectrometry (ESI-MS) spectra were obtained on a Waters SQD2 with Waters H-Class UPLC equipped with a standard ESI source. Tissues were cut into sections using a cryostat (Leica, Germany) and examined using a Nikon ECLIPSE 80i microscope (Nikon Instruments Inc, USA). Confocal microscopy images of tissue sections were acquired on a FV3000 confocal laser scanning microscope (Olympus Corporation, Japan). NIR-I fluorescence and chemiluminescence imaging were performed on an IVIS spectrum imaging system (PerkinElmer, Inc, USA). NIR-II fluorescence and chemiluminescence imaging were carried out on a NIR-II imaging system (Shanghai Didi-United Biotech) at a power of 4 W with the detector of InGaAs, the light filter default to 1000 nm. Blood and urine samples were collected using heparinized capillary tubes and metabolic cages, respectively. MALDI-TOF analysis was performed on a Bruker ultraflex TOF/TOF instrument. All the images and elements in Figs. 1a, b, c, 5a, g, 6a, 7a, 8a, e, f, g and Supplementary Figs. 1a, 14a were created by PowerPoint, and no permission proof or license was required.

Synthesis of compound 1

Compound 1 was prepared according to the literature procedure⁶³. To a solution of 3,5-dimethoxybenzyl bromide (916 mg, 4 mmol) and compound 21 (800 mg, 5 mmol) in dry THF (20 mL) was added sodium hydride (240 mg, 10 mmol) at 0 °C, then the reaction mixture was stirred at room temperature overnight under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with EtOAc for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the intermediate as a colorless oil (1.18 g, yield of 95%). A solution of the intermediate (620 mg, 2 mmol) and PCC (473 mg, 2.2 mmol) in DCM (20 mL) was stirred at room temperature overnight under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 1 as a yellowish oil (604 mg, yield of 98%). ¹H NMR (400 MHz, CDCl₃) δ 6.45 (d, J = 2.4 Hz, 2H), 6.36 (t, J = 2.0 Hz, 1H), 4.43 (s, 2H), 3.78 (s, 6H), 3.49 (s, 2H), 1.28 (s, 6H), 1.24 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 170.58, 160.77, 141.22, 105.07, 99.44, 82.51, 77.81, 73.03, 55.25, 41.17, 37.00, 28.61, 23.47, 20.94. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₈H₂₈O₄ :308.20; Found 308.41.

Synthesis of compound 2

Compound 2 was prepared according to literature procedure⁶³. To a solution of compound 1 (308 mg, 1 mmol) in dry THF (10 mL) was added lithium diisopropylammonium (2 M in THF, 1.5 mL) at − 78 °C for 2 h under an Ar atmosphere. The reaction mixture was quenched with cold H₂O (15 mL) and extracted with EtOAc for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 2 as a yellowish oil (227 mg, yield of 74%). ¹H NMR (400 MHz, CDCl₃) δ 6.71 (d, J = 2.4 Hz, 2H), 6.37 (t, J = 2.4 Hz, 1H), 4.96 (s, 1H), 3.90 (d, J = 8.8 Hz, 1H), 3.78 (s, 6H), 3.69 (d, J = 8.8 Hz, 1H), 1.92 (s, 1H), 1.39 (s, 3H), 1.19 (s, 3H), 0.92 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 160.30, 149.86, 137.97, 125.29, 107.95, 100.50, 83.12, 55.34, 47.15, 32.42, 27.43. ESI-MS (m/z): [M+Na]⁺ Calcd for C₁₈H₂₈O₄ :331.20; Found 331.42.

Synthesis of compound 3

Compound 3 was prepared according to the literature procedure⁶³. To a solution of compound 2 (308 mg, 1 mmol) in dry DCM (10 mL) was added silver trifluoromethanesulfonate (258 mg, 1 mmol) and stirred for 30 min, followed by the addition of 1,1-dichlorodimethyl ether and stirred for 2 h under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 3 as a white solid (210 mg, yield of 68%). ¹H NMR (400 MHz, CDCl₃) δ 10.27 (s, 1H), 7.15 (d, J = 2.4 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H), 5.39 (s, 1H), 5.09 (s, 1H), 3.90 (d, J = 4.8 Hz, 6H), 3.68 (dd, J = 20.4, 8.8 Hz, 2H), 1.37 (s, 3H), 1.26 (s, 3H), 0.86 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 189.98, 165.37, 165.24, 146.31, 117.96, 106.93, 97.04, 88.96, 88.59, 79.69, 55.98, 55.63, 49.06, 40.75, 26.44, 23.83. ESI-MS (m/z): [M + Na]⁺ Calcd for C₁₉H₂₈O₅ :359.19; Found 359.50.

Synthesis of compound 4

Compound 4 was prepared according to the literature procedure⁶³. A solution of compound 3 (336 mg, 1 mmol), BBr₃ (500 mg, 2 mmol) and dry DCM (5 mL) was stirred at 0 °C for 2 h under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with EtOAc for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 4 as a yellow solid (226 mg, yield of 78%). ¹H NMR (400 MHz, CDCl₃) δ 11.97 (s, 1H), 9.83 (s, 1H), 6.35 (d, J = 4.4 Hz, 2H), 3.91 (s, 2H), 1.35 (s, 6H), 1.07 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 159.28, 156.72, 147.76, 138.89, 126.05, 108.17, 99.79, 83.41, 56.34, 55.62, 47.17, 32.56, 31.70, 27.03. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₇H₂₂O₄ :291.15; Found 291.26.

Synthesis of compound 5

A solution of compound 4 (290 mg, 1 mmol), Cs₂CO₃ (325 mg, 1 mmol), compound 25 (188 mg, 1 mmol) and DMF (3 mL) was stirred at room temperature for 12 h under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with EtOAc for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 4 as a yellow solid (137 mg, yield of 36%). ¹H NMR (400 MHz, CDCl₃) δ 10.35 (s, 1H), 6.72 (s, 1H), 6.63 (d, J = 2.0 Hz, 1H), 6.59 (s, 1H), 3.91 (s, 1H), 2.62 – 2.56 (m, 2H), 2.47 (t, J = 5.6 Hz, 2H), 1.74 (dd, J = 6.7, 3.2 Hz, 4H), 1.57 (s, 6H), 1.06 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 200.80, 166.52, 160.75, 141.15, 105.14, 99.45, 83.89, 78.22, 73.01, 55.27, 50.07, 41.15, 37.05, 30.48, 25.66, 23.56, 23.30. HRMS (m/z): [M]⁺ Calcd for C₂₄H₂₈O₄ :380.1988; Found 380.1623.

General procedure for the synthesis of compound HCLs

A solution of compound 5 (380 mg, 1 mmol), EtOH (10 ml), AcONa (82 mg, 1 mmol) and indole derivatives (1 mmol) was stirred at 60 °C for 2 h under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the intermediate. A solution of intermediate (0.1 mmol), methylene blue (5 mg, 0.015 mmol) and methanol (10 mL) was stirred and inserted an air pump needle into the mixed solution to blow O₂ in an ice bath for 2 h under white light. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure without purification to afford HCLs.

Compound HCL1

Purplish blue solid (368 mg, yield of 68%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.60 (d, J = 15.2 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.69 (d, J = 16.4 Hz, 1H), 7.55 (s, 2H), 7.49 (d, J = 8.0 Hz, 1H), 6.99 (s, 1H), 6.92 (d, J = 10.4 Hz, 1H), 6.54 (d, J = 14.8 Hz, 1H), 3.89 (s, 3H), 2.72 (d, J = 20.0 Hz, 4H), 1.86 (s, 2H), 1.79 (s, 6H), 0.88 (s, 17H). ¹³C NMR (101 MHz, DMSO) δ 160.83, 155.18, 143.05, 141.91, 135.29, 132.19, 132.06, 129.82, 129.13, 126.54, 122.96, 116.69, 115.07, 114.41, 112.77, 102.53, 67.88, 50.11, 40.64, 40.43, 40.22, 40.01, 39.80, 39.59, 39.38, 38.56, 32.57, 30.27, 28.83, 28.65, 27.88, 27.88, 24.13, 23.72, 22.86, 20.63, 14.36, 11.27. HRMS (m/z): [M]⁺ Calcd for C₃₆H₄₂NO₅⁺ :568.3507; Found 568.0858.

Compound HCL2

Purplish blue solid (482 mg, yield of 78%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.62 (d, J = 8.0 Hz, 1H), 8.44 (d, J = 8.4 Hz, 1H), 8.34 (d, J = 8.8 Hz, 1H), 8.25 (d, J = 11.6 Hz, 2H), 8.10 (d, J = 8.8 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.81 (s, 2H), 7.74 (d, J = 15.2 Hz, 1H), 7.35 (s, 1H), 3.97 (s, 3H), 2.96 (s, 4H), 2.03 (s, 2H), 1.97 (s, 6H), 0.88 (s, 17H). ¹³C NMR (101 MHz, DMSO) δ 188.13, 182.37, 148.54, 147.95, 143.97, 139.11, 136.72, 136.58, 135.45, 133.65, 133.09, 132.51, 132.29, 132.17, 131.60, 130.95, 130.00, 129.74, 129.64, 129.48, 129.06, 126.08, 125.93, 124.65, 116.27, 113.09, 105.24, 103.59, 97.26, 57.60, 57.49, 32.63, 26.39, 26.21, 20.49. HRMS (m/z): [M]⁺ Calcd for C₄₀H₄₄NO₅⁺ :618.3214; Found [M + H]⁺ 619.3244.

Compound HCL3

Purplish green solid (294 mg, yield of 51%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.09 (d, J = 14.8 Hz, 1H), 8.75 (d, J = 7.6 Hz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.09 (s, 1H), 7.99 (s, 1H), 7.88 (s, 1H), 7.57 (s, 1H), 7.43 – 7.35 (m, 1H), 7.23 (s, 1H), 7.14 – 7.07 (m, 1H), 6.99 (d, J = 14.8 Hz, 1H), 4.57 (s, 3H), 1.87 (s, 4H), 1.56 – 1.50 (m, 2H), 1.23 (s, 17H). ¹³C NMR (101 MHz, DMSO) δ 152.08, 148.07, 143.45, 141.53, 129.56, 128.90, 128.61, 123.46, 120.28, 114.95, 111.48, 61.49, 53.67, 51.82, 35.49, 35.29, 32.44, 32.05, 29.45, 28.12, 27.64, 27.42, 26.77, 25.55, 24.90, 24.07, 20.06, 12.25. HRMS (m/z): [M]⁺ Calcd for C₃₇H₃₈NO₅⁺ :576.2477; Found 576.5732.

Compound HCL4

Purplish brown solid (206 mg, yield of 33%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.19 (d, J = 7.2 Hz, 1H), 9.10 (d, J = 14.8 Hz, 1H), 8.97 (d, J = 8.4 Hz, 1H), 8.63 (s, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.26 (d, J = 6.4 Hz, 1H), 8.14 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 5.2 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 3.6 Hz, 1H), 7.61 (s, 1H), 7.56 (m, 1H), 7.31 (d, J = 15.2 Hz, 1H), 3.80 (s, 3H), 2.07 (s, 4H), 1.65 (s, 2H), 1.03 (s, 17H). ¹³C NMR (101 MHz, DMSO) δ 172.76, 143.56, 142.74, 142.51, 141.56, 141.09, 130.05, 129.11, 126.75, 125.68, 125.03, 123.04, 122.72, 122.54, 116.55, 112.02, 110.90, 102.16, 50.72, 50.66, 49.50, 48.61, 29.32, 28.07, 27.97, 26.35, 26.06, 25.96, 24.78), 24.62, 24.38, 23.85, 21.56. HRMS (m/z): [M]⁺ Calcd for C₄₁H₄₀NO₅⁺ :626.2744; Found 625.1776.

Compound HCL5

Purplish brown solid (157 mg, yield of 24%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (d, J = 13.2 Hz, 1H), 8.24 (dd, J = 16.0, 7.8 Hz, 2H), 7.78 (s, 2H), 7.54 (dd, J = 13.6, 6.3 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.28 (s, 1H), 7.23 (d, J = 7.6 Hz, 1H), 7.13 – 7.08 (m, 1H), 6.73 (s, 1H), 6.39 (d, J = 12.4 Hz, 1H), 4.06 (s, 3H), 2.32 (s, 4H), 1.27 (s, 2H), 1.05 (s, 17H). ¹³C NMR (101 MHz, DMSO) δ 178.22, 160.86, 160.79, 153.90, 142.72, 141.77, 133.41, 129.34, 129.24, 127.89, 127.62, 123.29, 116.69, 115.03, 114.50, 113.76, 105.07, 103.91, 101.75, 76.45, 73.80, 70.66, 68.48, 60.90, 50.99, 50.61, 49.03, 44.76, 28.88, 28.03, 27.96, 25.86, 25.21, 23.97, 20.40. HRMS (m/z): [M]⁺ Calcd for C₄₁H₄₀NO₅S⁺ :658.2622; Found [M + H]⁺ 659.2445.

Synthesis of compound 8

A solution of compound 6 (62 mg, 0.1 mmol), compound 22 (41 mg, 0.1 mmol), K₂CO₃ (27 mg, 0.2 mmol), Cs₂CO₃(33 mg, 0.1 mmol) and DCM (3 mL) was stirred at room temperature for 2 h under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure to afford the intermediate. It could be directly put into the next step of the reaction without purification. A solution of the intermediate (0.1 mmol), NaOMe (27 mg, 0.5 mmol) and DCM (3 mL) was stirred at room temperature for 1 h under an Ar atmosphere. The reaction mixture was quenched with NH₄Cl (50 mL) and extracted with DCM for three times. Compound 7 was afforded by reduced-pressure concentration. A solution of compound 7 (41 mg, 0.05 mmol), SH-PEG₂₀₀₀-alkyne (85 mg, 0.06 mg), sodium ascorbate (20 mg, 0.1 mmol), CuSO₄ (16 mg, 0.1 mmol), H₂O (2 mL) and THF (2 mL) was stirred at room temperature for 2 h. The reaction mixture was quenched with H₂O (50 mL) and extracted with EtOAc for three times. The crude product was concentrated by cooling under reduced pressure and purified by HPLC to afford compound 8 as a blue solid. ¹H NMR (400 MHz, D₂O) δ 8.52 (d, J = 13.6 Hz, 1H), 7.65 (d, J = 7.2 Hz, 1H), 7.50 (dd, J = 17.6, 10.2 Hz, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.15 (s, 1H), 7.02 (d, J = 11.6 Hz, 2H), 6.36 (d, J = 15.2 Hz, 1H), 4.22 (s, 15H), 3.89 (s, 16H), 3.68 (s, 600H), 3.29 (t, J = 6.4 Hz, 4H), 2.70 – 2.58 (m, 12H), 1.81 (d, J = 6.0 Hz, 3H), 1.71 (d, J = 14.4 Hz, 6H), 0.87 (d, J = 15.2 Hz, 17H). ¹³C NMR (101 MHz, DMSO) δ 177.16, 162.76, 162.20, 161.54, 154.60, 144.99, 142.30, 142.19, 141.93, 141.93, 135.65, 134.97, 134.68, 129.72, 129.32, 127.18, 126.23, 124.87, 123.26, 115.13, 114.88, 114.26 – 114.07, 113.43, 103.98, 102.41, 81.85, 73.39, 69.23, 63.95, 57.77, 53.66, 51.03, 50.63, 36.25, 31.23, 30.22, 28.77, 28.51, 28.55 – 27.84, 28.06, 27.61, 26.31, 26.02, 25.26, 24.08, 20.48, 19.57, 18.21, 13.29, 1.64. MALDI-TOF MS found: ~ 2951.631.

Synthesis of compound 10

A solution of compound 9 (63 mg, 0.1 mmol), compound 23 (30 mg, 0.1 mmol), compound 24 (20 mg, 0.1 mmol), triphosgene (90 mg, 0.3 mmol), DMAP (25 mg, 0.2 mmol) and DCM (5 mL) was stirred at 0 °C for 2 h under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure to afford compound 10 (100 mg, yield of 85%. It could be directly put into the next step of the reaction without purification. ¹H NMR (500 MHz, DMSO-d₆) δ 8.58 (d, J = 12.5 Hz, 1H), 8.29 (d, J = 7.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.11 (s, 1H), 7.80 (s, 1H), 7.77 (s, 1H), 7.54 (d, J = 4.5 Hz, 2H), 7.38 (d, J = 8.5 Hz, 1H), 7.27 (s, 1H), 7.25 (s, 1H), 7.23 (s, 1H), 7.10 (s, 1H), 6.73 (d, J = 8.5 Hz, 2H), 6.52 – 6.47 (m, 1H), 6.41 (d, J = 13.5 Hz, 1H), 4.09 (s, 3H), 2.66 – 2.58 (m, 6H), 2.33 (s, 4H), 1.70 (s, 2H), 1.62 (s, 2H), 1.53 (s, 2H), 1.05 (s, 17H). ¹³C NMR (101 MHz, DMSO) δ 190.75, 184.98, 151.22, 150.59, 146.60, 141.72, 139.35, 139.27, 139.18, 138.09, 136.24, 135.72, 135.14, 134.91, 134.81, 134.22, 133.66 – 133.45, 132.64, 132.27, 132.09, 131.18, 128.68, 128.52, 127.25, 118.87, 115.68, 107.83, 106.20, 43.77, 39.54, 36.48, 35.26, 35.17, 31.70, 31.52, 30.85, 30.13, 29.03, 28.84. HRMS (m/z): [M]⁺ Calcd for C₆₃H₆₅F₃N₃O₁₂S₂⁺ :1176.3800; Found [M + H]⁺ 1177.4300.

Synthesis of compound CAR

A solution of compound 8 (0.1 mmol), compound 10 (0.1 mmol), Et₃N (0.5 mL) and THF (3 mL) was stirred at room temperature for 3 h under an Ar atmosphere. The reaction mixture was concentrated under reduced pressure to afford the intermediate. A solution of intermediate (0.1 mmol), methylene blue (5 mg, 0.015 mmol) and methanol (10 mL) was stirred and inserted an air pump needle into the mixed solution to blow O₂ in ice bath for 2 h under white light. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined water layer was concentrated under reduced pressure and purified by HPLC to afford compound CAR. ¹H NMR (400 MHz, DMSO-d₆) δ 8.63 (s, 1H), 8.18 (d, J = 8.0 Hz, 14H), 7.68 (s, 5H), 7.56 (s, 2H), 7.49 (d, J = 11.6 Hz, 1H), 6.97 (s, 2H), 6.55 (d, J = 14.8 Hz, 1H), 4.23 (s, 119H), 3.91 (s, 36H), 3.66 (dd, J = 8.4, 4.8 Hz, 70H), 3.41 (s, 39H), 3.24 (s, 32H), 1.95 (s, 70H), 0.88 (s, 40H). ¹³C NMR (126 MHz, DMSO) δ 188.12, 183.99, 182.38, 165.71, 158.87, 158.70, 148.60, 147.96, 144.00, 142.71, 139.11, 136.74, 136.58, 135.47, 133.64, 133.11, 132.53, 132.31, 132.27, 132.19, 131.62, 130.95, 130.02, 129.74, 129.66, 129.49, 129.23, 128.91, 126.08, 125.92, 124.65, 116.28, 113.09, 105.23, 103.61, 84.10, 75.60, 71.54, 71.54, 66.23, 65.98, 62.41, 62.18, 58.78, 57.61, 57.56, 47.67, 41.18, 40.15, 37.20, 36.93, 33.87, 32.60, 32.79 – 32.15, 32.47, 29.09, 28.93, 28.64, 28.40, 28.22, 28.17, 28.07, 27.52, 26.41, 26.22, 23.17, 21.27, 20.77, 20.63, 20.39, 20.26. MALDI-TOF MS found: ~4158.

Synthesis of compound LIR

A solution of compound 9 (63 mg, 0.1 mmol), trifluoromethane-sulfonic anhydride (30 mg, 0.1 mmol), pyridine (0.5 mL) and DCM (3 mL) was stirred at room temperature for 2 h under an Ar atmosphere. The reaction mixture was concentrated under reduced pressure to afford the intermediate. A solution of intermediate (0.1 mmol), methylene blue (5 mg, 0.015 mmol) and methanol (10 mL) was stirred and inserted an air pump needle into the mixed solution to blow O₂ in an ice bath for 2 h under white light. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The purification using column chromatography on silica gel was performed to afford the compound LIR (52 mg, yield of 66%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.03 – 8.74 (m, 2H), 8.42 (s, 1H), 8.25 (s, 1H), 8.12 (s, 2H), 7.93 (s, 2H), 7.69 (s, 2H), 7.40 (s, 2H), 7.10 (s, 1H), 4.69 (s, 3H), 3.55 (s, 2H), 2.80 (s, 2H), 1.87 (d, J = 32.4 Hz, 4H), 1.50 (s, 6H), 0.88 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 178.22, 160.87, 160.83, 153.90, 145.71, 142.72, 141.77, 133.41, 129.29, 129.29, 127.92, 127.62, 123.28, 116.70, 115.02, 114.51, 113.75, 105.06, 103.93, 101.74, 76.45, 73.77, 70.65, 68.52, 60.93, 50.98, 50.60, 49.04, 44.75, 28.88, 28.02, 27.95, 25.86, 25.20, 23.96, 20.40. HRMS (m/z): [M]⁺ Calcd for C₄₂H₃₉F₃NO₇S₂⁺ :790.2115; Found [M + H]⁺ 791.2142.

Synthesis of compound KIR

A solution of compound 8 (0.1 mmol), compound 24 (20 mg, 0.1 mmol), Et₃N (0.5 mL) and THF (5 mL) was stirred at room temperature for 2 h under an Ar atmosphere. The reaction mixture was concentrated under reduced pressure to afford the intermediate. A solution of intermediate (0.1 mmol), methylene blue (5 mg, 0.015 mmol) and methanol (10 mL) was stirred and inserted an air pump needle into the mixed solution to blow O₂ in an ice bath for 2 h under white light. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The purification using column chromatography on silica gel was performed to afford the compound KIR. ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (d, J = 14.8 Hz, 1H), 7.81 (s, 1H), 7.78 (s, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 7.2 Hz, 1H), 7.23 (s, 1H), 7.12 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 14.8 Hz, 1H), 5.02 (d, J = 7.6 Hz, 1H), 4.51 (t, J = 7.2 Hz, 3H), 4.10 (s, 10H), 3.80 (s, 9H), 3.58 (d, J = 18.4 Hz, 822H), 3.29 (s, 9H), 3.11 (s, 3H), 1.81 (d, J = 6.8 Hz, 13H), 0.88 (s, 18H). ¹³C NMR (126 MHz, DMSO) δ 178.19, 170.34, 160.85, 160.81, 156.58, 156.43, 153.90, 145.68, 142.71, 141.75, 133.45, 129.34, 129.29, 127.87, 127.62, 123.29, 116.68, 115.04, 114.49, 113.74, 105.05, 103.89, 101.75, 81.84, 76.45, 73.79, 70.64, 69.29, 69.22, 68.47, 63.95, 63.66, 60.89, 50.98, 50.59, 49.03, 44.73, 44.23, 30.19, 27.98, 27.94, 25.85, 25.21, 24.09, 20.40. MALDI-TOF MS found: ~ 3147.782.

Synthesis of compound 11

Compound 11 was prepared according to literature procedure⁶⁴. A solution of 2,3,3-Trimethylindolenine (160 mg, 1 mmol) and methyl iodide (288 mg, 2 mmol) was stirred at room temperature for 6 h under an Ar atmosphere. The mixture was added to the diethyl ether (50 mL), the solid was filtered and washed with diethyl ether for three times to afford compound 11 (170 mg, yield of 98%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.92 (dd, J = 6.0, 3.2 Hz, 1H), 7.85 – 7.81 (m, 1H), 7.64 – 7.61 (m, 2H), 3.98 (s, 3H), 2.78 (s, 3H), 1.53 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 196.47, 142.09, 129.77, 129.29, 123.81, 115.64, 54.43, 35.41, 22.22, 14.93. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₂H₁₆N⁺ :174.13; Found 174.17.

Synthesis of compound 12

A solution of 1,1,2-Trimethyl-1H-benzo[e]indole (209 mg, 1 mmol) and methyl iodide (288 mg, 2 mmol) was stirred at room temperature for 6 h under an Ar atmosphere. The mixture was added to the diethyl ether (50 mL), the solid was filtered and washed with diethyl ether for three times to afford compound 12 (197 mg, yield of 88%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.37 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 8.8 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.10 (d, J = 8.8 Hz, 1H), 7.81 – 7.76 (m, 1H), 7.73 (dd, J = 8.0, 1.2 Hz, 1H), 4.09 (s, 3H), 2.87 (s, 3H), 1.75 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 196.38, 139.97, 136.97, 133.48 – 132.68, 130.98, 130.22, 128.86, 127.59, 123.90, 113.67, 55.74, 35.74, 21.78, 14.68. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₆H₁₈N⁺ :224.14; Found 224.17.

Synthesis of compound 13

Compound 13 was prepared according to the literature procedure⁶⁴. A solution of benz[cd]indol-2(1H)-one (169 mg, 1 mmol), methyl iodide (288 mg, 2 mmol), THF (10 mL) and sodium hydride (96 mg, 4 mmol) was stirred at 0 °C under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with EtOAc for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 13 (168 mg, yield of 92%). ¹H NMR (400 MHz, CDCl₃) δ 8.06 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.73 – 7.67 (m, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.47 (dd, J = 8.4, 7.0 Hz, 1H), 6.90 (d, J = 6.8 Hz, 1H), 3.45 (s, 3H). ¹³C NMR (126 MHz, MeOD) δ 138.89, 134.83, 131.20, 130.88, 129.65, 120.52, 32.96. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₂H₉NO :183.07; Found 184.13.

Synthesis of compound 14

Compound 14 was prepared according to the literature procedure⁶⁴. A solution of compound 13 (183 mg, 1 mmol), THF (5 mL) and methylmagnesium bromide (2.5 mL) was stirred at 50 °C under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 14 (134 mg, yield of 74%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.87 (d, J = 76.4 Hz, 2H), 8.47 (d, J = 23.6 Hz, 2H), 8.07 (d, J = 60.4 Hz, 2H), 4.22 (s, 3H), 3.20 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 130.64, 128.91, 128.57, 128.44, 126.70, 125.02, 124.10, 120.25, 104.62, 26.28. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₃H₁₂N⁺ :182.10; Found 182.08.

Synthesis of compound 15

Compound 15 was prepared according to the literature procedure⁶⁵. A solution of indolin-2-one (133 mg, 1 mmol), o-bromobenzaldehyde (368 mg, 2 mmol), Cs₂CO₃ (495 mg, 1.5 mmol) and DMSO (10 mL) was stirred at 120 °C under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with EtOAc for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 15 (113 mg, yield of 52%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.68 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.53 (d, J = 6.0 Hz, 1H), 7.44 (t, J = 8.4 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H), 6.80 (t, J = 7.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ 168.56, 143.59, 135.29, 134.10, 133.43, 131.84, 131.08, 130.85, 129.67, 128.44, 123.61, 123.03, 121.76, 120.99, 110.76. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₅H₉NO :219.07; Found 221.11.

Synthesis of compound 16

Compound 16 was prepared according to the literature procedure⁶⁵. A solution of compound 15 (219 mg, 1 mmol), THF (5 mL) and methylmagnesium bromide (2.5 mL) was stirred at 50 °C under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the intermediate compound. A solution of the intermediate compound (1 mmol), THF (5 mL) and methylmagnesium bromide (2.5 mL) was stirred at 50 °C under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure. It could be directly put into the next step of the reaction without purification. ¹H NMR (500 MHz, DMSO) δ 7.82 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.55 (t, J = 7.5 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 6.88 (t, J = 7.5 Hz, 1H), 3.22 (s, 3H), 1.99 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 130.64, 128.91, 128.57, 128.44, 126.70, 125.02, 124.10, 120.25, 26.28. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₇H₁₄N⁺ :232.11; Found [M+Na]⁺ 255.65.

Synthesis of compound 17

Compound 17 was prepared according to the literature procedure⁶⁵. A solution of compound 13, N-Bromosuccinimide (178 mg, 1 mmol) and MeCN (5 mL) was stirred at room temperature under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with EtOAc for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 17 (179 mg, yield of 79%). ¹H NMR (400 MHz, CDCl₃) δ 8.15 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 7.2 Hz, 1H), 7.80 (t, J = 6.4 Hz, 1H), 7.68 (d, J = 7.6 Hz, 1H), 6.76 (d, J = 7.6 Hz, 1H), 3.43 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.45, 139.64, 131.10, 130.11, 129.59, 128.66, 126.86, 125.93, 124.97, 113.98, 105.60, 26.32. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₂H₈BrNO :260.18; Found 261.27.

Synthesis of compound 18

A solution of compound 17 (261 mg, 1 mmol), thiophene (168 mg, 2 mmol), K₂CO₃ (270 mg, 1 mmol), Pd(OAc)₂ (22.4 mg, 0.1 mmol) and DMAC (10 mL) was stirred at 120 °C under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with EtOAc for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 18 (111 mg, yield of 42%). ¹H NMR (400 MHz, CDCl₃) δ 8.38 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 6.8 Hz, 1H), 7.76 (d, J = 7.2 Hz, 1H), 7.59 (d, J = 7.2 Hz, 1H), 7.42 (d, J = 5.2 Hz, 1H), 7.28 (d, J = 2.8 Hz, 1H), 7.20 (dd, J = 5.2, 3.6 Hz, 1H), 6.93 (d, J = 7.2 Hz, 1H), 3.47 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 170.16, 167.43, 140.43, 139.09, 130.23, 130.18, 129.82, 128.69, 127.38, 127.10, 127.01, 126.31, 126.17, 125.22, 125.14, 106.69, 41.91. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₆H₁₁NOS :265.06; Found 266.19.

Synthesis of compound 19

Compound 19 was prepared according to the literature procedure⁶⁵. A solution of compound 18 (265 mg, 1 mmol), THF (5 mL) and methylmagnesium bromide (2.5 mL) was stirred at 50 °C under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure. It could be directly put into the next step of the reaction without purification.

Synthesis of compound 20

Compound 20 was prepared according to literature procedure⁶⁵. A solution of compound 1-Methoxy-1-(trimethylsilyloxy)-2-methyl-1-propene (870 mg, 5 mmol), trimethylacetyl chloride (660 mg, 5.5 mmol), TiCl₄ (1.13 g, 6 mmol) and dry DCM (20 mL) was stirred at 0 °C under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 20 (178 mg, yield of 96%). ¹H NMR (400 MHz, CDCl₃) δ 3.72 (s, 3H), 1.39 (s, 6H), 1.19 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 52.51, 50.15, 43.50, 26.44, 25.07, 21.60, 19.95. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₀H₁₈O₃ :186.02; Found 187.24.

Synthesis of compound 21

Compound 21 was prepared according to literature procedure⁶³. A solution of compound 20 (744 mg, 4 mmol) and LiAlH₄ (444 mg, 12 mmol) in DCM (20 mL) was stirred at room temperature overnight under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure to afford compound 21 as a colorless oil (627 mg, yield of 98%). It could be directly put into the next step of the reaction without purification. ¹H NMR (400 MHz, CDCl₃) δ 3.52 (d, J = 10.8 Hz, 1H), 3.43 (d, J = 10.4 Hz, 1H), 3.30 (s, 1H), 2.99 (s, 2H), 1.04 (s, 9H), 1.04 (s, 3H), 1.03 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 85.41, 74.70, 40.44, 37.28, 28.48, 26.04, 25.51, 20.68. ESI-MS (m/z): [M + H]⁺ Calcd for C₉H₂₀O₂ :160.15; Found 183.15.

Synthesis of compound 22

Compound 22 was prepared according to the literature procedure⁶⁵. A solution of compound β-D-Glucosamine pentaacetate (389 mg, 1 mmol), HBr/HOAc (3.2 mL) and HOAc was stirred at 0 °C under an Ar atmosphere. The reaction mixture was quenched with sodium bicarbonate solution (50 mL) and extracted with DCM for three times. The crude product was concentrated, and it could be directly put into the next step of reaction without purification to afford compound 22 (359 mg, yield of 88%). ¹H NMR (400 MHz, CDCl₃) δ 5.85 (s, 1H), 5.71 (d, J = 8.8 Hz, 1H), 5.17 (dd, J = 21.2, 11.2 Hz, 2H), 4.29 (d, J = 9.2 Hz, 1H), 4.14 (d, J = 14.4 Hz, 1H), 3.84 (d, J = 9.6 Hz, 1H), 2.09 (s, 3H), 2.05 (s, 6H), 1.94 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.23, 170.68, 170.16, 169.56, 169.29, 92.57, 72.74, 72.64, 67.89, 61.71, 52.94, 23.14, 20.88, 20.64. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₄H₂₀BrNO₈ :409.04; Found 410.29.

Synthesis of compound 23

Compound 23 was prepared according to the literature procedure⁶⁶. A solution of 2,6-tert-butyldimethylsilyl-4-methylphenyl trifluoro-methanesulfonate (528 mg, 1 mmol), TBAF (574 mg, 2.2 mmol) and THF (5 mL) was stirred at room temperature under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with EtOAc for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 23 (288 mg, yield of 96%). ¹H NMR (400 MHz, CDCl₃) δ 7.38 (s, 2H), 4.75 (s, 4H), 2.40 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.42, 141.19, 135.16, 124.07, 53.29, 21.02. ESI-MS (m/z): [M-H]⁻ Calcd for C₁₀H₁₂F₃O₅S 300.03; Found [M + K]⁻ 338.43.

Synthesis of compound 24

A solution of tert-butyl (6-aminohexyl) carbamate (216 mg, 1 mmol), maleic anhydride (100 mg, 1.1 mmol), NaOAc (82 mg, 1 mmol) and Ac₂O (5 mL) was stirred at 120 °C under an Ar atmosphere. The reaction mixture was quenched with H₂O (50 mL) and extracted with DCM for three times. The combined organic layer was concentrated under reduced pressure. The purification using column chromatography on silica gel was performed to afford the compound 24 (133 mg, yield of 68%). ¹H NMR (400 MHz, CDCl₃) δ 6.71 (s, 2H), 3.52 (t, J = 7.2 Hz, 2H), 2.35 (t, J = 7.2 Hz, 2H), 1.64 (dtd, J = 22.4, 15.1, 7.6 Hz, 6H), 1.35 (dd, J = 15.6, 8.4 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 179.65, 170.91, 134.08, 37.58, 33.79, 28.15, 26.10, 24.08. ESI-MS (m/z): [M + H]⁺ Calcd for C₁₀H₁₆N₂O₂ :196.12; Found [M+Na]⁺ 219.224.

Synthesis of compound 25

A solution of cyclohexanone (98 mg, 1 mmol), PBr₃ (1 mL), DCM (5 mL) and DMF (1.5 mL) was stirred at 0 °C under an Ar atmosphere. The reaction mixture was quenched with sodium bicarbonate solution (50 mL) and extracted with DCM for three times. The crude product was concentrated, and it could be directly put into the next step of reaction without purification to afford compound 25 (165 mg, yield of 88%). ¹H NMR (400 MHz, CDCl₃) δ 10.02 (s, 1H), 2.79 – 2.71 (m, 2H), 2.31 – 2.24 (m, 2H), 1.80 – 1.73 (m, 2H), 1.73 – 1.65 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 193.69, 143.67, 135.23, 38.80, 24.97, 24.24, 21.06. ESI-MS (m/z): [M + H]⁺ Calcd for C₇H₉BrO :187.98; Found 188.05.

Preparation of stock solution

CAR (10 µM) was dissolved in PBS (10 mM, pH 7.4) to obtain a stock solution after filtration by using a syringe filter (0.22 μm). H₂O₂, HClO, and O₂⁻ stock solution was prepared by directly diluting H₂O₂, NaClO, and KO₂, respectively. •OH was generated by Fenton reaction between H₂O₂ and Fe(ClO₄)₂. Stock solutions of NaCl, KCl, MgSO₄, glutathione, β-galcatosidase, gamma-glutamyl transferase and N-acetyl-β-D-glucosaminidase were prepared with distilled water.

Sensing capability

The CAR solution (20 µM) was incubated with KO₂ (80 µM) and NAG (40 mU) in PBS (10 mM, pH = 7.4). Absorption and fluorescence spectra of the solution were measured on a UV-VIS and fluorescence spectrophotometer. NIR-I fluorescence images were acquired using the IVIS spectrum imaging system with the excitation at 675 ± 10 nm, the emission at 720 ± 10 nm and the acquisition time of 0.1 s. NIR-II fluorescence images were acquired using the NIR-II imaging system with the excitation at 808 nm, the emission at 1000 ± 10 nm and the acquisition time of 0.1 s. Chemiluminescence images were acquired under bioluminescence mode with an open filter and an acquisition time of 30 s.

Selectivity studies

The CAR solution (20 µM) was incubated ROS (80 µM), metal ions (80 µM) and enzymes including glutathione (1.0 U), β-galcatosidase (1.0 U), gamma-glutamyl transferase (1.0 U) and N-acetyl-β-D-glucosaminidase (40 mU) in HEPES buffer (50 mM, pH 7.4) or PBS (10 mM, pH 7.4) at 37 °C for 120 min. Fluorescence and chemiluminescence enhancements of CAR were measured after incubation.

The limit of detection (LOD)

Chemiluminescence intensities of CAR (20 µM) were determined after the addition of different concentrations of KO₂. The LOD (Limit of Detection) was calculated based on the equation: LOD = 3σ/k, where σ is the standard deviation of emission intensity of blank, and k is the slope of the plot of emission intensities against the concentration of KO₂.

Chemiluminescence kinetic profiles

KO₂ (80 µM) was added into the CAR solution (20 µM) in PBS (10 mM, pH = 7.4). Chemiluminescence intensities were continuously acquired and plotted as a function of time.

Enzymatic kinetics assays

CAR (10, 20, 30, 60 and 90 µM) were incubated with NAG (10 mU) at 37 °C. The cleavage compound was quantified using HPLC after incubation. The initial reaction velocity (µM min⁻¹) was calculated, plotted against the concentration of CAR and fitted to a Michaelis-Menten curve. The kinetic parameters were calculated by the following equation: V = Vmax[S] / (Km + [S]), where V is the initial velocity, and [S] is the substrate concentration.

Thermal stability

BD-OTf or AD-OTf (20 µM) was dissolved in FBS solution and placed in 13 tubes with the same concentration and stored at room temperature in dark. Chemiluminescence intensities were recorded for each sample after the addition of KO₂ (80 µM) at different storage time points.

pH Stability

CAR solution (20 µM) was incubated in different buffer solution with a pH range from 4.0 to 8.5 at 37 °C for 3 h. Fluorescence intensity of CAR was measured on IVIS spectrum imaging system or NIR-II imaging system after incubation.

Histological examination

All tissues were fixed with 4% paraformaldehyde (PFA), dehydrated in ethanol solution, embedded in paraffin, and cut into 10 μm thick sections for H&E staining. Paraffin was removed by washing with xylene, and sections were incubated with hematoxylin for 4 min and eosin for 2 min, followed by washing with distilled water. Stained sections were examined using the FL Automated Imaging System + EVOS® on-stage incubator. Mouse peritoneum was snap frozen in OCT (Tissue-Tek) and stored at − 20 °C until sectioning. Frozen samples were cryosected (7-8 μm) using a freezer equipped with a cryotape transfer system and a tungsten blade. Sections were dried at room temperature for 2-4 h and then frozen at − 20 °C until staining. Prior to staining, sections were fixed with 100% acetone at − 20 °C for 10 min, dried at 37 °C for 5 min, and closed with 10% goat serum (Boster 17I27C04) for 90 min at room temperature, followed by washing with PBS. Sections were then first incubated with rat anti-mouse Ly-6G (Gr-1) (Proteintech, 65140-1-lg, 1:200) primary antibody overnight, washing with PBS to remove unbound antibody. Followed by goat anti-rat-lgG H&L (Alexa Fluor®488) (Abcam, ab150157, 1:200) secondary antibody for 1 h and washed three times with PBS for 5 min at room temperature. These washing steps were also performed between each staining step. After staining, all sections were stained with 50 μL DAPI (Solarbio C0065) for 5 min at room temperature and washed 4 times with PBS for 5 min. Slides were mounted with neutral balsam (Sinopharm Chemical Reagent Co, Ltd.). The images were then imaged using an SP5 upright or SP8 inverted confocal microscope (Olympus FV3000) with a 20x objective. Images were processed using Olympus Micro software (FV31S-SW) and analyses with Image J software.

Tissue-penetration studies

BD-OTf solution (40 µM), HCL1-OTf solution (40 µM) and HCL5-OTf solution (40 µM) in PBS (10 mM, pH 7.4) were place into the wells of a black 96-well plate. Chicken tissue with the desired thickness was covered on the top of the wells. After the addition of KO₂ (80 µM), chemiluminescence images were acquired using the IVIS spectral imaging system in bioluminescence mode with an open filter and an acquisition time of 30 s. The NIR-II chemiluminescence acquisition of time was 30 s. The SBR was calculated as SBR. The fluorescence images were acquired at an excitation wavelength of 400 ± 10 nm, 670 ± 10 nm or 808 nm and an emission wavelength of 450 ± 10 nm, 720 ± 10 nm or 1000 ± 10 nm with an acquisition time of 0.1 s. The SBR was calculated as SBR = fluorescence intensity (or chemiluminescence intensity)/background, where background is the signal intensity of the adjacent tissue acquired during imaging. The SBRs were plotted as a function of tissue depth.

TEM Sample Preparation

CAR (1 µM) was incubated with KO₂ (4 µM) in phosphate-buffered solution (1 × PBS, pH = 7.4), the carbon-coated side of the grid was gently immersed in the dispersion, the photo was taken by a Hitachi TEM system. Magnification = 40000, Accelerating Voltage = 100000.

Computational details

The geometric optimization and vibrational frequency analysis were performed using density functional theory (DFT) at the B3LYP/6-311 G(d,p) level with Grimme’s D3(BJ) dispersion correction (GD3BJ) in the Gaussian 16 program package. The SMD implicit solvation model with DMSO as the solvent was applied during these calculations. Optimized structures were confirmed as true minima by the absence of imaginary frequencies. Excited-state properties of the first excited state were investigated using time-dependent density functional theory (TD-DFT) at the same level. Frontier molecular orbitals were visualized using the IQmol molecular viewer.

Pharmacokinetic studies

All experimental mice were approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University (SYSU) with the approval number SYSU-IACUC-2022-001938. 8-week-old male mice were selected to be injected with CAR, LIR and KIR (2.5 µmol kg⁻¹ body weight) intravenously. Before injection, the tail of the mice was cut and blood was drawn with heparin capillary. After injection of CAR, LIR and KIR, blood was taken at 1, 4, 9, 16, 30, 60, 120, 240, 720 and 1440 min respectively. The collected blood samples were stored in an ice box to prevent coagulation, and then centrifuged at 2247 x g for 15 min. CAR, LIR and KIR in the blood were quantified using HPLC and plotted as a function of time to calculate the elimination half-life value (t_1/2β).

Renal clearance studies

Male mice were i.v. injected with CAR (2.5 µmol kg⁻¹ body weight), LIR (2.5 µmol kg⁻¹ body weight) and KIR (2.5 µmol kg⁻¹ body weight) and placed in metabolic cages. Feces was collected at 7 d post-injection, and urine was collected at 24 h post-injection, diluted in PBS and centrifuged 2247 x gfor 10 min and filtered by 0.22 µm syringe filter. CAR, LIR and KIR in the urine were quantified using HPLC. Then, determine the UV absorption and fluorescence spectrum in urine, and then sacrificed the mice and collect the main organs, homogenized in PBS buffer (10 mM, pH = 7.4) and centrifuged 2247 x g for 15 min to remove insoluble components. The supernatant containing extracted molecules were taken for HPLC analysis.

Biocompatibility studies

Major organs including heart, liver, spleen, lung and kidneys were collected from mice after 24 h injection of PBS, CAR, FL1R, FL2R, LIR and KIR (2.5 µmol kg⁻¹ body weight), then placed into 4% paraformaldehyde for histological examination.

Establishment of liver ischemia-reperfusion models in living mice

Male mice were randomly selected and partial hepatic ischemia-reperfusion model was prepared through carefully orchestrated series of three main steps: opening the abdomen after the mice was entirely anesthetized and dissecting the interlobular ligaments with using microvascular clamp to interrupt the arterial and portal venous blood supply to the left lateral and median liver lobes for 15 min, 30 min, 45 min or 60 min then reperfusion for 4 h. The control groups were treated with PBS (0.2 mL) or NAC (10 mg kg⁻¹ day⁻¹ during the study, intraperitoneal injection) or sham-operation.

Real-time in vivo chemiluminescence and fluorescence imaging of HIRI in living mice

Real-time chemiluminescence and fluorescence imaging was conducted at 4 h reperfusion after i.v. injection of CAR (2.5 µmol kg⁻¹ body weight) or LIR (2.5 µmol kg⁻¹ body weight) and KIR (2.5 µmol kg⁻¹ body weight). Chemiluminescence images were acquired under bioluminescence mode with an open filter and an acquisition time of 30 s. Fluorescence images were acquired using the IVIS spectrum imaging system with the excitation at 670 ± 10 nm, the emission at 720 ± 10 nm and the acquisition time of 0.1 s. Fluorescence and chemiluminescence intensities of liver and kidney in living mice were analyzed by the ROI analysis using the Living Image 4.3 Software and Image J. Mice were euthanized after imaging at 60 min post-injection of probes. Major organs were collected and placed into 4% paraformaldehyde for histological examination.

In vivo stability

Collected urine was measured in PBS (10 mM, pH 7.4) by UV-Vis and fluorescence spectrophotometry, imaged with the IVIS spectral imaging system, and analyzed by HPLC as well as MALDI-TOF mass spectrometry.

Statistics analysis

The in vivo and ex vivo chemiluminescence and fluorescence intensities were quantified with ROI analysis using Living Image 4.3 Software. Data are mean ± standard deviation (S. D.) unless stated otherwise. Investigators were blinded to group allocation during experiments. Statistical differences between two groups were tested with a two-tailed Student’s t test, and more than three groups were determined by one-way analysis of variance followed by Tukey’s post hoc test. For all tests, P-values less than 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. All statistical calculations were performed using GraphPad Prism 6.0, including assumptions of tests used.

Animal handling

All male mouse experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-sen University (SYSU), with the approval number SYSU-IACUC-2023-B0504. To determine the clearance pathway and biocompatibility in living male mice, 8 weeks KunMing male mice (7 groups, three mice for each group, 2.5 μmol/kg body weight) were injected with CAR, KIR and LIR intravenously. All male mice were fed with non-fluorescent chow (Xietong Pharmaceutical Bioengineering CO., Ltd, XT19008). Housing conditions: 12 h dark/12 h light cycle, ambient temperature: 20–25 °C, Humidity: 45–55%.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.