Recent progress in H2S activated diagnosis and treatment agents

Hydrogen sulfide (H2S) is a key biosignal molecule in the human body. Endogenous H2S, as a gas delivery and protective agent in the body, is involved in a variety of physiological processes, including mediating vascular tone and neuromodulation. The production of abnormal H2S levels in the body is related to the occurrence of various diseases, so real-time monitoring of H2S in vivo is very important. However, traditional detection methods face enormous challenges in the in vivo detection of H2S owing to its high volatility and rapid catabolism. Optical probes developed in recent years with the advantages of high sensitivity, short response time, non-invasive nature and capacity for real-time monitoring can overcome the limitations of traditional detection methods and offer the possibility of real-time monitoring of H2S in cells and in vivo. In addition, the production of high concentrations of H2S is closely related to the formation of colon cancer, and H2S-activated treatment agents have been developed for use in this particular tumor microenvironment, which reduce the toxic side effects of traditional therapy on normal tissues and improves the treatment effect. This review summarizes the recent advances in H2S detection probes in vitro and in vivo, as well as H2S-activated tumor treatment agents.


Introduction
Hydrogen sulde (H 2 S) is an irritating gas with a smell of rotten eggs that has long been considered toxic. [1][2][3][4] Recent studies have shown that H 2 S is an endogenously unstable gas, which has been identied as a gas carrier, as well as nitric oxide (NO) and carbon monoxide (CO). [5][6][7] Endogenous H 2 S can be enzymatically produced by cystathionine g-lyase (CSE), cystathionine bsynthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3MST) in mammalian cells. 3,[8][9][10][11] These enzymes digest cysteine or cysteine derivatives and produce H 2 S in different organs. It has been shown that H 2 S is involved in many physiological processes, [12][13][14] such as regulating blood pressure, exerting antioxidant and anti-inammatory effects, and regulating the central nervous system, 15,16 respiratory and gastrointestinal systems. 17 The physiological concentration of H 2 S is 0.01-3 mM at the cellular level and 30-100 mM in the serum. 18 Abnormal levels of H 2 S in the body can induce several malignant diseases, including Alzheimer's disease, 19 diabetes, heart disease, Xiaodong Wang is a master's degree candidate at Shanghai Normal University in Professor Qiwei Tian's group. Her current research interest is H 2 Sactivated smart materials for application in tumor diagnosis and treatment. Lu An received her master's degree in Inorganic Chemistry from Shanghai Normal University in 2012. She is now a PhD candidate at Shanghai Normal University. Her current research interests focus on gas small molecule detection in vivo and its application in cancer diagnosis and treatment.
hypertension and other cardiovascular diseases. 20 Therefore, real-time detection of H 2 S levels is important for further study of its physiological and pathological roles in biological systems.
Traditional analytical methods for H 2 S mainly include colorimetry, 21 electrochemical analysis, 22 gas chromatography, 23 and sulde precipitation. 24 These methods need high-standard preparation of samples and collection of H 2 S from cells or tissues. [25][26][27] However, a fast H 2 S catabolism rate leads to uctuations in its concentration, further resulting in inaccurate measurement. 28,29 Therefore, the traditional methods have difficulty meeting fast, accurate, and real-time monitoring criteria for H 2 S levels in living systems. Optical detection methods are attracting increasing research interest owing to their high sensitivity, short response time, non-invasive nature, capacity for real-time monitoring and easy sample preparation. [30][31][32][33] Based on the good nucleophilic and reducing chemistry of H 2 S, researchers have been developing optical probes with high sensitivity, selectivity and biocompatibility for the detection of H 2 S in biological systems. These probes are based primarily on specic H 2 S-induced reactions, including azide reduction, [34][35][36] nitro reduction, 37,38 removal of quenchers (such as copper(II)), [39][40][41] and nucleophilic reactions, [42][43][44] to allow uorescence to be turned on for H 2 S detection at different biological levels.
In addition, there have been some reports that CBS is selectively up-regulated and the concentration of H 2 S is significantly increased in cancer tissues such as colon, breast and ovarian cancers. [45][46][47][48] H 2 S plays an important role in tumor proliferation and metastasis, and has become a new target for cancer treatment. 49 Traditional cancer treatment methods mainly include surgical resection, chemotherapy, radiotherapy and other means. [50][51][52] These treatment methods not only have a low cure rate, but also have relatively large side effects. 53 Scientists are working to develop H 2 S-activated reagents for the treatment of cancer, on account of high concentrations of H 2 S in the tumor microenvironment. These mainly include: (i) H 2 Sactivated nanodrug carriers for delivering chemotherapeutic drugs to tumor sites, improving the therapeutic efficiency of cancer while reducing the toxic side effects on normal tissues; 54 (ii) H 2 S trapped in normal tissues aer intravenous injection, causing damage to normal tissues on light irradiation. The H 2 S-activated phototherapy agent only produces therapeutic effects at the tumor site, thereby reducing damage to normal tissues.
In this review, we summarize the recent developments of H 2 S-activated probes in the biomedical eld, including uorescent probes and photoacoustic probes for in vitro and in vivo applications. In addition, the application and advantages of H 2 S-activated reagents in cancer diagnosis and treatment are also discussed. We also reference the side effects of traditional therapy reagents in the treatment of tumors, and describe the requirements and challenges of H 2 S-activated reagents. Finally, the possible future application prospects of H 2 S-activated diagnostic and therapeutic reagents for cancer therapy are also discussed.

H 2 S-activated probes
Abnormal H 2 S levels in organisms are associated with the development of many diseases. 15 High-sensitivity probes for H 2 S concentrations in animals are very important; they can help us to understand the effects of H 2 S on various physiological and pathological processes, and to diagnose related diseases in a timely manner. Probes for H 2 S detection in vitro 55,56 and in vivo 57-59 are listed in Table 1 and described in detail below.

H 2 S probes in vitro
H 2 S intelligent optical probes with high sensitivity, high selectivity, high signal-to-noise ratio and stability are being developed. 60 Fluorescence imaging by uorescent probe staining is one of the most attractive molecular imaging techniques for H 2 S detection in living cells, tissues and living animals. 61 H 2 Sactivated uorescent probes are mainly based on the difference of emission wavelength before and aer response. 62 Although a lot of effort has been expended, uorescence imaging is limited by problems such as the low concentration of endogenous H 2 S and the presence of a large number of interfering molecules, including reduced glutathione, cysteine (Cys) and thiol-containing proteins, in complex living systems. Therefore, it is still a signicant challenge to develop highly sensitive and selective uorescent probes. Based on the nucleophilic and reductive properties of H 2 S, scientists have developed uorescent probes for H 2 S detection founded on the reduction of azides to amines, nucleophilic reactions and copper sulde precipitation. [63][64][65][66] Liu et al. 67 designed a H 2 S uorescent probe containing bis-electrophile to take advantage the nucleophilicity of H 2 S. The uorescence intensity of the disulde-containing probe increased dramatically (55-70-fold) when 50 mM H 2 S was presented in solution. In addition, the maximum intensity was reached in 1 h, suggesting that the reaction was fast. The uorescent probe is selective for H 2 S and does not react with other bio-thiols, such as cysteine and glutathione, at the same concentration (100 mM). A uorophore of dansyl azide (DNS-Az) with high quantum yield was prepared by Peng et al. 68 The azide is reduced to an amine by reduction with H 2 S to emit uorescence for rapid detection of H 2 S in vitro. The probe was very sensitive, with a detection limit of 1 mM in buffer/Tween and 5 mM in bovine serum. The reaction was complete in a few seconds, while the uorescence was enhanced immediately. No obvious response to the probe was observed for most of the tested anions at a concentration of 1 mM, which is a 40-fold higher concentration than that of sulde. Sasakura et al. 69 designed and synthesized a novel H 2 Sdetecting uorescent probe Cyclen-AF + Cu 2+ (HSip-1) based on the azamacrocyclic ring to form a stable metal complex with Cu 2+ . The paramagnetic Cu 2+ center could quench the uorophore's uorescence. When H 2 S binds to Cu 2+ , Cu 2+ is released from the azamacrocyclic ring, resulting in enhanced uorescence. The probe showed a large (50-fold) and immediate increase in the uorescence intensity upon addition of 10 mM H 2 S, whereas almost no uorescence increment was observed upon the addition of 10 mM GSH. Thus, HSip-1 is more selective for H 2 S than previously reported uorescent probes using 2,4-dinitrosulfonyl or azide groups.
For most single-window-response uorescent probes the experimental results change with the experimental conditions. 70 Ratiometric uorescent probes are able to overcome the interference due to experimental conditions. Bae et al. 71 reported a H 2 S-activated mitochondrially localized two-photon ratiometric uorescent probe, SHS-M2 (Fig. 1A), which has 6-(benzo[d]thiazol-2-yl)-2-(methylamino) naphthalene as the uorophore, 4-azidobenzyl carbamate as the H 2 S response site, and triphenylphosphonium salt as the mitochondria-targeting moiety. The thiolate-triggered reaction with the azide group would cleave the carbamate linkage and liberate the amino group, accompanied by a decrease in emission intensity at 420 nm and a gradual increase at 500 nm. The color also changes from blue to yellow. Thereby, the emission and the  cross-section of the ratiometric two-photon probe can be increased (Fig. 1B). The probe is more sensitive and the detection limit of H 2 S is 0.4 mM in vitro. The uorescence intensity of the SHS-M2 aer triggering by H 2 S (0.1 mM) is 5-8-fold higher than that with 10 mM glutathione (GSH) and 1 mM cysteine (Cys), which conrms the high selectivity for H 2 S over GSH and Cys. Two-photon microscopy ratiometric imaging of SHS-M2 as a probe can be used to study the relationship between CBS expression and H 2 S levels in cells and brain sections (Fig. 1C).
The main problem of current H 2 S probes is low detection sensitivity. Förster resonance energy transfer (FRET)-based uorescent probes can eliminate the effect of excitation backscattering on uorescence detection because of the large offset between donor excitation and acceptor emission. 72,73 In addition, two well-separated emission bands with comparable intensities can be used to ensure the accuracy of their strength and ratio. Some fast and accurate ratiometric uorescent probes for detecting H 2 S have been developed based on FRET. 74, 75 Zhao et al. 76 developed a self-assembled micelle aggregate NanoBODIPY uorescent probe with H 2 S-triggered FRET switch, which consists of a dynamic energy receptor semi-cyanine-BODIPY hybrid dye (BODInD-Cl) and a complementary energy donor (BODIPY1). In the absence of H 2 S, a specic FRET from BODIPY1 to BODInD-Cl occurs due to the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. In contrast, in the presence of H 2 S, the Cl on the aromatic ring in NanoBODIPY is replaced by the H 2 S via nucleophilic substitution and the absorption of the probe is shied from 540 to 738 nm, resulting in loss of FRET owing to the lack of overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor ( Fig. 2A). This results in a uorescence signal that simultaneously "turns on" the energy donor BODIPY1 and a uorescence signal that "closes" the energy acceptor BODInD-Cl. NanoBODIPY can sensitively and quickly detect H 2 S with a detection limit of 7 nM by ratiometric uorescence. The emission intensity gradually increased at 511 nm aer adding different concentrations of sodium hydrosulde (NaHS), accompanied by a loss of emission at 589 nm, and the response was complete within 140 s (Fig. 2B). Through competitive experimental studies, NanoBODIPY showed good selectivity for NaHS with minimal interference from other biologically relevant analytes in PBS buffer (Fig. 2C).
By a similar approach, Feng et al. 77 reported a FRET-based ratiometric uorescent probe composed of a coumarin-merocyanine dyad. Before the reaction with H 2 S, the emission wavelength of coumarin apparently overlaps with the absorption of merocyanine, and a resonance energy transfer process occurs, so that the probe displays the uorescence of the cyanine. In the presence of H 2 S, the merocyanine moiety undergoes a nucleophilic addition reaction with H 2 S, and the conjugated system is destroyed; as a result, resonance energy transfer cannot be achieved, and so the uorescence of coumarin is exhibited. The probe has a detection limit of as low as 40 nM. It can be used for mitochondrial endogenous and exogenous H 2 S detection; it shows a greater emission shi than other H 2 S probes, and so it exhibits higher selectivity and sensitivity.

H 2 S probes in vivo
Despite rapid progress in the development of H 2 S probes in the past few years, there are still many problems in the transition from solutions, cells and tissues to whole organisms. Tissue penetration, poor spatial resolution in deep biological tissues, uorophore stability at high excitation wavelengths and other issues have largely limited their application for in vivo H 2 S detection.
2.2.1 Fluorescent probes. The light sheet uorescence microscope (LSFM) is an imaging tool that connes excitation light to a sheet that coincides with the focal plane of a wide eld of view imaging system. 78 The LSFM can image larger samples than confocal microscopes while enabling rapid imaging. The LSFM combined with a H 2 S-responsive uorescent probe enables detection of H 2 S levels in vivo. 79 Hammers et al. 80 developed an azide-functionalized O-methylrhodol uorophore (MeRho-Az) for the detection of H 2 S in live zebrash (Fig. 3A). The xanthene core modied O-methylrhodol (MeRho) is locked in the non-uorescent spirolactone tautomeric form. The H 2 S reduction of azide regenerates the amine while releasing the uorescent open tautomer to produce an intense uorescence, and exhibits a rapid >1000-fold uorescence response. MeRho-Az can sensitively detect low concentrations of H 2 S, with a detection limit of 86 AE 7 nM. Owing to the pH insensitivity and photostability of MeRho-Az, it can be used for the detection of H 2 S in living organisms. The results showed that the uorescence signal was rapidly increased aer the addition of NaHS to MeRho-Az ( Fig. 3B and C). Then MeRho-Az was used to detect endogenous H 2 S in C6 rat glial cells by uorescence imaging. The uorescence signal of the C6 rat glial cells in the group treated with AP39 (H 2 S donor) plus MeRho-Az was higher than that for the group treated with MeRho-Az alone. In contrast, the uorescence signal of the group treated with AOAA (aminooxyacetic acid; H 2 S inhibitor) plus MeRho-Az was lower than that of the group treated with MeRho-Az alone. These results demonstrated that MeRho-Az can sensitively detect low concentrations of H 2 S (Fig. 3D).
Phosphorescent transition metal complexes have attracted much attention owing to their strong visible light absorption and emission, large Stokes shi, and stable photochemical properties. 81,82 A ruthenium(II) complex-based responsive luminescence probe (Ru-MDB) for H 2 S detection was studied by Du et al. 83 MBD is a masking moiety for the Ru-MDB complex H 2 S response. The metal-to-ligand charge transfer (MLCT) excited state of the Ru II complex is destroyed by an intramolecular lightinduced electron transfer photo-induced electron transfer (PET) process when the electron acceptor group MDB is linked (Fig. 4A). To utilize the nucleophilic properties of H 2 S, the new MDB masking group was linked to one of the bipyridine ligands of the Ru II complex through an ester bond that could be cleaved by H 2 S, resulting in an approximately 86-fold increase in luminescence intensity. The detection limit was measured to be 45 nM, which suggested high sensitivity of Ru-MDB for monitoring H 2 S in mice. The main characteristics of this probe enabled the monitoring of lysosomal H 2 S generation in live cells, and the visualization of exogenous/endogenous H 2 S in live Daphnia magna, zebrash and mice (Fig. 4B).
Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) showed reduced autouorescence, enhanced tissue penetration, and higher spatial resolution in vivo. 84 Xu et al. 85 designed a H 2 S-activated NIR-II@Si uorescent probe (Fig. 5A) that visualizes colorectal cancer. The probe encapsulates the H 2 S-responsive uorescent probe in the hydrophobic interior of the core-shell silica nanocomposite. The uorescent nanoprobes comprise two organic chromophores: boron-dipyrromethene (ZX-NIR) dye, which has a maximum emission shi from 600 nm to 900 nm in the presence of H 2 S to produce NIR-II emission, and aza-BODIPY (aza-BOD), the emission of which remains unchanged at 700 nm, as an internal reference ( Fig. 5B and C).The detection limit for H 2 S was measured to be 37 nM, indicating the high sensitivity of NIR-II@Si for ratiometric detection of H 2 S. This activatable H 2 S-specic targeting probe can be used for deep tissue imaging of H 2 S-rich colon cancer cells. Utilizing the advantages of NIR-II imaging, tumor sites can be selectively   detected, and visual monitoring of tumor models of colon cancer can be achieved (Fig. 5D).

Photoacoustic probes.
Fluorescence imaging is limited by problems such as poor tissue penetration and auto-uorescence, and few probes can be used for imaging in deep tissues and whole animals. In order to solve these problems, it is highly desirable to develop a probe with a new mode of imaging. Photoacoustic imaging combines the advantages of the high resolution of optical imaging and high penetration depth of ultrasound imaging. 86,87 It is a medical imaging diagnostic technology with broad clinical application prospects.
In the last few years, people in related elds have been working on developing photoacoustic probes for detecting H 2 S in vivo. Shi et al. 88 developed a H 2 S-activated Si@BODPA photoacoustic probe that encapsulates a semi-cyanine-BODIPY hybrid dye (BODPA) in the interior of a silica nanocomposite (Fig. 6A); thereby the probe has good water solubility and excellent biocompatibility. Conversion of BODPA to BOD-HS within the nanoparticles (NPs) by aromatic nucleophilic substitution in the presence of H 2 S results in high NIR absorption around 780 nm (Fig. 6B). Therefore, the Si@BODPA probe produces a strong photoacoustic signal output in the NIR region. The detection limit was measured to be 53 nM. The probe shows an extremely fast response and can detect transient changes in H 2 S. Si@BODPA allows direct photoacoustic tracking of endogenous H 2 S production in an HCT116 (human colon cancer cell) tumor-bearing mouse model. As shown in Fig. 6C, there was no photoacoustic signal from the normal sites of mice and the tumor site of the mice pre-treated with the CBS inhibitor aminooxyacetic acid (AOAA, 100 nmol) aer injection of Si@BODPA, while a photoacoustic signal was observed in the tumors of the mice without and with pretreatment with a CBS activator (S-adenosyl-L-methionine), indicating that Si@BODPA can be used for detection of H 2 S in vivo.
At present, photoacoustic probes for H 2 S detection mostly provide single-response photoacoustic signals, and the results will be affected by factors such as instrument, probe concentration and external environment. On the contrary, a ratiometric photoacoustic probe can eliminate the effects of the above factors by using the ratio of two separate wavelength photoacoustic response signals, thereby obtaining reliable experimental results. Ma et al. 89 developed a novel ratiometric photoacoustic nanoprobe for in vivo detection of H 2 S. The nanoprobe AzHD-LP consists of a liposome (LP) with a H 2 Sresponsive near-infrared dye (AzHD) encapsulated inside it (Fig. 7A). Aer the reduction of azide to amine in the AzHD-LP photoacoustic probe by H 2 S, the absorption peak appears red-shied. The absorption of AzHD-LP at 600 nm is reduced, while the absorption at 700 nm is increased, resulting in a ratiometric PA signal in the presence of H 2 S. The detection limit of AzHD-LP for NaHS in solution was determined to be 91 nM. Furthermore, aer AzHD-LP was conjugated to tumortargeting peptide c(RGDyK), detection of intratumoral H 2 S production in HCT116 colon tumor mice was achieved under excitation of 532 nm and 700 nm pulsed lasers (Fig. 7B).
In this section, uorescent probes and photoacoustic (PA) probes for H 2 S detection are introduced. Although uorescent probes are widely used in the detection of H 2 S, their applications in vivo are limited by the autouorescence and penetration depth. Photoacoustic imaging with high tissue penetration can be used to detect H 2 S levels in the living body and accurately locate a lesion. However, their sensitivity impedes their further application. As a result, it is necessary to develop better probes. NIR-II uorescence and NIR-II photoacoustic imaging 90,91 are emerging technologies that exhibit greater penetration depth and higher sensitivity. Therefore, the design of NIR-II  uorescence probes with weaker autouorescence and NIR-II PA probes is the way forward.

H 2 S-activated therapeutic reagents
Compared with the traditional treatment of colon cancer, targeted response therapy can reduce side effects and cause more obvious therapeutic effect. Overexpression of cystathionine-bsynthase (CBS) in tumor cells leads to an increase in H 2 S levels (0.3 to 3.4 mM), especially in colon tumor cells. 45 So, it will be more efficient to use H 2 S-activated therapy for colon cancer than other tumor microenvironment factors (pH, GSH, etc). Therefore, a series of H 2 S-activated therapeutic reagents have been designed on account of endogenous hydrogen sulde, which is highly expressed in colon tumors, including H 2 Sactivated chemotherapy, photodynamic therapy, and photothermal therapy ( Table 2).

Chemotherapy
Chemotherapy is currently the main method used in the clinical treatment of cancer. Current chemical drugs for cancer treatment include doxorubicin (DOX), 92 curcumin 93 and so on. Unfortunately, we have not yet broken through the bottleneck in nding chemical drugs with excellent anti-tumor effects. Since most chemotherapeutic drugs have poor water solubility and low bioavailability, systemic administration is very difficult. The key problem is that normal cells will be damaged when the drugs are administered intravenously, resulting in toxic side effects. Therefore, scientists have long desired to develop a drug carrier from which the release of chemotherapeutic drugs can be stimulated at the tumor site only. In order to increase the targeting effect on tumor tissues and improve the therapeutic effect, a hydrogen sulde-activated azide-functionalized biocompatible mesoporous silica nanoparticle (MSNP) was developed by Thirumalaivasan et al. 94 as a specic drug delivery system (Fig. 8A). Further, folic acid (FA) was attached to the surface of the MSNP to actively target cancer cells. In the presence of H 2 S, the ester bond in the DOX-loaded MSNP-N 3 -FA is cleaved, resulting in the release of DOX from the MSNP, while no DOX is released from the MSNP before being activated by H 2 S. The in vivo results based on HT-29 tumor mice suggested that the therapeutic effect of MSNP-N 3 -FA with DOX is greater than that of DOX or MSNP-N 3 -FA alone (Fig. 8B).
Similarly, Zhang et al. 95 used a series of N-(2-hydroxyethyl)-4azide-1,8-naphthalimide-ended amphiphilic diblock copolymer poly(2-hydroxyethyl methacrylate)-block-polymethylmethacrylate (N 3 -Nap-PHEMA-b-PMMA-N 3 ) polymer nano-micelles for loading DOX (Fig. 8C). Under the action of H 2 S, the charge on the surface of the micelles of these nanomaterials is reversed and the azide reduction reaction occurs. The surface charge of the micelles changes from negative to positive, which promotes the uptake of the materials by the cells and accelerates the release of DOX (Fig. 8D).
A pharmaceutical carrier should have excellent biocompatibility. Chen et al. 96 designed a H 2 S-activated protein cage (CuDOX NP) loaded with chemotherapeutic drugs. They used horse spleen apoferritin (apo-HSF) as a container for coppercomplexed doxorubicin to obtain a water-soluble nanocomposite. Breaking of the CuDOX coordination interaction by H 2 S under physiological pH conditions allows the DOX to be  slowly released from the protein cage without disrupting the structure of the protein. In vitro cell experiments showed that CuDOX nanoparticles activated by H 2 S can reduce the premature release of drugs, reduce the toxicity of DOX to normal cells, and enhance the anti-cancer effect.

Photodynamic therapy
Photodynamic therapy (PDT) is based primarily on the accumulation of non-toxic photosensitizers, oxygen and light to produce reactive oxygen species, particularly singlet oxygen ( 1 O 2 ), which selectively induces apoptosis and necrosis in cancer cells. 97 are taken out from the MOF node to obtain a photosensitizer, and the uorescence is recovered (Fig. 9B). This open-type uorescent MOF photosensitizer probe achieves effective cancer treatment through controlled release of photoactive ligands, and the experimental results showed signicant therapeutic effects (Fig. 9C). In addition, Wu et al. 100 reported a class of H 2 S-activated uorescent probes and photodynamic smart reagents using electrochromic materials (EMs) with organic p-electron structure (dicationic 1,1,4,4-tetraphenylbutadiene, 1 2+ ) as H 2 Sresponsive chromophores. EM1 2+ is doped into semiconductor polymer nanoparticles (SNPs) to form H 2 Sactivatable uorescent probes (1 2+ -SNPs) (Fig. 9D). Within 1 2+ -SNPs, EM1 2+ can effectively quench the uorescence of SNP by a uorescence resonance energy transfer (FRET) process. Subsequent reduction of 1 2+ to colorless 2 NPs by H 2 S eliminates the FRET process and restores uorescence. Further, tumor-targeting ligand folic acid modied uorescent probes (1 2+ -SNP830-FA) were used for tumor imaging in H 2 S-enriched mice. Tumor-targeting and H 2 S-activatable PSs (1 2+ -PSs-FA) using EM1 2+ were further developed by replacing the SNP with organic PS. 1 2+ -PSs-FA accumulates well at the tumor site. Aer H 2 S-specic activation, 1 2+ -PSs-FA produces ROS under the action of 808 nm laser irradiation. The reagent exhibits negligible phototoxicity to normal tissues and signicant tumor photodynamic therapy effects (Fig. 9E).
In addition, Wang et al. 101 have designed and synthesized a theranostic prodrug (TNP-SO) for H 2 S-activatable nearinfrared emission-guided on-demand administration of PDT. The theranostic probe consists of an H 2 S-activated NIR imaging probe and a sensitizing drug. These two units are connected by a short diglycolamine spacer. The newly obtained small molecule probe is encapsulated into the hydrophobic interior of a silica nanocomposite to produce a nanoprobe with good water solubility and photostability. The absorption of TNP-SO at 509 nm decreased as 677 nm NIR absorption increased aer being triggered by H 2 S. The NIR uorescence increased linearly with H 2 S concentration (0-20 mM), and the determined detection limit was 21 nM, indicating that Nano-TNP-SO has high sensitivity for H 2 S detection. Nanoprobes can also act as good photosensitizers for the efficient production of 1 O 2 . The in vivo results using this probe reveal that cancer imaging accurately guides the location of light exposure to produce the cytotoxic ROS required for on-demand cancer treatment, maximizing treatment efficiency and minimizing side effects.

Photothermal therapy
Photothermal therapy is a simple, safe, non-invasive treatment method that converts near-infrared laser energy into heat energy to achieve local high-temperature killing of tumor cells. 102 Near-infrared photothermal reagents based on photothermal therapy have attracted much attention. Traditional photothermal reagents have limitations such as non-specicity and toxicity. In order to solve these problems, photothermal reagents with intelligent response are required. Shi et al. 103 developed a H 2 S-activated second near-infrared self-assembling uorescent nanoprobe for guiding photothermal therapy of colon cancer (Fig. 10A). A self-assembled H 2 S response small molecule (SSS) was designed that contains three triethylene glycol monomethyl ether chain functionalized benzene rings as hydrophilic tails to guide the self-assembly of the SSS. The monochlorinated BODIPY core is the activatable unit based on thiol-halogen nucleophilic substitution of H 2 S. In the absence of H 2 S, the nanostructured photothermal agent (Nano-PT) produces minimal photothermal effects with absorption and emission at 540 and 589 nm, respectively. However, the H 2 S response results in high NIR absorption near 790 nm, which not only causes efficient photothermal energy conversion with 785 nm laser irradiation, but also produces bright luminescence in the NIR-II region (Fig. 10B). Using these excellent properties, the Nano-PT enables efficient photothermal ablation of imaging-guided colon cancer tumors (Fig. 10C).
An et al. 104 designed an intelligent diagnostic reagent for colon cancer based on the in situ reaction of cuprous oxide (Cu 2 O) with endogenous H 2 S at the colon tumor site (Fig. 10D). Highly expressed endogenous H 2 S in colon tumors reacts with cuprous oxide and produces copper sulde, which has strong near-infrared absorption, triggering photoacoustic and photothermal effects (Fig. 10E). The design of the in situ reaction at the tumor site reduces the damage to normal tissues during treatment and produces a signicant therapeutic effect (Fig. 10F).

Summary and outlook
Abnormalities in H 2 S levels are associated with the development of a variety of diseases, such as colon cancer, breast cancer and ovarian cancer. In order to achieve early prevention and diagnosis of related diseases, research aimed at producing highly sensitive and selective H 2 S probes has been promoted. Among the possible techniques available, optical detection methods have higher sensitivity than traditional H 2 S detection methods. The transition from a single wavelength uorescent probe to a more sensitive ratiometric uorescent probe reduces the effects of external environment and other factors. In order to achieve real-time monitoring of H 2 S in vivo, further development from a short-wavelength uorescent probe to a second near-infrared uorescent probe, and photoacoustic probe with high tissue penetration has taken place. More importantly, utilizing the special microenvironment with high expression of endogenous H 2 S at the colon tumor site, H 2 S-activated intelligent therapeutic agents have been developed. Compared with using the traditional reagents, this strategy reduces the damage to normal tissues and shows more obvious therapeutic effects. Although many H 2 S probes with high sensitivity and high selectivity have been developed so far, as well as H 2 S smart reagents for cancer treatment, it is still necessary to continue to explore probes with lower side effects before their clinical application.
We believe that the integration of diagnostic and therapeutic agents for H 2 S detection and related disease treatment has a broad development prospect. In our subsequent research we aim to: (i) develop diagnostic reagents that are easy to prepare, and have good stability and biocompatibility; (ii) combine a variety of methods for tumor diagnosis and treatment, to develop intelligent diagnostic reagents with multi-modal diagnosis and synergistic treatment-for example, combining uorescent probes with photoacoustic probes; 105 (iii) undertake an in-depth study of the side effects of various agents, as well as their potential toxicity. Only once the problems described in this review have been solved, can the reagents can be further applied to clinical use.

Conflicts of interest
There are no conicts of interests to declare.