Hydrogen Sulfide Responsive Phototherapy Agents: Design Strategies and Biological Applications

Hydrogen sulfide (H2S) is one of the critical gasotransmitters, which play important roles in regular physiological processes, especially in vital signaling pathways. However, fluctuations in endogenous H2S concentration can be linked to serious health problems, such as neurodegenerative diseases, cancer, diabetes, inflammation, cardiovascular diseases, and hypertension. Thus, it has attracted a great deal of attention in therapeutic applications, specifically in the field of phototherapy. Photodynamic therapy (PDT) and photothermal therapy (PTT) are two subclasses of phototherapy, which utilize either reactive oxygen species (ROS) or local temperature increase upon irradiation of a photosensitizer (PS) to realize the therapeutic action. Phototherapies offer unique advantages compared to conventional methods; thus, they are highly promising and popular. One of the design principles followed in new generation PSs is to build activity-based PSs, which stay inactive before getting activated by disease-associated stimuli. These activatable PSs dramatically improve the selectivity and efficacy of the therapy. In this review, we summarize small molecule and nanomaterial-based PDT and PTT agents that are activated selectively by H2S to initiate their cytotoxic effect. We incorporate single mode PDT and PTT agents along with synergistic and/or multimodal photosensitizers that can combine more than one therapeutic approach. Additionally, H2S-responsive theranostic agents, which offer therapy and imaging at the same time, are highlighted. Design approaches, working principles, and biological applications for each example are discussed in detail.


Hydrogen Sulfide (H 2 S)
Hydrogen sulfide (H 2 S), a colorless gas with an unpleasant odor, has been widely identified as a toxic substance for decades. 1−6 After H 2 S was recognized as the third gasotransmitter, in addition to carbon monoxide (CO) and nitric oxide (NO), its importance in biological systems was unveiled. 7−13 H 2 S can be produced through enzymatic or nonenzymatic pathways. 14,15 Enzymatic pathways begin with the conversion of homocysteine to cystathionine by the catalytic action of cystathionine β-synthase (CBS). Subsequent conversion of cystathionine to cysteine then takes place by cystathionine γ-lyase (CSE). Cysteine can finally be converted to H 2 S together with NH 4 + and pyruvate. While this pathway takes place in the cytosol, cysteine can be converted to 3mercaptopyruvate by aspartate aminotransferase (AAT) in mitochondria, which is subsequently transformed to H 2 S by 3mercaptopyruvate sulfurtransferase (MPST). 2,4,15,16 The nonenzymatic production pathways of endogenous H 2 S are not well-defined. However, recent studies have demonstrated that one route utilizes the catalytic action of iron and vitamin B6, while cysteine acts as a substrate. 17 H 2 S can also be generated from polysulfides present in garlic by human red blood 18 cells or from thiosulfate under hypoxic conditions, 19 but these pathways leading to H 2 S generation still require further investigation.
H 2 S is involved in a diverse range of biological processes, including glycolysis, 20,21 anti-inflammation, 22−24 migration, 25,26 cell proliferation, 27−29 neuromodulation, 30−32 apoptosis, 33,34 and angiogenesis 35,36 among others. In addition, it is associated with various pathological conditions, such as diabetes, 37−39 Alzheimer's disease, 40−43 Down syndrome, 44,45 cardiovascular diseases, 46,47 hypertension, 46−50 and cancer 51−53 (Figure 1). H 2 S is overexpressed in different types of cancer cells due to the elevated expression of enzymes responsible for its production in these cells. 51 Therefore, it is essential to monitor and characterize the role of H 2 S in biological activities, and it can also be utilized as a key initiator for therapeutic action.

Phototherapy
Photodynamic therapy (PDT) is a clinically approved, noninvasive treatment modality used for the treatment of cancer and a range of nonmalignant diseases by employing cytotoxic reactive oxygen species (ROS). 54−61 PDT holds unique advantages compared to conventional therapies including minimal side effects, no drug resistance, and activation of the immune system. 57,61−64 Typical PDT involves three key elements: an outer light source, a photosensitizer, and tissue oxygen. In the presence of light, the photosensitizer (PS) is transferred from the S 0 ground state to the S 1 singlet excited state. The excited PS then may follow the S 1 to S 0 relaxation pathway while emitting light known as fluorescence, or it may follow nonradiative relaxation, which releases heat. Furthermore, it may undergo intersystem crossing (ISC) and switch to the first triplet excited state, T 1 . At that point, a photosensitizer either generates free radicals as a result of electron or proton transfer from T 1 to biological substrates, favoring type I PDT, or transfers its energy to tissue oxygen ( 3 O 2 ), generating 1 O 2 through a type II mechanism. In addition, it may return to the ground state from the T 1 excited state through phosphorescence ( Figure 2). The majority of current PSs follow the type II pathway, and accordingly singlet oxygen is the major cytotoxic agent. It is worth mentioning that there is a growing interest in type I PSs as they can function effectively under hypoxic conditions, which is a difficult condition for 1 O 2 generating type II PSs. 57,65,66 Photothermal therapy (PTT), on the other side, induces local temperature increase upon irradiation of PSs. 67−70 PTT does not consume tissue oxygen; thus, it is highly attractive for the treatment of hypoxic tumors as in the case of type I PDT.
In the case of both PDT and PTT, cancer cell selectivity is still highly sought to fully eliminate the adverse effects on healthy cells. Phototherapies are known to have intrinsic selectivity, as the irradiation light can be directed to the lesion area. However, in most cases this type of selectivity is not sufficient, and more sophisticated PS designs are needed. To this end, new generation PSs that can be activated solely in cancer cells are highly attractive. These activity-based PSs stay in their OFF state in nonmalignant cells even under light irradiation and turn on their cytotoxicity after getting activated in cancerous  cells with a tumor-associated input such as biothiols, enzymes, and reactive oxygen species. H 2 S is among the most attractive analytes and has been utilized in numerous designs. The activity-based approach is also followed in the design of molecular sensors (activity-based sensors, ABS) to detect and image analytes of interest selectively. In such designs, the activity and concentration of the target analyte are strongly correlated to the response of activity-based probes. On the other hand, traditional binding-based designs exhibit an output signal upon binding or interaction with a specific target. Although they report on the concentration of the target, such as an enzyme, they do not provide information about their activity as opposed to activity-based agents. 71−73 Activity-based phototherapy agents propose a notable success in cancer targeting, as they mostly remain in their off-state in normal cells. However, it is also possible that selected analytes can be expressed in different regions of the body or the serum itself. H 2 S, specifically, is involved in various physiological processes as explained in detail. Therefore, activation of the probe in different regions might damage vital organs in the body upon light exposure. One possible solution to address this issue is promoting activation of the agents within the target lesion by intratumoral or peritumoral injection. However, the ultimate goal of the new generation of photosensitizers is to trigger activation in the targeted zone, even after intravenous injection. Therefore, additional approaches have been employed to enhance the targeting and selectivity. One option in this direction is to utilize cell surface receptors (e.g., biotin, folate, human epidermal growth factor, etc.) that are differentially expressed in cancer cells. 74−78 To this end, PSs are modified with specific groups that can bind to the receptor of interest selectively. Tumor-homing peptides, which are capable of penetrating the cell membrane, recognizing tumor-related antigens or proteins, and binding to specific cell surfaces, are highly popular. The most well-known tumor-homing peptide, RGD, is composed of a tripeptide chain (Arg-Gly-Asp) and binds to integrins α V β 3 and α V β 5 . 74,79,80 NGR is also composed of a tripeptide chain (Asp-Gly-Arg) and exhibits specific selectivity toward aminopeptidase N. 74,81 Chlorotoxins, a small group of peptide toxins derived from scorpion venom, have been employed as targeting units in various studies, but they are particularly used for glioblastoma. 74,80 In addition to the peptide family, glucose metabolism can be targeted via glucose transporters. 75,77,82 Proteins such as transferrin and apolipoproteins have also been employed as targeting moieties. 74,83,84 Furthermore, a range of vitamins have been utilized as cancer targeting ligands. 75 In recent years, the combination of fluorescence imaging and phototherapy, in the form PDT, PTT, or multimodal PDT + PTT, on a single PS has emerged as a striking approach. These theranostic PSs enable the selective treatment and imaging of cancer cells at the same time. There are several reviews covering recent advancements in the field of activatable photosensitizers and multimodal phototheranostic agents. 54,55,57,60 Fluorescent probes that aim to detect biothiols have also been summarized in several review papers. 85−87 Herein, the scope of this review is to introduce current H 2 S responsive phototherapy and phototheranostic agents and their applications in both in vitro and in vivo models.

General Mechanisms for H 2 S-Induced Activation
In the design of H 2 S-responsive phototherapeutic agents, the common approach is to mask the PS core with a H 2 S-cleavable caging unit to quench the photo-cytotoxicity of the PS. Selective removal of the cage group then activates the therapeutic action by modulating the photophysical properties of the PSs. This modulation can be satisfied through different mechanisms including photoinduced electron transfer (PeT), intramolecular charge transfer (ICT), aggregation-induced emission (AIE), and energy transfer processes. 88 Selective removal of the masking units and activation of the probes with H 2 S are commonly based on nucleophilic reactions, reduction, and sulfidation. Reduction-based activation mostly targets azide, nitro, nitroso, or azo groups. For instance, H 2 S-mediated azide to amine reduction leads to 1,6-elimination, which subsequently results in the release of the active scaffold while activating the ICT process. 89 High nucleophilicity of H 2 S is mostly utilized to trigger nucleophilic aromatic substitution, disulfide bond cleavage, or Michael addition reactions. 90−92 Nitrobenzene rings, for example, generally tend to undergo nucleophilic aromatic substitution, leading to the release of active phototherapeutic agents, which possess altered photophysical properties due to the modulation of PeT and/or ICT processes. 7-Nitro-1,2,3-benzoxadiazole (NBD) is another well-known group that is cleaved upon nucleophilic attack of H 2 S. 93 Sulfidation is mostly satisfied as a result of reaction taking place between H 2 S and Cu 2+ in which copper sulfide (CuS) is precipitated. 94 Removal of Cu 2+ by H 2 S changes the photophysical properties of the agents. Each of these mechanisms is discussed in detail through specific examples in the following sections.

H 2 S-RESPONSIVE PHOTODYNAMIC THERAPY (PDT) AGENTS
Considering the relationship between H 2 S and tumorigenesis, a variety of H 2 S activatable phototherapy agents have been developed. In this section, H 2 S-responsive PDT agents are introduced. In 2017, Ma et al. developed a novel photosensitizer that was extensively based on metal−organic framework nanoparticles (MOF NPs) in which the advantages of both nanostructures and the inherent features of crystalline MOFs were combined. 95 The metal nodes of this network were constructed using Cu 2+ ions, as these paramagnetic ions would interact with H 2 S and activate, otherwise quenching the emission of the MOF NP molecules ( Figure 3). This nanoscale copper−zinc MOF NP (NP-1) was prepared via a hydrothermal microemulsion with the formula of {Cu 2 (ZnTcpp)·H 2 O}. After HS − treatment, the emission signal of NP-1 was increased, in a concentration dependent manner, around 610 and 660 nm with two shoulder peaks upon excitation at 420 nm. NP-1 was generating controllable 1 O 2 as it is only activated in the presence of H 2 S while its generation was negligible in the absence of H 2 S. Selective activation of NP-1 was also confirmed in HepG2 cells (human hepatocellular liver carcinoma cells) under confocal microscopy using propidium iodide (PI) and calcein-AM. Exogenous treatment with NaHS activated the probe, and strong red fluorescence was recorded on the red channel while the calcein-AM channel remained blank. As NP-1 stayed in its "on" state, singlet oxygen generation was initiated upon light exposure, which eventually caused cell death. As a result, a strong fluorescence intensity was obtained from PI ( Figure 4). Additionally, the in vivo antitumor efficacy of NP-1 was evaluated on cancer cells having different levels of H 2 S expression. Tumor growth was not inhibited in HepG2 and LoVo (human colorectal adenocarcinoma cell) cells, which exhibit low levels of H 2 S. However, the tumor growth was suppressed successfully in the H 2 S overexpressing HCT116 cells.
Following the previous study, Wu et al. engineered electrochromic H 2 S activatable probes for imaging and PDT. 96 The authors acknowledged the previous work but stated that MOF NPs lack selective targeting of cancer cells and NIR light absorption, which restricts PDT action in vivo. 1,1,4,4-Tetra-aryl butadiene (EM 1 2+ ), an organic π-electron structure with a sharp absorption band at 500 and 850 nm and H 2 S responsive characteristic, was modified to get activatable fluorescence probes (1 2+ -SNP580, 1 2+ -SNP700, and 1 2+ -SNP830) and PSs. EM 1 2+ doped with semiconducting polymers initially showed no fluorescence due to fluorescence resonance energy transfer (FRET), but its emission was recovered once reduced to diene EM 2 in the presence of H 2 S. All the fluorescence probes selectively and sensitively responded to H 2 S and the fluorescence response of 1 2+ -SNP830 fluctuated in accordance with the treating enhancer, inducer, or inhibitor ( Figure 5). 1 2+ -SNP830 and 1 2+ -SNP580 responded to endogenous H 2 S in RAW264.7 macrophages and were primarily localized on lysosomes ( Figure 5). Besides, the one working in the near-infrared region, 1 2+ -SNP830, accurately measured H 2 S content in human plasma. 1 2+ -SNP830 also monitored hepatic H 2 S activity in vivo. Folic acid was employed to enhance targeting abilities of 1 2+ -SNP830 along with the DSPE-PEG2000/DSPE-PEG2000-FA extension. Once 1 2+ -SNP830-FA was prepared using the nanoprecipitation method, it monitored H 2 S-rich HT29 and HCT116 colorectal cancer cells efficiently together with H 2 S content in KB tumor-bearing mice. As a final approach, R6G and NIR775, whose absorption signals were similar to those of 1 2+ , were selected as controllable 1 O 2 generating scaffolds to prepare 1 2+ -PSs-FA. A sensitive turn-on response was detected for H 2 S with a very low limit of detection (LOD) value, approximately 19 and 39 nM at 555 and 780 nm, respectively. As shown by experiments with singlet oxygen sensor green (SOSG), in the presence of NaHS, 1 2+ -PSs-FA was activated, and its 1 O 2 generation was initiated upon 808 nm laser irradiation, while negligible response was observed from the sensor in the absence of NaHS. In vivo studies have shown that intravenously injected 1 2+ -PSs-FA was localized primarily to tumors and activated with endogenous H 2 S. After laser irradiation at 808 nm, tumor growth was inhibited notably because of effective PDT action, while no toxicity was observed in major organs. Furthermore, once tumors were stimulated by exogenous L-Cys, a vital substrate in the H 2 S production pathway, the PDT efficacy was enhanced.
In 2019, Wang et al. proposed a theranostic prodrug platform (TNP-SO), a combination of a H 2 S-responsive imaging agent (NIR-BSO) and a photosensitive drug (3I-BOD) ( Figure 6). 97 Due to low water solubility of TNP-SO, the core was encapsulated in water-dispersible silica nanocomposites, named as nano-TNP-SO. After H 2 S treatment, nano-TNP-SO exhibited a red-shift from 537 to 677 nm in the absorption signal resulting in 137-fold enhancement in the emission signal at 712 nm after irradiation at 640 nm. Nano-TNP-SO was activated selectively in H 2 S-rich HCT116 cells and exhibited light induced toxicity but remained inactive in HepG2 cells. Besides, nano-TNP-SO displayed minimal dark toxicity in HCT116 cells. Ten days after treatment, nano-TNP-SO inhibited tumor volume with 82% efficiency.
A H 2 S/GSH dual responsive activatable PS (aPS), TDBP, was developed by Huang et al. in 2022. 98 TDBP was composed of an active 5,10,15,20-tetra(4-hydroxyphenyl)porphyrin (THPP) core and a H 2 S/GSH responsive quencher ( Figure  7). TDBP was activated in the presence of H 2 S or GSH, which restored its fluorescence at 660 nm, as well as its ROS generation ability. Upon activation by endogenous H 2 S or GSH, TDBP exhibited photocytotoxicity in HCT116 cells, but negligible cytotoxicity was observed in normal human hepatocyte LO2 cells under light irradiation or under dark conditions.
In 2022, the BODIPY core was combined with tetraphenylethene (TPE) units and H 2 S sensitive moiety to construct H 2 S responsive PS (DB2T) by Quan et al. (Figure 8). 99 DB2T  responded to H 2 S selectively with low LOD (6.39 nM) and restored its fluorescence at 579 nm upon excitation at 534 nm.
In vitro studies revealed that DB2T displayed selective activation in cells as it was activated in HCT116 cells but remained nonfluorescent in HepG2, PC12, or HUH-7D cells. Additionally, DB2T exhibited photo-cytotoxicity in HCT116 because of efficient ROS generation. DB2T switched on its fluorescence selectively with endogenous H 2 S and successfully monitored the tumor region in HCT116-tumor-bearing living nude mice (Figure 9). In contrast, DB2T was activated only in the presence of exogenous H 2 S in zebrafish.
An example of a nanoplatform offering trimodal synergistic therapy, PDT, PTT, and chemodynamic therapy (CDT), was devised by Yang et al. in 2022. 100 The nanocomplex NP-Cu was fabricated using dibenzocyclooctyne (DBCO) functionalized lysine (D-K) and an azamacrocylic ring (4A/Cu) along with a photosensitizer, chlorin e6 (Ce6), and hypoxia-activated prodrug banoxantrone (AQ4N) ( Figure 10). As Cu 2+ quenched the emission of Ce6, initially the NP-Cu remained in its off state, but introduction of H 2 S turned PDT to its onstate, and in harmony, endogenous H 2 S reacting with Cu 2+ generated CuS particles, a potent PTT agent. Synergistically, consumption of oxygen due to PDT promoted hypoxia and activated AQ4N, which resulted in CDT and eventual trimodal therapy ( Figure 10). In vitro studies indicated that NP-Cu was activated by endogenous H 2 S in HCT116 cells selectively, turned on therapeutic channels of trimodal therapy, and displayed selective toxicity. In accordance with the in vitro   101 The type I AIE PS (TDCAc) was encapsulated with a H 2 S donor, (NH 4 ) 2 S, and then further constructed on a hydrogel system (TSH). Temperature was elevated upon light irradiation at 660 nm and released previously encapsulated TDCAc aggregates along with (NH 4 ) 2 S. While TDCAc aggregates localized at mitochondria due to ionic interactions, H 2 S was generated from (NH 4 ) 2 S in an acidic tumor microenvironment. This inhibited catalase activity, which resulted in continuous radical formation, with the assistance of a labile iron pool, due to TDCAc induced H 2 O 2 generation. The design approach followed here did not rely on H 2 S activation but proposed a way to utilize H 2 S in synergistic therapy for enhanced PDT efficacy.
In 2022, Zhang et al. presented a novel nanoplatform, ZNPPs, with H 2 S responsive and depleting characteristics for real-time monitoring of H 2 S activity in biological systems and delivering PDT action ( Figure 11). 102 ZM1068-NB was first synthesized by following a six-step synthetic route, then ZNNPs and ZNNPs@FA were prepared using a nanoprecipitation method with the assistance of mPEG 5000 -PCL 3000 and mPEG 5000 -PCL 3000 -FA.
The active sites on these nanoprobes underwent substitution reactions with endogenous H 2 S and generated NIR shift in the fluorescence channel from 1070 to 720 nm along with a shift in the ratiometric photoacoustic signals from 900 to 680 nm. H 2 S introduction to ZM1068-NB resulted in a blue shift in the absorption maximum to 650 nm along with activated 1 O 2 generation upon 660 nm laser irradiation, which ultimately induced cell death. Photophysical characterization studies confirmed that ZNNPs not only responded to H 2 S selectively but also exhibited high linear correlation response within the range of 0 to 500 μM NaHS. ZNNPs were later successfully applied for the monitoring of endogenous H 2 S in HCT116 cells as well as photoacoustic imaging. Fluorescence signal was shown to decrease in HCT116 cells treated with ZnCl 2 (40 μg/mL), a H 2 S quencher, or DL-propargyl glycine (50 μg/mL), a CSE inhibitor. In contrast, the fluorescence intensity increased upon treatment with L-Cys (24 μg/mL), an upregulator for H 2 S, or exogenous NaHS (1 mM) ( Figure  12). Besides, the hepatic H 2 S level in mouse liver and endogenous H 2 S in the injured brain of mice was successfully evaluated using ZNNPs both through fluorescence and photoacoustic imaging. ZNNPs@FA inhibited tumor progression in HCT116 tumor-bearing BALB/c mice, and tumor size reduction up to 89.3% was reported. On the other hand,

ACS Bio & Med Chem Au pubs.acs.org/biomedchemau Review
ZNNPs@FA was found to deplete intracellular H 2 S, thus preventing proliferation in HCT116 and contributing to PDT. Recently, our group introduced an iodinated resorufin corebased H 2 S-responsive phototheranostic agent (RHS) for selective treatment and imaging of neuroblastoma. 103 RHS itself was initially inactive as ICT was blocked. Selective removal of the masking unit on the hydroxyl group by H 2 S released the active Res-I core ( Figure 13) and restored its emission at 606 nm upon excitation at 586 nm, while activating the PDT action.
RHS possessed high singlet oxygen quantum yield (Φ Δ = 0.42) and selective response to H 2 S, compared with other biologically relevant analytes. Selective activation of RHS in SH-SY5Y neuroblastoma cells was shown under confocal microscopy using N-ethylmaleimide (NEM) as a H 2 S inhibitor and NaHS as an inducer (Figure 14). RHS exhibited light   In 2023, Jia et al. incorporated a H 2 S sensitive competent (NBD) to phenazinium derived methylene violet 3RAX dye to construct a H 2 S depletion aided PDT platform (3RAX-NBD) ( Figure 15). 93 In the presence of H 2 S, the probe underwent a slight blue shift from 560 to 540 nm and resulted in amplified singlet oxygen generating ability along with enhanced fluorescence at 610 nm upon irradiation at 550 nm. 3RAX-NBD displayed no dark toxicity but photoinduced cytotoxicity in the concentration region of 0−50 μM in 4T1, HeLa, and MCF-7 cancer cells. PDT efficiency of the probe was later evaluated in 4T1 tumor-bearing BALB/c mice, and the tumor size was reduced by 91.3% under 550 nm laser irradiation, while no reduction in tumor size was noted in the control groups.

H 2 S-RESPONSIVE PHOTOTHERMAL THERAPY (PTT) AGENTS
In 2018, Cu 2 O nanoparticles were prepared by An et al. for H 2 S activatable photoacoustic (PA) imaging and PTT. 104 The oxide nanoparticles were converted to copper sulfide by H 2 S, enhancing PTT and activating PA. Under 808 nm laser irradiation, the sulfidized nanoparticles exhibited a concentration-dependent increase in the temperature and the PA signal. Photothermal conversion efficiency of the particles after NaHS treatment was determined to be 15.6%. Cu 2 O particles were selectively activated in HCT tumor-bearing mice, and tumor distribution was monitored through PA channels.
Remarkably, the tumor completely disappeared on the 16th day as a result of PTT action in the S-adenosyl-L-methionine treated group; however, tumor volume increased gradually in positive and negative control groups ( Figure 16). In 2018, Shi et al. constructed an example of a nanostructured NIR-II fluorescence-guided PTT agent with H 2 S responsive characteristics (nano-PT) using a monochlorinated BODIPY scaffold (SSS) that was extended with a hydrophilic tail in order to mediate self-assembly ( Figure 17). 105 Red shift in the absorption spectrum from 540 to 790 nm upon NaHS treatment enhanced photothermal conversion and increased the temperature of the solution containing nano-PT by 32°C upon 785 nm NIR laser irradiation. Consistently, PTT was reported to rely on the concentration of the probe or analyte and the corresponding laser power. PTT efficacy of the nano-PT was proven successfully under confocal microscopy using calcein-AM and PI treated H 2 S rich HCT116 cells.
Remarkably, in vivo studies indicated that after 10 min of NIR laser exposure, the temperature in the tumor region of HCT116 tumor-bearing mice rose to nearly 60°C; as opposed to that, slight temperature change was observed in control groups.
A nanoplatform (NPs@BOD/CPT) was established by the same group using a BODIPY derived (InTBOD-Cl) probe ( Figure 18) for the aim of simultaneous combined therapy and imaging. 106 The nanoplatform was assembled by coencapsulation of the BODIPY derived theranostic and camptothecin-11 (CPT-11), a chemotherapeutic drug. The thermal shift of the NPs@BOD/CPT under 785 nm laser exposure was modest in the absence of H 2 S, but introduction of H 2 S led to significant photothermal conversion and a concomitant temperature rise. At the eutectic melting point (39°C), previously encapsulated CPT-11 was released and aided the therapy. In accordance with the design principle, NPs@BOD/ CPT was turned on by H 2 S in HCT116 cells and released CPT-11 under NIR light irradiation. Similar on-demand photocontrolled drug release was observed in the in vivo studies. As a result, tumor growth was suppressed in HCT116  In 2019, the probe Au@Cu 2 O was proposed by Tao et al. to realize PTT and PA imaging of H 2 S-rich cancer cells, where the localized surface plasmon resonance (LSPR) coupling effect between the Au nanosphere seed and the sulfidized Cu 2 O layer (Cu 9 S 8 ) would enhance the photoacoustic contrast and promote PTT. 107 The synthesis was carried out by coating the Cu 2 O layer on Au nanosphere seeds while using polyvinylpyrrolidone (PVP) as a surfactant. During kinetic studies, the Au@Cu 2 O system was reported to have an absorption maximum at 808 nm that displayed an increased intensity even at low concentrations of NaHS (0.08 mM), while providing an improved photoacoustic contrast change. Similarly, upon irradiation, Au@Cu 2 O was reported to cause dramatic change in temperature even with a low concentration of NaHS (0.014 mM). The in vivo experiments performed with HCT-bearing mice revealed that the groups treated with Au@ Cu 2 O and S-adenosyl-L-methionine (SAM) + Au@Cu 2 O displayed high photoacoustic contrast when compared with the control groups, where the PA contrast of the SAM + Au@ Cu 2 O group was reported to have surpassed that of the Au@ Cu 2 O-only mice. Likewise, during the photothermal therapy trials, the tumor tissue was reported to have been eradicated in SAM + Au@Cu 2 O mice, while a minor change in the relative   (Figure 19). In 2020, a PTT-enhanced CDT probe (EA-Fe@BSA) was devised by Tian et al. to hasten the H 2 S-mediated Fe(III)/ Fe(II) cycle, which enhances the yields of ROS that are generated via Fenton and Fenton-like reactions while synergistically improving the CDT efficacy via PTT ( Figure  20). 108 The synthesis of these nanoparticles was carried out by mixing an aqueous solution of FeCl 3 with bovine serum albumin (BSA), followed by the addition of ellagic acid at room temperature. The in vitro studies carried out with HCT116 cells revealed that the NPs were able to promote the synthesis of hydroxyl radicals to great extents in the presence of NaHS and H 2 O 2 . The ROS-generating capacity of NPs was also seen to be improved with moderate heating. Upon irradiation of EA-Fe@BSA with an 808 nm laser, a concentration-dependent photothermal effect was detected with a conversion rate of approximately 31−32%. Additionally, the longitudinal (T 1 ) and transverse (T 2 ) relaxation times of the EA-Fe@BSA system proved it to be a viable T 1 -weighted MRI contrast agent that can provide concentration-dependent imaging of cells. The in vivo trials conducted with HCT116 mice were able to display the localization of NPs through MRI and their therapeutic effects. Total tumor ablation was achieved in the NPs + S-adenosyl-L-methionine (SAM) + laser group, while considerable tumor shrinkage was observed in the NPs + laser group. However, the laser-only, NPs-only, and NPs-SAM groups were not able to obtain such decreases  A multifunctional cascade activated theranostic nanosystem (AB-DS@BSA-N 3 ) bearing diallyl trisulfide (DATS), a H 2 S donor, and a H 2 S sensitive azide functional group was synthesized by Zheng et al. in 2020. 109 In the tumor microenvironment, reductive GSH, one of the biothiols that is overexpressed in cancer, can release H 2 S from DATS, and consequently the released H 2 S was used for either gas therapy or reduction of azide (−N 3 (−)) to ammonium ion (−NH 3 (+)), promoting PTT together with PA due to prolonged tumor retention. Under 808 nm laser irradiation, the temperature was increased in a dose dependent manner, and AB-DS@BSA-N 3 exhibited selective photothermal toxicity in Hep2 cells with slight toxicity in control groups. In vivo studies showed that while the tumor can be monitored successfully through PA or fluorescence channels using AB-DS@BSA-N 3 , the tumor was completely suppressed after NIR laser irradiation in Hep2 tumor-bearing mice.
In 2021, H 2 S responsive water-soluble MoO 3 nanoparticles were designed and developed by Wang et al. for PTT and PA imaging. 110 The nanoparticles were prepared with an average size of 9 nm through a one-pot process encompassing ultrasonication and oxidation ( Figure 21). Initially, MoO 3 nanoparticles remained in their off-state, but addition of NaHS generated polyoxometalates (POMs) via redox reaction ( Figure 21) and activated both NIR-I and NIR-II channels at 760 and 1080 nm in a dose dependent manner. A similar trend was recorded in thermal and PA channels. In good correlation with the characterization, MoO 3 nanoparticles displayed photothermal cytotoxicity in HCT116 and 4T1 cells after  laser irradiation. Also, PTT was activated by endogenous H 2 S in HCT116 tumor-bearing mice and resulted in suppression of the tumor. Photothermal conversion efficiency was determined to be higher with NIR-II irradiation as survival rate was lower in NIR-II irradiation in both cases. Additionally, PA images were captured successfully through both channels. Similarly, the signal-to-noise ratio was higher in the NIR-II region.
A MOF that consisted of trimesic acid, copper, and curcumin (Cur@HKUST-1@PVP) was first introduced by Tian et al. in 2022 to treat colon cancer cells by combining   111 During the synthesis of this therapeutic agent, curcumin was loaded into the HKUST-1 network, and the improper release of curcumin was blocked with the surface ligand polyvinylpyrrolidone (PVP). Upon reaction with gradually increasing concentrations of H 2 S, the absorption intensity of Cur@HKUST-1@PVP at 980 nm was shown to increase. It was also demonstrated that the photoacoustic intensity of H 2 S-treated Cur@HKUST-1@ PVP displayed a 5-fold increase compared to the control groups, and upon irradiation of the NaHS incubated cores, a temperature rise (10−21°C) was observed. The in vivo studies of Cur@HKUST-1@PVP demonstrated the colon cancer cell selectivity of the probe as the same 5-fold increase in photoacoustic intensity was only observed at tumor sites. Following its irradiation, the probe was also reported to completely eradicate the colon tumor cells in 16 days in vivo, while HKUST-1 and curcumin control groups only managed to suppress tumor growth ( Figure 22).

CONCLUSION AND OUTLOOK
To close, we summarize here the recent advances in H 2 Sresponsive PDT and PTT agents by covering both small organic molecule and nanoparticle based designs (Table 1). It is a well-established fact that an abnormal level of H 2 S is directly associated with a wide variety of pathological states. Thus, it appears as an attractive biomarker that can be utilized in activity-based phototherapy applications. As evidenced from the current literature examples presented in this review, there is a growing interest toward development of H 2 S-responsive and multifunctional phototherapy agents. However, some critical limitations such as off-target activation of H 2 S activatable PSs, phototoxicity of the caged PSs, high reactivity of H 2 S with other biological molecules due to its high nucleophilicity and redox activity, and interference of other endogenous biothiols still need to be addressed. There are also some other drawbacks arising from the chronic problems of phototherapies such as low cytotoxicity of single mode PTT agents, poor performance of PSs in aqueous environments and under hypoxic conditions, and limited penetration of the excitation light through tissues. Although recent advances in the field of both small molecule and nanomaterial based PSs offer judicious solutions to these challenges, there is still room for development to improve the therapeutic outcome. In this direction, we anticipate that new H 2 S-responsive agents that can combine phototherapy and other modes of therapeutic actions such as chemotherapy, radiotherapy, and sonodynamic therapy will start to increase in the coming years, which will improve the efficacy of the therapy. Additionally, one should expect that these multimodal agents may also enable precise imaging by utilizing fluorescence in the NIR II region and PA, magnetic resonance (MR), or positron emission tomography (PET) techniques. Furthermore, there is no doubt that organelle targeted photosensitizers are promising candidates to achieve high cytotoxicity in phototherapy applications. It is very likely for a H 2 S-responsive agent to be activated in normal cells, as H 2 S also plays important roles in physiological processes. To this end, new generation H 2 S-responsive agents should have improved selectivity. This can be achieved by dual-locked agents, which can be activated in the presence of H 2 S and another related biomarker. Furthermore, it should not be surprising to see more examples soon where H 2 S itself is utilized as a cytotoxic agent. Finally, special attention needs to be paid to the pharmacokinetics and pharmacodynamics characteristics of H 2 S activatable agents in animal models to pave the way for clinical translation.