Applications of Activatable Organic Photoacoustic Contrast Agents

Jiawei Liu; Jing Wang; Qi Wang; Quli Fan; Wei Huang

doi:10.7536/PC20072

Progress in Chemistry ›› 2021, Vol. 33 ›› Issue (2) : 216-231 DOI: 10.7536/PC20072

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Applications of Activatable Organic Photoacoustic Contrast Agents

Jiawei Liu ¹
, Jing Wang ¹
, Qi Wang ¹^,²^,^*
, Quli Fan ¹^,^*
, Wei Huang ³

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Abstract

Photoacoustic(PA) imaging, as a new type of imaging technique that offers strong optical absorption contrast and high ultrasonic resolution, shows great application prospects in the early disease diagnosis for its characteristics of deep tissue penetration and high spatial resolution. However, traditional “always on” PA contrast agents have many disadvantages such as low signal-to-noise ratio, poor selectivity and specificity. In contrast, activatable PA contrast agents, where the imaging signal can be changed in response to pathologic parameters, have shown decreased background signal and improved selectivity and specificity in early disease diagnosis. Moreover, these contrast agents can obtain pathological parameters and information of various diseases at the molecular level by rational design to their structures, providing important guidelines for the optimization of treatment options. Therefore, activatable PA contrast agents hold greater promise in clinical practice than traditional “always on” PA contrast agents. In this review, we describe the recent advances in the development of activatable PA contrast agents. The design mechanisms and proof-of-concept applications of these activatable PA contrast agents are summarized in detail. The use of these activatable probes to detect different pathologic parameters(such as metal ions, enzymes, reactive nitrogen and reactive oxygen species) is highlighted. Finally, current challenges and future perspectives in this emerging field are also analyzed.

Contents

1 Introduction

2“Always on”versus activatable PA imaging

3 Applications of activatable PA imaging contrast agents

3.1 Metal ions

3.2 Enzymes

3.3 RONS

3.4 pH

3.5 Gasotransmitters

3.6 Glutathione(GSH)

3.7 Hypoxia

3.8 Others

4 Conclusion and outlook

Graphical abstract

Keywords

photoacoustic imaging / contrast agents / tumor microenvironment / activatable probes / biological imaging

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Jiawei Liu, Jing Wang, Qi Wang, Quli Fan, Wei Huang. Applications of Activatable Organic Photoacoustic Contrast Agents. Progress in Chemistry, 2021, 33(2): 216-231 DOI:10.7536/PC20072

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1 Introduction

Biomedical imaging is an important research method to analyze the structure and various physiological functions of biological tissues. At present, the common imaging technologies usually use light wave, ultrasound and nuclear magnetic resonance as the media of imaging detection, such as fluorescence (FL) imaging, photoacoustic (PA) imaging, computed tomography (CT) imaging, ultrasound (US) imaging and magnetic resonance (MR) imaging^[1⇓~3]^{[4⇓⇓⇓⇓⇓~10]}^[11,12]^[13,14]^[15]. As a new non-invasive, non-ionizing and non-radiation biomedical imaging method, PA imaging has become one of the research hotspots in the field of biomedical imaging. At present, the clinical application of PA imaging has been carried out in the brain, thyroid and breast^[16]. The principle of PA imaging is as follows: after the light absorber (contrast agent) in the biological tissue receives the irradiation of short pulse laser, the absorbed light energy is converted into heat energy, and due to the instantaneous rise of local temperature,Pressure waves (sound waves) are formed by the thermoelastic expansion of the tissue, and these sound waves are collected by an external ultrasonic detector, and image reconstruction is performed according to the arrival time to obtain imaging information^[17]. Because photoacoustic imaging combines the advantages of both optical and acoustic imaging, it can achieve high contrast and high resolution imaging of deep tissues.

Contrast agents used in PA imaging are generally divided into endogenous contrast agents and exogenous contrast agents. Although endogenous contrast agents have been successfully used in tumor, hemoglobin concentration, cardiovascular disease and oxygen saturation detection and functional imaging, they have the problems of poor imaging contrast and limited detection depth, so scientists are focusing on the development of exogenous contrast agents with higher performance^[18]. Organic molecules have important application value in the field of biological tissue imaging due to their good biocompatibility, easy modification of structure, easy adjustment of optical properties, excellent light absorption and heat generation properties^{[19⇓⇓~22]}. Among them, activatable contrast agents can specifically respond to the microenvironment markers of pathological tissues, and have the characteristics of low background noise, high sensitivity and specificity, and real-time correlation between signal and disease status, so the design and development of activatable organic photoacoustic contrast agents with near-infrared (NIR) absorption has become an important trend in this field^[23⇓~25]^[25⇓~27]. In view of the important research value of activated PA probes, this paper reviews the related research of activated organic PA contrast agents, and summarizes the application examples of contrast agents in the detection and diagnosis of pathological tissue microenvironment markers such as metal ions, enzymes, reactive oxygen and reactive nitrogen (RONS), pH and so on.The representative research achievements in this field in recent years were summarized systematically, and the development trend of activated PA contrast agents in the future was prospected.

2 Signal always on versus activated PA imaging

In recent years, PA imaging has been increasingly used in the biomedical field. PA imaging can effectively and accurately present the structure and function of biological tissues, and provide necessary information for the study of their morphological structure, physiological characteristics, pathological characteristics and metabolic functions. Excellent PA imaging should have two characteristics: (1) the PA signal of the lesion is maximized; (2) background signal minimization. Studies have shown that the traditional PA contrast agent (signal intensity opening type) can be passively or actively targeted to maximize the enrichment in the lesion tissue, thereby effectively improving the PA signal intensity of the lesion tissue. However, this type of PA contrast agent will produce a strong PA signal after entering the organism whether it reaches the target tissue or not, which leads to high background signal and low signal-to-noise ratio of imaging, and easily leads to "false positive" diagnostic results, which greatly limits the practical clinical application of PA imaging. At present, the design and construction of activatable PA contrast agents has become one of the best solutions to solve these problems. This kind of contrast agent can react with a large number of markers (metal ions, enzymes, RONS, pH, gas signal molecules, etc.) In the pathological tissue microenvironment, thus causing specific changes in the PA signal of the contrast agent.Therefore, it has the advantages of specific targeting of lesion tissues, low background interference and high signal-to-noise ratio of imaging, and can obtain pathological parameters and pathological mechanisms of different diseases from the molecular level, which provides important guidance for the optimization of treatment programs. In addition, compared with signal-gated probes, activated PA probes have the advantages of faster imaging rate and lower detection limit, so they have a broader application space in the biomedical field (Figure 1).

3 Application of activated PA imaging contrast agent

As we all know, normal physiological microenvironment is very important for the stable operation of organisms. Studies have shown that there are significant differences between the physiological microenvironment of pathological tissues and that of normal tissues, and these differences are closely related to many diseases, such as tumors, cardiovascular diseases, Alzheimer's disease, tissue injury and inflammation.The design and development of PA imaging contrast agents activated by pathological microenvironmental markers bring new opportunities for specific and accurate diagnosis of pathological tissues.

3.1 Metal Ions

Metal ions are essential cofactors in many biological processes, such as energy metabolism and storage, signal transduction and nucleic acid processing.Therefore, the development of efficient and stable detection methods for metal ions is of great significance for medical imaging diagnosis, environmental monitoring and food safety^[28]. At present, PA imaging has been used to detect some biologically relevant metal ions, including Cu and MeHg, Hg, Ca, Li, UO₂, Pd and so on^2+[29~33]^+[34]^2+[35]^2+[36]^+[37]^2+[38]^2+[39]. The detection principle is that the empty orbitals of the outermost unfilled electrons of the atomic orbitals of metal ions (especially some transition metals) can form coordination bonds with "N" and "O" with lone pair electrons, thus adjusting the optical properties of the probe. According to the results of the interaction between the probe molecules and the metal ions, the probes can be divided into three types: (1) the metal ions directly coordinate with the atoms of the chromophores; (2) metal ion coordination induces specific reactions; (3) The binding of metal ions to ligands other than chromophores indirectly affects the PA signal of the chromophores.

(1) The metal ion is directly coordinated to the atom of the chromophore. Lin et al. reported a signal-opening Cu²⁺ responsive probe (LET-2) consisting of a heptamethylaminocyanine IR823 derivative and a diallylethylenediamine (DPEN) moiety (metal ion chelator)^[33]. When DPEN was coordinated with Cu²⁺, its absorption peak was red-shifted from 625 nm to 715 nm (Fig. 2A), and a strong PA signal was generated at 715 nm. The detection limit calculated from the corresponding PA value at 715 nm was 10.8×10^-9mol/L (Fig. 2B). They believe that the reason for the change of optical properties is that the electron density of DPEN partial amine is reduced after the coordination of Cu²⁺. In addition, they used the probe to image the Cu²⁺ in the liver of mice, further verifying its ability to monitor the Cu²⁺ in vivo.

Zhang et al. Designed a series of Cu²⁺ activated probes RPS1-RPS4, in which aniline derivatives in the molecular structure can specifically chelate with Cu²⁺ to form free radicals, resulting in enhanced absorption and PA signals in the near infrared region^[30]. The probe RPS1 can rapidly respond to the Cu²⁺ within a few seconds, and has high selectivity and low detection limit (90. 9 nmol/L). Due to the low relative molecular mass (less than 438 Da) of the probe, the probe can effectively cross the blood-brain barrier, thereby realizing the visual detection of the Cu²⁺ in the brain of an Alzheimer disease (AD) mouse through PA imaging. In addition, Jokerst et al. Used the coordination reaction of porphyrin macrocyclic compounds for PA imaging detection of uranyl cations^[38]. Westmeyer et al. reported the first cell permeable and selective Ca²⁺ sensor^[36]. Cai et al. Designed a ratiometric Pd²⁺ probe based on cyanine dyes^[39].

(2) The coordination of metal ions induces specific reactions. Chen et al. designed and constructed a novel ratiometric PA imaging probe (NRh-IR-NMs) for efficient detection of Cu²⁺, which consists of a Cu²⁺ sensitive dye (NRh) and a Cu²⁺ inert dye (IR) (fig. 3A)^[31]. The coordination of Cu²⁺ with NRh changed the amide structure, and the PA signal at 716 nm was enhanced, while the intensity of PA from IR at 834 nm remained unchanged. The proportional signal of ΔPA₇₁₆/ΔPA₈₃₄ could be used for the detection of Cu²⁺. Chan et al. Designed a ratiometric probe APC-2 for Cu²⁺ detection by introducing 2-picolinate into aza-BODIPY molecule^[32]. In the presence of Cu²⁺, probe APC-2 was hydrolyzed to the phenolate product (Fig. 3B), with a red shift of the absorption peak from 697 nm to a reduced signal in the 767 nm,PA₆₉₇ and an increased signal in the PA₇₆₇. The reason for the red shift of the absorption peak of the probe is that the electron-withdrawing picolinate group leaves and the resulting phenolate enhances the electron-donating ability of the oxygen atom. Huang et al. Reported a ratiometric probe (LP-hCy7) for the detection of MeHg⁺ in vivo. Under the action of MeHg⁺, hCy7 was converted to hCy7 'by cyclization (Fig. 3C), and the absorption peak was red-shifted from 690 nm to 860 nm, so the concentration of MeHg⁺ could be detected by the ratio change of PA₈₆₀/PA₆₉₀^[34]. They used zebrafish as a model for PA imaging experiments, and found that the PA ratio after incubation with MeHg⁺ was 10.44 times that of the normal group.

(3) The binding of a metal ion to a ligand other than a chromophore. Clark et al. designed a probe for monitoring the concentration of Li⁺ consisting of a Li⁺ carrier VI (L), a protonated chromophore (CH⁺), and a charge balancer R^- (fig. 4A)^[37]. When the Li⁺ is recognized and extracted into the hydrophobic polymer core of the probe, it leads to the deprotonation of the CH⁺, thus changing the PA signal intensity of the probe. Kopelman et al. designed a PA probe for K⁺ in a similar way. When K⁺ is added, the ionophore binds to the K⁺, the chromophore is deprotonated, and the absorption peak at 540 nm increases, while the absorption peak at 660 nm decreases (Fig. 4B)^[40]. The chromophore in this type of probe is essentially a pH indicator that protonates or deprotonates as the concentration of the metal ion changes.

3.2 Enzyme

Enzymes are a class of biological macromolecular catalysts, which are indispensable to the metabolic processes in cells. Abnormal enzyme activity is closely related to the pathology of various diseases, and the precise detection of enzyme activity has become the focus of research in recent years^[41⇓~43]. A general strategy for that design of enzyme-activated probe is: (1) attaching an enzyme-activating moiety (typically a short peptide chain or protein) to a chromophore; (2) The material undergoes an enzymatic reaction and induces a change in its properties. The main ways to change the properties of the materials are: (1) self-assembly of molecules caused by the removal of part of the branched chain; (2) leaving the electron-withdrawing group to establish a new energy transfer process; (3) cutting the connection between the two chromophores; (4) that redox reaction change the electron-withdrawing/donating properties of the group.

(1) self-assembly of that molecule is initiate by cutting off part of the branch chain. The molecules can self-assemble by non-covalent force such as π-π stacking after the hydrophilic chain is cleaved by the enzyme. The dyes activated by PA signal in this way generally have the properties of aggregation-induced luminescence quenching (ACQ), mainly including phthalocyanine, porphyrin and cyanine derivatives. Liang et al. reported an example of an alkaline phosphatase (ALP) -triggered nanoparticle self-assembled probe 1p^[44]. Probe 1p was composed of cyanine dye IR775 and a short peptide chain with phosphate group. When the phosphate was cleaved by ALP, the nanoparticles self-assembled in the tumor, and the PA signal at 795 nm was enhanced by 6.4 times. In order to increase the accumulation and retention of the Probe in the lesion tissue, Wang et al designed and synthesized a smart response probe Probe 1 with active targeting capability^[45]. The molecule consists of the chromophore purpurin 18 (P18), the enzyme-responsive peptide chain Pro-Leu-Gly-Val-Arg-Gly (PL GVRG), and the targeting ligand Arg-Gly-Asp (RGD) (Fig. 5A). When the probe enters the body, it is first recognized by the α_vβ₃ receptor on the cell membrane, and then the gelatinase selectively cleaves the PLGVRG chain. Due to the change of hydrophobicity and steric hindrance, the probe self-assembles in situ to form nanofibers, which prolongs the retention time in the tumor. After incubation of the probe with gelatinase (15 ng·mL^-1) in the agar model for 1 H, the PA signal at 730 ± 1 nm was enhanced 3.5-fold (Fig. 5B). In PA imaging experiments in the U87 xenograft mouse model, 8 H after injection, the mean PA intensity at the tumor site was 16-fold higher in Probe 1 than in Probe 2 (control) (Fig. 5C). In addition, when the anhydride group in the P18 molecule is hydrolyzed, the hydrophilicity of the molecule is enhanced, which is beneficial to the dissociation and excretion of the nanofibers. Based on the design idea of Probe 1, Wang et al. Also reported two activation-type probes, which can recognize and respond to gelatinase in bacterial infection and cysteine protease (ATG4B) in tumor, respectively^[46,47].

In addition to the self-assembly based on the non-covalent bond interaction after enzymatic hydrolysis, the self-assembled nanoprobes involving covalent bonds have also been reported. Gambhir et al. designed a furin-activated probe, ESOR-PA01, in which the cyano group of 2-cyano-6-aminobenzothiazole (CABT) and the 1,2-aminothiol group of cysteine were condensed to form oligomers and further self-assembled into nanoparticles after activation in the tumor microenvironment (Fig. 6a)^[48]. Cell imaging showed that the PA signal produced was 2.6 times higher than that in furin-deficient cells (fig. 6B). In vivo imaging showed that mice with MDA-MB-231 tumors (over-expressing furin) detected a significant increase in PA signal after injection of the ESOR-PA01 probe (Fig. 6 C). Ye et al. Designed a probe for cysteine proteinase-3 recognition by using this condensation reaction, but the difference was that the enzymatic reaction was followed by an intramolecular condensation reaction to obtain a cyclic product^[49].

And (2) leave that electron-withdrawing group to change the charge property of the chromophore and realize the amplification of the PA signal. Pu et al. Reported a urokinase (uPA) -activated probe, P-Dex (Fig. 7A), that can be used to differentiate between invasive and non-invasive breast cancer^[50]. After the enzymatic cleavage of the amide bond and the spontaneous 1,6 elimination reaction, the dye was in an "unblocked" state, the electron-donating ability of the oxygen atom was enhanced, and the PA signal at 690 nm was significantly enhanced (Fig. 7B). By hydrolyzing the phosphate group in LET-3 probe with alkaline phosphatase (ALP), Lin et al. Enhanced the electron-donating property of the oxygen atom in the probe and increased the PA intensity by 27 times^[51].

(3) Cleavage of the link between two chromophores by enzymes is also a common method for designing enzyme-activated probes. Gambhir et al. designed a probe for matrix metalloproteinase (MMP-2 and MMP-9) activation, B-APP-a, in which the quencher BHQ3 (A1) was linked to a cell-penetrating peptide, polyarginine, and the fluorescent molecule Alexa750 (A2) was linked to a non-cell-penetrating polyglutamic acid chain, and the two amino acids were linked by the peptide sequence Pro-Leu-Gly-Leu-Ala-Gly (PLGLAG)^[52]. After activation, A1 enters the cell, A2 leaves, and the difference between the PA₆₈₀ (from A1) and the PA₇₅₀ (from A2) increases. In PA imaging of mouse thyroid tumors, the signal intensity difference was enhanced by 1.7 times after 140 min of injection. Lu et al. Linked the quencher and fluorophore through DNA aptamer, and the prepared probe achieved ratiometric response to thrombin^[53].

(4) that redox reaction change the electron-withdrawing/donating properties of the group. Cai et al. designed a probe IR1048-MZ that responds to nitroreductase in hypoxic environment. After the nitro group in the probe is reduced, the absorption at 980 nm is enhanced and the PA signal is turned on. The probe achieves tumor PA imaging with centimeter-level deep tissue penetration^[54].

3.3 RONS

Reactive oxygen species and reactive nitrogen species (RONS) play key roles in biological metabolism and intercellular signaling^[55⇓~57]. ROS is a kind of one-electron reduction products of oxygen, including

O 2 · -

, H₂O₂, ^·OH, ¹O₂, HOCl/^-OCl and so on. RNS refers to NO and its higher oxides, including

NO 2 ·

, ONOO^-, NO₃^-, etc. The molecules and mechanisms of RONS response have been reviewed, and here we will discuss how the oxidation of RONS alters the PA signal of the probe^[58⇓~60]. According to the results of oxidation reaction, it can be divided into two categories: (1) oxidation and induced decomposition of hydrophilic chains, which promotes the self-assembly of probe molecules; (2) oxidation of the chromophore or its adjacent groups to directly modify the optical properties.

(1) oxidizing and decompose hydrophilic chains to promote that self-assembly of probe molecule. Pu et al. Developed a semiconductor macromolecule activatable probe PCBP and applied it to RONS-targeted enhanced PA imaging in vivo by in-situ self-assembly^[61]. The probe consists of a near infrared absorbing phthalocyanine core, a RONS-responsive self-eliminating segment and a hydrophilic poly (ethylene glycol) (PEG) arm. When the molecule reacts with H₂O₂ or ONOO^-, the aryl borate ester is firstly oxidized, the pinacol borate ester leaves, the phenol is decomposed through the rearrangement of the quinonomethyl compound, and the hydrophilic PEG chain is cleared at the same time, and the hydrophobic phthalocyanine self-assembles under the hydrophobic force and π-π stacking force (Fig. 8A). After H₂O₂ treatment, the radius of nanoparticles increases from 20 ± 2 nm to about 350 nm, and the PA intensity at 700 nm increases by a maximum of 1. 4 times. In the subcutaneous 4 T1 xenograft tumor model, the ΔPA₇₀₀ of mice pretreated with D, L-methionine- (S, R) -thioimine (BSO), a drug that increases ROS levels, was 2.0-fold higher than that of untreated mice 8 H after injection (Fig. 8 B), suggesting that elevated ROS can activate the probe and produce stronger PA signaling.

(2) Oxidation of chromophores or their adjacent groups can be divided into two subcategories according to the degree of oxidation: (I) no degradation of the structure; (ii) degradation of the structure.

(I) The structure does not degrade. Liu et al. designed a H₂O₂ activated nanoprobe Lipo @ HRP & ABTS. In the presence of H₂O₂, the colorless ABTS will be oxidized to a

ABTS · +

with strong near-infrared absorption (Fig. 9a), and the absorption and PA intensity at 800 nm are significantly enhanced^[62]. Zhang et al. Reported a probe for the detection of

O 2 · -

, BDP-DOH, in which the ortho-phenolic hydroxyl group on the BODIPY molecule was easily oxidized (Fig. 9B), resulting in a decrease and increase in absorbance at 680 and 750 nm, respectively, resulting in an increase in the ratio of PA₇₅₀/PA₆₈₀^[63].

Near-infrared Ⅱ (NIR-Ⅱ, 1000-1700 nm) imaging has become a research hotspot in recent years due to its advantages of deep penetration and high contrast.Xing et al reported a H₂O₂ activated probe SHT, and realized photoacoustic imaging of tumors in NIR-Ⅱ region^[64]. Under the conditions of H₂O₂ and low pH, 3,3 ', 5,5' -tetramethylbenzidine (TMB) in the probe self-assembled with its divalent cation oxide to form a charge-transfer nanocomplex, which resulted in an enhanced absorption band and PA signal at 750-1200 nm. The activated probe SHT has a deep tissue penetration capability with clear imaging contrast (10 dB signal-to-noise ratio) even at a tissue depth of 2 cm. In the imaging experiment of 4 T1 tumor-bearing mice, the PA signal at the tumor site increased gradually with time and reached the peak at 4-6 H after injection, and a significant difference in intensity between tumor and normal tissue PA images was observed (p < 0.001). In addition, it is worth mentioning that the fluorescence imaging and chemiluminescence imaging of activated NIR-Ⅱ have made great progress in recent years, but there are still few reports on activated NIR-Ⅱ organic PA probes, so this kind of probes will be an important trend in the future^[23]^[65].

(ii) degradation of the structure. Pu et al. Reported a ratiometric probe SOA-NIR775 responsive to ClO^-, which is composed of an amphiphilic dye SOA that can be degraded by ClO^- and an inert component NIR775, for the decomposition of chromophores induced by RONS oxidation^[66]. Under the action of ClO^-, the sulfur atom in the SOA molecule is oxidized and gradually degrades the entire chromophore, resulting in a decrease in the corresponding absorption intensity, while the absorption intensity of NIR775 remains unchanged. The ratio value of the activated PA₇₈₀/PA₆₈₀ is 8.3 times that of the initial inactive state.

Cyanine dyes can be decomposed by some RONS. Chen et al. Composed a cyanine dye IR790s, a perylene diimide (PDI) derivative grafted with cisplatin prodrug and iron ions to form a ratiometric nanoprobe PDI-IR79s-Fe/Pt (Fig. 10a)^[67]. In vivo, nanoparticles first release cisplatin to activate NOX (nicotinamide adenine dinucleotide phosphate oxidase), which catalyzes the formation of H₂O₂ and superoxide, and then the Fe³⁺ converts H₂O₂ into

· OH

through Fenton reaction. The generated radicals decompose IR790s and cause a decrease in the absorption intensity of IR790s at 790 nm, while the absorption intensity from PDI molecules at 680 nm remains unchanged, allowing ratiometric PA imaging to track RONS production and tumor treatment efficacy. In the in vivo experiment, the ratio of ΔPA₆₈₀/ΔPA₇₉₀ was 1.7 times higher after injection of PDI-IR790s-Fe/Pt nanoparticle (5 mg·mL^-1,200μL)24 h than after 2 H of injection (Fig. 10B), indicating a good ability to monitor RONS. In addition, probes based on the principle of oxidized cyanine dyes have also been reported in some literatures^[68,69].

In the aspect of inducing the decomposition of adjacent groups of chromophores, Pu et al. Reported a kind of ONOO^- responsive probe OSN-B1. When RONS cleaves the phenylboronic acid pinacol ester of BBD molecule in the probe (Fig. 11A), the PA intensity at 680 nm remains unchanged, the PA signal at 750 nm is significantly enhanced, and the ratio of probe OSN-B1 in the PA₇₅₀/PA₆₈₀ is enhanced by 7 times compared with that before activation^[70]. In addition, they found that doped borane could effectively block the oxidation of BBD molecules by H₂O₂, thus increasing the specificity of probe detection. In our research group, a probe OEG-Aza-BODIPY-BAPE was constructed by connecting benzeneboronic acid pinacol ester and Aza-BODIPY through quaternization^[71]. Upon H₂O₂, the quaternary ammonium on aza-BODIPY changes to a tertiary amine (Fig. 11B), resulting in a red shift in absorption from 720 to 825 nm. The mean concentration of H₂O₂ in the tumor was estimated to be 60 μmol/L by the ratio change of the PA₈₂₅/PA₇₂₀. The quaternization connection mode can well eliminate the interference of the ONOO^- to the detection and ensure the specific response of the probe molecule to the H₂O₂. Uchiyama et al used H₂O₂ to remove the S-benzyl thiocarbonate moiety in the Probe molecule (Probe 8), and the weak aromatic phthalocyanine containing phenolic hydroxyl group was tautomerized into a quinoid structure with strong aromaticity, which showed a strong absorption peak and PA signal at 880 nm^[72].

3.4 pH

The regulation of pH plays an important role in maintaining the balance of human internal environment, and the charge state of macromolecules and proteins is also closely related to pH^[73]. Changes in pH can also induce a range of diseases, including cancer, ischemia, kidney disease and intrauterine diseases. Therefore, monitoring the change of acidity in the body microenvironment is of great significance for the early prevention and treatment of diseases. The detection of pH is essentially a reaction of H⁺/OH^-, which can be divided into three types: (1) protonation/deprotonation; (2) Acidic environment induces chemical bond breakage; (3) participating in certain reactions as a synergistic condition.

(1) Protonation/deprotonation. Protonation usually occurs on atoms that contain lone pair electrons, including nitrogen, oxygen, and so on^[74⇓~76]^[77,78]. Lin et al. Designed a pH-responsive PA probe LET-4 for monitoring gastric acid secretion^[74]. The near-infrared dye IR-pH was protonated under acidic conditions (Fig. 12A), resulting in an increase in the intensity of the probe absorption at 808 nm and the PA signal, and the intensity of the PA₈₀₈ signal at pH = 3 was 3.45 times greater than that at pH = 7. Pu et al. reported a ratiometric Probe, Probe 3, consisting of an inert component, F-DTS, and a pH-responsive boron-dipyrromethene (pH-BDP) molecule for in vivo pH imaging (fig. 12B)^[77]. In acidic environment, the hydroxyl group of pH-BDP is protonated, which leads to the decrease of the absorption intensity at 750 nm and the PA signal.The PA₆₈₀/PA₇₅₀ at pH = 5. 5 is 1. 8 and 3. 1 times of that at pH = 6. 4 and 7. 4, respectively. In addition, the PA signal was amplified by the photo-induced electron transfer (PET) between F-DTS and pH-BDP.

(2) The self-assembly of nanoparticles can be triggered by the breaking of chemical bonds induced by acidic environment. Xing et al. Designed a pH-responsive near-infrared nanoprobe NP1 based on dextran. Due to the aggregation of H, IR-783 has two absorption peaks at 708 and 785 nm^[79]. Acidification resulted in cleavage of the bond (fig. 13A), dissociation of the IR-783 monomer, and an increase in the ratio of PA₇₈₅/PA₇₀₈ (fig. 13B). Chang et al. introduced the easily hydrolyzed citraconamide on the surface of melanin-like nanoparticles (MelNPs), which aggregated in acidic environment and enhanced the PA signal by 8.1 times compared with neutral environment^[80].

(3) participating in certain reactions as a synergistic condition. MnO₂ can react with H₂O₂ to form Mn²⁺ under acidic conditions, and our research group designed a PA probe PBP@MnO₂NPs responsive to tumor microenvironment based on this reaction (Fig. 14A)^[81]. In the probe, a polymer (PBP) grafted with a BODIPY molecule is used as an inert component and can be used to encapsulate MnO₂ nanoparticles. Under the action of acidic environment and H₂O₂, the absorption peak of MnO₂ (680 nm) gradually decreases, while the absorption peak of PBP (825 nm) is almost unchanged. In vivo imaging experiments, the ΔPA₈₂₅/ΔPA₆₈₀ ratio after 24 H of injection was enhanced by a factor of 3.7 compared to the signal ratio after 2 H of injection (Figure 14 B).

3.5 Gas Signal Molecule

Gaseous signaling molecules exist in a wide range of organisms and play an important role in the regulation of physiological and pathological processes. Hydrogen sulfide (H₂S) plays a role in vasodilation, angiogenesis, antioxidant, anti-inflammatory, and central nervous system regulation. Abin H₂S can lead to a variety of diseases, including Down's syndrome and Alzheimer's disease^[82]. The detection of H₂S molecules is generally based on their nucleophilic and reductive reactions with probes. He et al. Designed a ratiometric PA probe (HS-CyBz) and used it to image H₂S in vivo^[83]. The enol cyanine derivative obtained by nucleophilic substitution of the ester group in the probe molecule by the HS^- is further converted to the keto form (fig. 15A), and the ratio of the PA₈₂₅/PA₇₇₅ is increased. Our research group designed probes CyCl-1 and CyCl-2 (Fig. 15B) based on the nucleophilic reaction of HS^- with cyanine dyes, in which the maximum absorption and PA peak of probe CyCl-1 was blue-shifted from 800 nm to 720 nm after the Cl atom was substituted, and the change of ln(ΔPA₇₂₀/ΔPA₈₀₀) could be used to monitor the concentration of H₂S in mice^[84].

Xing et al. designed a ratiometric probe AzHD-LP based on the reduction of azide by H₂S. When the azide of AzHD molecule in the probe was reduced to amino group (Fig. 15C), the absorption peak gradually shifted to red from 600 nm, and the ratio of 700 nm,PA₇₀₀ to PA₅₃₂ increased^[85]. In the experiment of H₂S imaging in the brain tissue of Alzheimer's disease mice, the ratio of PA₇₀₀/PA₅₃₂ in the normal group increased by 6.5 times, while that in the disease group increased by only 1.2 times, indicating that the content of H₂S in the brain of normal mice was significantly higher than that of the disease mice.

Nitric oxide (NO) contributes to the relaxation of vascular smooth muscle and vasodilation, and is a neurotransmitter of memory. However, the abnormal production of NO is also the cause of many diseases, such as lysosomal storage diseases and neurodegenerative damage^[86]. The functional groups used for NO-specific reaction include o-phenylenediamine, aromatic secondary amine and dihydropyridine^[87]. Zhang et al. designed a PA probe DATN activated in an NO/acidic environment, which is composed of a stimulus-responsive molecule NRM, an o-phenylenediamine derivative, and an inert component NIM^[88]. In the presence of NO, the diamine group in the NRM molecule is converted to a triazole group, resulting in an increased electron-accepting capacity (Fig. 16A). Based on the principle of intramolecular charge transfer (ICT), an obvious absorption peak at 680 nm was observed after the activation of the molecule, while the absorption peak at 950 nm from the reference NIM remained unchanged, so the ratio of PA₆₈₀/PA₉₅₀ could be used to monitor the NO content in tumor inflammation. They found that the probe showed a more significant photoacoustic signal enhancement under two-factor (NO and acid) activation, 9.8 times that of NO alone and 132 times that of acid alone.

Chan et al. designed five NO-responsive PA probes based on BODIPY, in which APNO-5 molecule combines with NO through N-methylamine to form N-nitroso product (Fig. 16 B), and this probe has the best response to NO^[89]. In the experiment of endogenous nitric oxide detection in the rat lipopolysaccharide-induced inflammation model, the PA₆₈₀/PA₇₇₀ value was 1.3 times higher than that of the control group.

3.6 Glutathione (GSH)

Glutathione (GSH), as one of the reducing substances, plays an important role in regulating the balance of redox state in the body^[90,91]. Our research group designed a GSH-responsive PA probe IR806-PDA based on the reduction of disulfide bond by GSH. The probe is composed of an amphiphilic cyanine derivative. When the disulfide bond in the molecule is broken, the sulfhydryl group is exchanged with the secondary amine (Fig. 17A), which reduces the absorption at 658 nm and increases at 820 nm. At the same time, the size of the nanoparticles decreases from 25.9 ± 2.3 nm before activation to 2.98 ± 1.1 nm^[92]. In the imaging experiment of the subcutaneous HeLa xenograft tumor model, the ΔPA₈₂₀/ΔPA₆₈₀(0.53±0.01) after 4 H of injection was 2.94 times that after 0.5 H (0.18 ± 0.03) (Fig. 17B).

Chen et al. Reported a ratiometric probe CR-POM activated by GSH in tumor microenvironment, which was synthesized by self-assembly of an organic dye CR and a molybdenum-based polyoxometalate POM cluster, and the NIR absorption intensity of the two components showed opposite trends after reacting with GSH^[93]. The absorption at 700 nm decreases after CR molecules are reduced. When Mo (Ⅵ) in POM was reduced to Mo (Ⅴ), the absorption intensity at 866 nm increased, and the ratio of PA₈₆₆ to PA₇₀₀ increased.

3.7 Hypoxia

Hypoxia often occurs in many diseases, including cancer, and when oxygen supply is insufficient, it can lead to impairment of physiological functions. Chan et al. designed a probe for hypoxia detection, HyP-1. The N-oxide on HyP-1 can be biologically reduced in an anoxic environment (Fig. 18 A), resulting in an enhanced PA signal at 770 nm^[94]. Wu et al. Introduced an azo group that can be broken in a hypoxic environment into the fluorophore (Fig. 18B). When the azo chain was decomposed, the probe molecule (NR-azo) showed an absorption peak at 680 nm and a PA signal^[95]. In addition, the nitrogen mustard (active drug) obtained by decomposition can be used as a therapeutic drug to inhibit tumor growth, and the probe realizes the integration of tumor imaging and treatment.

3. 8 Other

In some pathological microenvironments (such as temperature and polarity), the probe molecule can keep its molecular structure unchanged, and the PA signal can be changed by regulating its excited state through the microenvironment. Such probes can dynamically reflect physiological information.

Zheng et al. reported a temperature-responsive PA probe by attaching a light-harvesting porphyrin to a phospholipid tail and inserting it into a lipid matrix,There is a strong absorption peak of Q_y at 824 nm due to the J-aggregation between porphyrin molecules, but when the temperature is higher than the phase transition temperature, the absorption peak of Q_y is blue-shifted to 750 nm, and the intensity of PA at this wavelength is enhanced^[96].

Tang et al. designed a probe, ER-P, which responds to the polarity of the endoplasmic reticulum, for the diagnosis of liver injury caused by diabetes^[97]. Based on the intramolecular charge transfer (ICT) effect, the absorption peak of the probe has a significant red shift in polar solvents, so the polarity of the medium can be quantified by the ratio of PA₇₀₀/PA₈₀₀.

4 Conclusions and outlook

PA imaging combines the advantages of optical and acoustic imaging to provide higher spatial resolution and deeper tissue penetration. In recent years, the activated PA contrast agent has received extensive attention and rapid development, because it can overcome some shortcomings of the traditional PA contrast agent, such as low signal-to-noise ratio, poor selectivity and specificity. Activated probes can only cause the change of PA signal after reacting with specific targets, so they have higher imaging signal-to-noise ratio and can establish real-time correlation with pathological processes at the molecular level, and have been applied to the detection and imaging of metal ions, enzymes, RONS, pH and gas signaling molecules. Of course, there is still a huge room for improvement of activated PA probes, which is also an important development direction of probe design in the future. These developments include: (1) development of single-component ratiometric probes. The ratiometric probe can perform self-calibration of the signal intensity, and the single-component probe does not have the problem of "leakage" of the two-component dye molecules, so that the quantitative analysis can be performed more accurately. (2) designing a reversible PA probe. The reversible probe can dynamically reflect the physiological tissue parameters, thus avoiding the problem of signal interference caused by the probe leaving the target site after activation. Such probes are generally designed based on reversible reactions, such as protonation-deprotonation, oxidation-reduction, and coordination-dissociation. And (3) trigger that self-assembly of the nanoparticles aft activation. Probes self-assemble to form larger particles in a specific environment, which can improve the retention ability, while unactivated molecules are metabolized. And (4) designing probes which are activated by double or multiple factors, namely "double locks" or "multiple locks". Some groups can be recognized and reacted in a variety of environments, for example, some ester groups can be hydrolyzed by both esterase and active species, which greatly interferes with the detection. Multiple responses need to match multiple parameters and therefore have higher accuracy. And (5) introduce a targeting group. Actively targeted nanoprobes can increase the enrichment of specific sites, thus further improving the signal-to-noise ratio of imaging. And (6) the absorption wavelength is in the near infrared II region (NIR-II). PA imaging requires external light source excitation, and the smaller the absorption and scattering of excitation light by tissues and bodies, the higher the image resolution and clarity. Although activatable PA probes are still in the basic research stage, we believe that in the near future, more sensitive and intelligent PA probes will emerge and be applied to the diagnosis and treatment of human clinical diseases.

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