Nanoparticle-Based Activatable Probes for Bioimaging.

Toxicity concerns of nanoparticles are non-negligible, which have led to their limited clinical translation to date. Systematic evaluation of the toxicity and metabolic behavior of nanomaterials in the body is essential to impel their clinical transition and extend their applications in disease theranostics. Ideally, the probes should accumulate in the targeted tissues or organs, and have higher sensitivity and intensity with the low side effect. After imaging, these probes should be cleared quickly and safely. This is a non-trivial task, and here are some of the major challenges. Tumor imaging with or without treatment modality is a major use of nanoprobes. For tumor imaging, there are two strategies for nanoprobes to accumulate the target region: active targeting and passive targeting. Active targeting can be achieved by using specific ligands (antibodies or peptides) that can be grafted to the nanoparticle surface and specifically bind to overexpressed receptors at the target site. The enhanced permeability and retention (EPR) effect is well known as the passive targeting mechanism. This effect based on two factors: 1) heavy energy and oxygen demand in fast-growing tumors leads to extensive angiogenesis and hyper-permeabilized neo-vasculatures, which cause leakage of circulating macromolecules and small particles; and 2) the lack of effective lymphatic drainage system in the tumor, which leads to the macromolecule or nanoparticle accumulation. In the last decade, the EPR effect was considered to be the main mechanism to overcome the lack of targeting of traditional small molecule drugs. However, after more than 30 years since the discovery of the EPR effect, the clinical evidence of the EPR effect is largely missing. The success rate of nanomedicine clinical translation for cancer treatment is low despite some successes such as Doxil, Abraxane, and Vyxeos. Less than ten anticancer drug delivery systems and imaging nanoprobes (e.g., silica-based Cornell dot, gold-based Auroshell) are currently in phase III or IV clinical trials, and only ten have received regulatory approval in Europe and the United States, seven of which are liposomal formulations. This is primarily due to the poor delivery efficiency of nanoparticle formulations to the cancer site. Recent studies showed the EPR effect is dependent on tumor types, while fast-growing tumors such as carcinomas are very vascular and therefore have more porous/leaky blood vessels; slow-growing tumors such as sarcomas are not extremely vascular. [ 115 ] However, this still needs to be verified in humans. Recently, original research revealed that the dominant mechanism of gold nanoparticle transport in the tumor is an active process and not a passive process such as EPR. In this study, the frequency of endothelial gaps in the tumor vasculature of the U87-MG glioblastoma xenograft model, the supposed sites where EPR happens, was analyzed using TEM and three-dimensional (3D) microscopy. Based on mathematical modeling using 3D images of tumor vasculature, and measuring the accumulation of nanoparticles in tumors post-intravenous injection, they found that the number of gaps observed via TEM is not sufficient to explain the accumulation of nanoparticles in tumors. They found trans-endothelial pathways are the dominant mechanism of nanoparticle extravasation into tumors. This has been validated in an additional three different mouse models and three different types of human tumors. This study is especially relevant to nanoprobes that are mainly inorganic nanoparticles. However, we have to keep in mind that substantial differences exist among tumor types, anatomical locations, and stages from patient to patient, and one also needs to consider that escaping the systemic circulation into the tumor is only one of many barriers faced by cancer nanomedicines, e.g. , high intratumoral pressure and dense extracellular matrix. Nevertheless, these results challenged our current rationale of using EPR effects to develop cancer nanomedicine and suggest that understanding these active pathways will unlock strategies to enhance tumor accumulation. [ 116 ] Although the clinical demonstration of the EPR effect is still required, recent advances help us to design smart probes for clinical settings. In addition to consideration of passive targeting such as EPR effects, the active targeting strategy should be considered. Typically, antibody, peptide, folic acid, transferrin, etc. are put on the surface of the nanoprobe for active targeting ability. This needs to consider the effect of the protein corona, which may affect active targeting. When NPs are introduced into the physiological environment, unspecific adsorption of proteins will form a layer on the particle surface, named “protein corona” (PC). [ 117 ] The PC is highly dynamic, and it is dependent on the characteristics of the NP (i.e., size, charge, and surface engineering), proteins (i.e., molecular weight, isoelectric points, structure, and folding), conditions (i.e., time, temperature, concentration), and type of biological sample (i.e., plasma, urine, tissue lysate, etc.). Proteins in the PC also vary under individual disease states (e.g., diabetes, rheumatism, cancer, obesity, hemodialysis, hyperfibrinogenaemia, hypercholesterolemia, hemophilia, and pregnancy), as well as specific disease conditions (e.g., cancer stage or grade and tumor heterogeneity). For nanoprobes, PEG is typically used to reduce the binding of opsonins and proteins and prolong the circulating time, and currently is the most common strategy to reduce PC formation and aggregation. In addition, zwitterionic polymers are other alternatives for reducing PC formation. Beyond compromising the targeting efficiency of NPs, PC also can be used to achieve active targeting. By modifying the liposomal surface with a short nontoxic peptide, the NPs obtained specific interactions with apolipoproteins. After adsorption of plasma apolipoproteins, the PC exposed the receptor-binding domain of apolipoproteins to achieve brain-targeted delivery. [ 118 ] In general, understanding and control the PC rather than eliminate that provides a new impetus for active targeting. The clearance of nanoparticles follows two main pathways in metabolic processes: 1) urinary excretion and 2) hepatobiliary and feces excretion. [ 119 , 120 ] Nanoparticles with sizes exceeding 6 nm could be rapidly taken up by the RES (liver and spleen). Nanoparticles with small sizes (<5.5 nm) are quickly excreted in the urine by the kidney because their sizes fall below the threshold required for kidney filtration. [ 121 ] Some nanoparticles with large sizes could be cleared by the kidneys after their degradation during the prolonged circulation in the body. [ 122 ] However, the clearance of NPs in a broad sense is very complicated. It is associated not only with the size of the particles but also with surface modification. The solution synthesis of colloidal NPs requires suitable surface capping agents to prevent the NPs from uncontrollable growth, and at the same time, increase the colloidal stability of the NPs. Towards biomedical applications, biocompatible surface coatings are required not only for colloidal stability in physiological environments but also provide reactive groups to further couple bioligands to enhance the accumulation of the particle probes in the region of interest. Surface coatings will inevitably interact with biomolecules such as lipids, sugars, and especially proteins that may subsequently adsorb onto the surface of NPs to form the PC, as we mentioned above. PC may affect the clearance of them from the mononuclear phagocyte system. Inorganic nanoparticles, especially gold nanoparticles, are non-biodegradable and are stable enough as a study object to understand the clearance of nanoparticles in vivo . Using a series of GS-AuNPs with different core sizes (2.5 nm and 1.7 nm NPs and atomic precisely sub-nanometer clusters), Du et al. found that the glomerular filtration barrier can behave as an atomically precise bandpass filter to significantly slow down the renal clearance of smaller particles in the sub-nanometer size regime while maintaining the renal clearance at a comparable level in the range of 1–3 nm. [ 123 ] They found that Au nanoclusters (AuNCs) smaller than 1 nm showed a decrease in renal clearance efficiency. This is because smaller AuNCs are more easily and physically retained by the glycocalyx of the glomeruli, similar to the separation principle used in gel filtration or size-exclusion chromatography. In another study, polyethylene glycol-block-poly(ε-caprolactone) (PEG-PCL) and PEG-block-poly(D, L-lactide) (PEG-PDLLA) micelles were used to evaluate the blood clearance kinetics. [ 124 ] A pair of FRET dyes, Cy5 and Cy5.5, was conjugated to the polymer to obtain PEG-PCL-Cy5 and PEG-PCL-Cy5.5. When these two polymers were assembled to micelles, a strong FRET effect would occur. In contrast, the disassembly of the micelles led to the low efficiency of FRET. In vivo experiments demonstrated that bloodborne proteins (particularly albumin) induced the most (~80%) PEG-PCL and PEG-PDLLA micelles to quickly dissociate into unimers, which were sequestered by Kupffer cells, while intact micelles were difficult to be cleared. An active nanoprobe for MRI was also used to study renal clearance. Tang group developed monodisperse Fe 2 O 3 supraparticles, which are self-assembled 3–5 nm-sized Fe 2 O 3 NPs. The carboxyl groups of citrate molecules among the nanocrystals played an essential role in the self-assembly. In the presence of GSH and H + , the disassembly and degradation of supraparticles increase r1 value (from 0.19 to 1.20 mM −1 S −1 ) and decrease r2/r1 value (from 20.79 to 1.59). After intravenous tail vein injection, the intensity of MRI signals at the kidneys and liver is enhanced in 10 min and almost completely recover after twelve hours. The MRI signals at the bladder could be detected at the same time as that at the liver and kidneys; however, the intensities are much higher than that of the two organs. The results demonstrated that the Fe 2 O 3 supraparticles are mainly excreted by renal clearance. [ 125 ] Overall, the development of rapidly cleared nanoparticles is an important task that needs to be adequately explored for biomedical applications, especially for the translation of such nanoprobes into clinical trials. In addition to the sensitivity and specificity, the clearance of the activatable nanoprobes should be paid more attention. Conversely, these activatable probes may also provide a powerful tool to understand clearance behavior. The clearance of nanoparticles from the blood vessel is the end of the imaging agent but not the end of nanomaterials. 90% of intravenously administered radio-labeled fullerenes are retained for at least a week, with 97.0% lodging in the liver and spleen. The toxicity of these materials should be considered before they were mostly excreted. In addition to activatable nanoprobes, numerous new nanomaterials are designed and produced continuously with novel physicochemical properties. Few have been evaluated for their toxicity systemically. The toxicity of nanomaterials can be attributed to the chemical composition, size, shape, surface coating, which are accompanied by different surface energy, redox status, surface defects, charge. Over the year, we have studied the toxicological profiles of metal oxide nanoparticles including transition-metal oxides (TMOs, e.g., Co 3 O 4 , Mn 2 O 3 ) versus rare-earth oxide (REO) NPs (e.g., Gd 2 O 3 , Y 2 O 3 ) and identified their toxicity mechanisms as well as the structure-activity relationships ( Figure 7 ). [ 126 ] For example, MOx with a high dissolution rate (ZnO, CuO, V 2 O 5 , etc.) could shed toxic ions to induce oxidative stress, inflammatory cytokines, and cell death. TMOs with conduction band energy overlapping the biological redox potential could induce oxidative stress and apoptotic cell death by inducing the activation of caspases 3 and 7. In contrast, REOs could transform into sea urchin structures in the acidic environment of lysosomes after cellular uptake, which triggers lysosomal damage and activation of the NLRP3 inflammasome, production of IL-1β, and GSDMD-mediated pyroptosis in macrophages, with cell swelling, membrane blebbing, and increased membrane permeability. High aspect ratio MOx could also trigger lysosomal damage and NLRP3 inflammasome activation, which can lead to chronic lung inflammation and fibrosis. Fumed silica mostly attaches to the cell membrane, causing membrane perturbation and potassium efflux, which lead to NLRP3 inflammasome activation and IL-1β production. MOx with cationic surface coating could induce proton sponge effects in lysosomes after cellular uptake, which can trigger cell death. [ 126 – 129 ] For toxicity evaluations of nanoprobes, it is recommended to use the mechanisms-based high-throughput screening (HTS) and a predictive toxicological approach, which will make predictions on the physicochemical properties of nanomaterials that may result in pathology or disease in vivo . [ 130 ] To establish this approach, four elements are essential: 1) a well combinational nanoparticle library, 2) rigorous physicochemical characterizations of nanomaterials, 3) development of in vitro HTS approaches to assess biological effects of nanomaterials quantitatively, 4) establishment of quantitative structure-activity relationships (SAR) with in vivo results. [ 131 ] In this predictive toxicological paradigm, HTS will be used to achieve rapid hazard ranking among a batch of nanoprobes. [ 128 , 129 , 132 , 133 ] The cells will include epithelial cells, endothelial cells, liver cells including Kupffer cells and hepatocytes, etc. The parameters will include oxidative stress, inflammatory cytokine production, DNA damage, and cell death. The in vitro HTS results will be validated in vivo in rodent animal models. The results from the in vitro and in vivo studies will be used to build nanomaterial structure and activity relationships (nano-SAR), which is helpful to design safer nanoprobes for biomedical applications. [ 126 , 131 , 134 – 136 ]

Though a large number of imaging nanoprobes have been designed and reported every year, few of them have been applied in clinical practice. Currently, the clinically approved nanomaterials are mainly MRI and SPECT contrast agents. MRI form pictures by detecting the relaxation signals of (water) proton spin in the strong magnetic field excited by radiofrequency waves. The proton spins return to their equilibrium states via two independent relaxation processes of T1 (spin-lattice) and T2 (spin-spin). The chemical agents that can accumulate the recovery of these two relaxations are called T1 contrast agents and T2 contrast agents, respectively. Though MRI is a powerful tool to image anatomical structures or blood flow, the sensitivity to differentiate between healthy and unhealthy tissue remains to be improved by using the contrast agent. The most commonly used intravenous contrast agents are T1 contrast agents, which are based on gadolinium chelates. [ 5 ] For example, Gd-DTPA (diethylene triamine pentaacetate acid, DTPA) can be used to help to image the breast, spine, liver, heart, brain, the soft tissue of joints, and inside bones. [ 110 – 112 ] Some studies have pointed out the concern of gadolinium-associated nephrogenic systemic fibrosis (NSF) [ 113 ] , and the FDA has issued warnings to limit the usage of gadolinium-based contrast agents in patients with renal failure. [ 114 ] In addition, the FDA expressed concerns on gadolinium retention in patients’ bodies, including the brain, for months to years after receiving these drugs. However, the FDA has not identified adverse health effects from retained gadolinium in the brain. As a safer alternative, SPION has received considerable attention due to their excellent T2 effect. The first clinically available brand is Feridex®/Endorem® (ferumoxide), a colloidal SPION associated with dextran, which was approved by the FDA in 1996 as a T2 contrast agent. Then, Resovist®/Cliavist® (Ferucarbotran), SPION coated with carboxydextran, is approved in the European market. However, these two T2 contrast agents are discontinued now due to a lack of clinical users. Combidex®/Sinerem®/Ferrotran® (Ferumoxtran-10) is an ultrasmall superparamagnetic iron-oxide (USPIO) coated with dextrans, which is still undergoing clinical trials. However, a pivotal study failed to demonstrate a consistent and statistically significant benefit for sensitivity that confirm non-inferiority concerning specificity; therefore, their clinical development was stopped. Feraheme®/Rienso® (ferumoxytol) is a USPIO, which is FDA-approved for intravenous iron-replacement therapy in anemic patients with chronic kidney disease. Interestingly, the paramagnetic properties of ferumoxytol also allow it to be used as an MRI contrast agent. The colloidal NPs, such as sulfur and stannous fluoride, are widely used to carry radioisotope to enhance SPECT imaging. 99m Tc is the most common radioisotope. Sulfur colloid (Technecoll®), albumin nanocolloid (Nanocoll®), rhenium sulfide nanocolloid (Nanocis®), and tin colloid (Hepatate®) have been approved for clinical practice. The intravenous nanoparticles that are currently undergoing clinical trials are summarized in Table 4 . The imaging model of these nanoprobes includes MRI/US, MRI, PET/MRI, PET, SPECT/CT, and optical imaging. None of them is an activatable probe.

In this review, recent achievements on activatable molecular imaging nanoprobes, especially for tumor-associated microenvironment detection, including optical imaging, MR imaging, and PA imaging, were summarized. Compared with small molecular imaging probes, nanoprobes are considered as a promising platform to design responsive mechanism upon different stimuli, including hypoxia, acidic condition, specific enzymes, redox status, etc., because their large specific surface area offers a big room to modify their functional moieties. Although a large number of achievements have been reported in this area, considerable challenges remain in the development of activatable nanoprobes for imaging in vivo . For example, redox responsive nanoprobes could be expanded beyond GSH/GSSG to other redox couples for nanoprobe design. [ 15 , 137 ] This will facilitate the diagnosis and/or therapy not only for the tumors but also for inflammation and other diseases with high specificity and sensitivity. Furthermore, hypoxia is increasingly recognized to play critical roles in the pathogenesis of other major causes of mortality, including myocardial ischemia, metabolic diseases, chronic heart and kidney diseases, and reproductive diseases such as preeclampsia and endometriosis. It is expected more nanoprobes will be developed for diseases other than tumors, which will allow us to understand the etiology and develop treatments. Also, for enzyme responsive nanoprobes, due to the vast number of enzymes that play important roles in many disease processes, there is a lot of room to design new enzyme-responsive nanoprobes, which will facilitate the diagnosis and treatment of a wide variety of diseases in the future. In addition, because the progression of diseases including tumors is associated with multiple physiological factors in the microenvironment, designing multi-modal nanoprobes are meaningful for fundamental studies and clinical applications. Although studies have demonstrated the potential application of stimulus-responsive imaging nanoprobes in the clinic, including the image-guided surgery, drug control release, evaluating therapeutic efficacy, and photothermal therapy, continued efforts are still needed to develop strategies for targeted delivery, incorporating anticancer agents with the nanoprobes, and constructing multi-stimuli responsive smart diagnostic and theranostic nanoplatforms. Finally, the toxicity evaluations and structure-activity relationships should be carried out to predict the potential adverse outcomes and facilitate the clinical translation of these nanoprobes.

Healthy cells and tissues need to maintain a crucial and delicate redox balance between oxidants, including reactive oxygen species (ROS) or reactive nitrogen species (RNS), and antioxidants, including glutathione (GSH), superoxide dismutase (SOD), catalase, cytochrome c, etc. [ 14 ] Among them, reduced GSH and oxidized GSH (GSSG) play a major role, and they act in concert with other redox-active compounds to regulate and maintain cellular redox status. The ratio of GSH to GSSG is a major indicator of the redox potential within cells, where an increased GSSG-to-GSH ratio is indicative of oxidative stress. In addition, there are also other redox couples, including NAD(P)H, cytochrome c, thioredoxin, ascorbate, etc. that help to maintain the redox balance. [ 15 , 16 ] However, in various diseases, the redox state is usually disrupted. Previous studies had revealed that compared to normal tissue, the tumor microenvironment exhibit higher GSH levels [ 17 – 19 ] with overproduced ROS. [ 20 ] The abnormal redox balance is also linked with many other pathological conditions, including inflammation [ 21 ] , diabetes [ 22 ] , atherosclerosis [ 23 ] , stroke [ 24 ] , etc. Disulfides transform to thiols in the presence of reducing agents, including GSH, and the resulting thiol groups can reversibly re-form disulfide bonds upon oxidation. The mild reaction conditions of thiol-disulfide exchange render it an appealing approach to construct disulfide-containing materials. Disulfides have been incorporated into material systems in the form of disulfide-containing crosslinkers. The cleavage of these crosslinkers leads to the release of the fluorophore to switch on the signal. Zhang et al. developed a multifunctional probe that was consisted of a gold/platinum star-shaped core (Au/Pt star) conjugated with a GSH-sensitive disulfide bond (S-S), a targeting ligand (rHSA-FA), a near-infrared fluorophore (IR780), and glucose oxidase (GOx). [ 25 ] As the disulfide linker was cleaved by intracellular GSH, the IR780 molecules could be released for photothermal therapy & photodynamic therapy (PTT&PDT) as well as bio-imaging. GOx could effectively catalyze intracellular glucose by consuming oxygen to generate H 2 O 2 and enhance the local acidity levels. The Pt layer exhibited peroxidase-like property to catalyze H 2 O 2 , producing toxic •OH to induce oxidative damage in tumors. The probes can simultaneously achieve GSH-sensitive fluorophore release, real-time imaging, and synergetic cancer therapy with PTT/PDT features. Liu et al. conjugated a hydrophobic fluorophore to form a core and linked two hydrophilic polymers that can quench fluorescence by redox-responsive disulfide bonds serving as the corona. [ 26 ] The cleavage of disulfide bonds will expose fluorophores to the surrounding proteins at the tumor tissue, leading to fluorescence “turn on” and the release of loaded Docetaxel simultaneously. This redox-triggered light-up nano-micelles offer the opportunity to monitor the pharmacokinetic process of the nano-micellar system and estimate the drug content in vivo . The transformed disulfides can also be used to design ROS responsive probes by crosslinking. A GSH-modified, lanthanide-based, fluorescent downconversion nanoparticles (DCNPs), which was in the second near-infrared window (NIR-II), can accumulate in the inflamed area and enhance fluorescence signal intensity due to the GSH oxidation, increased size, and lower excretion rate. [ 27 ] The degradation or self-aggregation of probes can alter the effect on the relaxation rate of water protons to turn the MRI signal “off” and “on” via the disulfide bond cleavage in an abundant GSH microenvironment. Iron oxide nanoparticles (NPs) are excellent T2 contrast agent, and several superparamagnetic iron oxide nanoparticles (SPION) has been approved by the Food and Drug Administration (FDA) for clinical use. [ 28 ] The T2 effect of iron oxide NPs is largely dependent on size. Therefore, based on a linker that contains peptides and disulfide bonds, the Gao group developed GSH-triggered NP aggregating systems to increase tumor contrast. [ 29 ] In this probe, poly(ethylene glycol) (PEG) ligand was first to bind to Fe 3 O 4 NPs surface by a diphosphate group ( Figure 2 ). The maleimide group on the other side of the PEG ligand was used to covalently attach an Arg-Gly-Asp (RGD) peptide and a self-peptide linked through a disulfide bond. After cleavage by the GSH in the tumor environment, the thiol group remaining on RGD moiety crosslinks the particles through interparticle reaction with the remaining maleimide residues from adjacent particles. In situ crosslinking of the responsive probes leads to prolonged retention of particles within tumors and increases the tumor contrast by a factor of more than 3 in vivo in contrast to non-crosslinkable Fe 3 O 4 nanoprobes. Besides, the single-photon emission computerized tomography (SPECT) imaging capacity is also largely enhanced owing to improved retentions of the crosslinked nanoprobes. The cleavage of disulfides under the reducing environment (e.g., GSH) can also be used to control 19 F NMR signal intensity. A “smart” probe that allows GSH-controlled assembly and legumain (Lgmn)-controlled disassembly could turn the 19 F NMR signal “off” and later “on” for the sequential detection of GSH and Lgmn. By this design, the probe was successfully applied for the detection of Lgmn activities in zebrafish at low doses (1.5 g/kg) in vivo under a high magnetic field strength at 14.1 T. [ 30 ] In another example, a self-assembled 1-ICG NP has been prepared with negligible fluorescence, an obvious “off” state. The GSH-induced cleavage would trigger the dissociation of them into ultra-small nanoparticles, leading to the activation of 19 F signals. ICG molecules can absorb NIR light to increase the temperature in the microenvironment, which can accelerate the movement of molecules, leading to the complete disassembly of these loose nanoparticles into small and water-soluble molecules, bringing on a distinct second round of amplification of 19 F NMR signals. Signal-to-noise ratio (SNR) of 19 F MRI signals observed at the tumor site could be about 3.9 for the first step and reach 8.3 after irradiation with an 808 nm laser at 1.5 W/cm 2 for 6 min. [ 31 ] The cleavage of disulfide can even be used to build a redox responsive fluorescence/ 19 F-MRS/ 1 H-MRI triple-functional probe for tri-modality imaging of reducing the biological environment. [ 32 ] The probe demonstrated tri-modal detection of reductive biothiol species, with a 70-fold turn-on ratio in fluorescence, a dramatic ~30-fold enhancement in 19 F-MRS, and a significant 68% reduction in the longitudinal r1 relaxivity (23.6 ± 0.2 vs. 7.6 ± 0.3 mM −1 s −1 at 0.5 T). It is known that Mn-O bonds can fastly biodegrade in the mild reducing microenvironment. [ 33 ] Khatik et al. combined Fe 3 O 4 and mesoporous silica (SiO 2 ) that were loaded with Mn 2+ ions to develop a GSH reactive T1/T2 dual MRI probe. [ 34 ] Compared with the control group, the relaxivity value (r1= 5.12 mM −1 s −1 , r2= 265.32 mM −1 s −1 ) were more than two folds at 3.0 T. Also, based on the Mn-O redox response, Wei et al. constructed a honeycomb MnO 2 nanosponge-sustained autocatalytic DNAzyme (ACD) machine for the robust MRI of the tumor and the concomitant in vivo miRNA imaging. [ 35 ] The reduction by GSH can also affect the metal optical properties of element nanomaterials. For example, MnMoOX nanorods typically do not have optical absorbance in the NIR window. However, they exhibit strong NIR absorption owing to the reduction of Mo VI in the initial MnMoOx to Mo V by GSH, which triggers the transformation of nanorods to ultrasmall nanodots. Moreover, MnMoOX nanorods with GSH-responsive NIR absorbance could also be employed to achieve tumor-specific PTT. [ 36 ] Oxidation-responsive materials mainly target ROS, such as hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals. At mild conditions, H 2 O 2 leads to a rearrangement of the oxonium group and the decomposition of the benzopyrylium group while releasing fluorophore. [ 37 ] Also, sulfonic esters [ 38 ] , boronate ester [ 39 ] , benzil [ 40 ] , and α-ketoamide moiety [ 41 ] can be used to design ROS oxidation-responsive materials. Some fluorophores show redox-dependent absorbance at a certain wavelength, which can be used to construct redox responsive nanoprobes. Based on fluorophores, the Tang group developed a sensitive redox-responsive PA nanoprobe to achieve GSH/H 2 O 2 simultaneous detection in vivo . [ 42 ] Similarly, Ai et al. construct a unique probe to monitor the multispectral optoacoustic tomography (MSOT) signal variations with ROS and RNS-sensitive NIR cyanine fluorophores and upconversion nanoparticles. [ 43 ] The probe can track multiple radicals under two independent wavelength channels, and more significantly, precisely validate their complex dynamics and correlations with the redox-mediated pathophysiological procession in vivo . They further integrated MSOT with upconversion nanoparticle (UCNP)-mediated luminescence imaging. The spectrum of multiplexing upconversion luminescence (UCL) response and the absorption shift of the H 2 O 2 -sensitive cyanine dye was deliberately designed to overlap. ROS not only induced a convertible absorption blue-shift of the probe through the chemo-specific reaction, which can be captured by MSOT, but also induced multispectral luminescence changes, which showed the opposite trend of the UCL signal variation comparing with the one observed in MSOT. Thus, the probe provides a spectrally opposite dual-modal ratiometric output that allows a more sensitive and accurate evaluation of local disease biology and drug treatment responses based on the self-calibrating feature of the reverse-ratiometric PA and UCL imaging technology. [ 44 ] The intracellular and extracellular pH of normal tissues is kept constant in a range of 7.35–7.45. [ 45 ] However, the acid-base balance can be disrupted to complicate the course of widely diverse diseases as well as trauma in many parts of the body. For example, as cancer grows, it rapidly outgrows its blood supply, resulting in a significantly lower oxygen concentration compared with that in healthy tissues, called hypoxia. When tumors are under hypoxia, the upregulation of glycolysis and reduction in oxidative phosphorylation leads to increased lactic acid production, producing an acidic environment in most solid tumors. [ 46 ] The pH disruption is also found in the inflammatory processes. [ 47 , 48 ] Taking advantage of the pH differences under disease conditions, several strategies could be used to design pH-responsive probes. The pH-sensitivity can be attributed to either the protonation of ionizable groups or acid-cleavage of chemical bonds and result in physical or chemical changes (such as swelling, shrinking, dissociation, and degradation). [ 49 , 50 ] The protonation of ionizable groups, which is usually reversible, can directly result in optical property changes. Utilizing the reversible protonated process of polyaniline, a broad and pH-responsive ratiometric sensor for PA imaging was developed by loading polyaniline on the surface of Au triangular NPs. The NP was shown to be ultrasensitive to pH, and response time was as fast as 0.6 s and durability as long as 24 h, which was repeatable for longitudinal monitoring. [ 51 ] Similarly, a self-assembled charge-transfer nanocomplex, which can accurately respond to pH change in the physiological range, was designed to be a new class of pH-sensitive photoacoustic contrast agent that operates in the NIR-II window. [ 52 ] Oxazine-containing polyheterocycles with aggregation-induced emission (AIE) characteristics display remarkable fluorescence changes to protonation and deprotonation. Based on these unique photophysical properties, in vivo mapping of intestinal pH of freshwater Cladocera Moina macrocopa was achieved, which showed an increased pH gradient approximately from 4.2 to 7.8 along the foregut, midgut, and hindgut. [ 53 ] The protonation can also result in physical changes and indirectly change optical or acoustic properties. Ding et al. developed a luminescence resonance energy transfer (LRET) system using NIR Ag 2 S nanodots (NDs) as energy acceptors and UCNPs as energy donors [ 54 ] . The protonation and deprotonation of the amino and carboxyl groups on the surface of Ag 2 S (NDs), which lead to the combination and dissociation from UCNPs, can efficiently affect the LRET process between UCNPs and Ag 2 S NDs. Thus, a ratiometric detection and imaging of pH in tumor cells and zebrafish in NIR windows have been achieved. An acid-induced swelling increases the size of a polymersome-based and perfluorocarbon-encapsulated nanoprobe (from 178 to 437 nm), which can be used for ultrasonic imaging with a lower vaporization threshold for the perfluorocarbon, and eventual release of doxorubicin for deep tissue chemotherapy. [ 55 ] Beyond acid-induced size change, the Gao group demonstrated the working mechanism of proton responsive transistor-like nanoparticles. In addition, based on the transition from positron-emitting neutral copolymer micelles to polycationic polymers, the polymers labeled with the positron-emitting isotope 64 Cu can achieve the non-invasive detection of small occult diseases (10–20 mm 3 or 3–4 mm) in the brain, head and neck, and breast by PET imaging. [ 56 ] Acid-cleavage is widely used to construct pH-activatable probes for MRI by releasing metal ions. As Mn 2+ with five unpaired 3d electrons is known to be a T1-shortening agent, the decomposition of MnO 2 nanoparticles under a mildly acidic environment is widely used to develop pH-activatable probe for MRI. Recently, Yang et al. design a pH-activatable hollow manganese dioxide nano-platform that combines imaging and drug release. [ 57 ] This probe could dissemble under reduced pH within the tumor microenvironment (TME) to release loaded therapeutic molecules (chlorine e6 (Ce6) and doxorubicin (DOX)), and in the meantime induce decomposition of tumor endogenous H 2 O 2 to relieve tumor hypoxia. As a result, a remarkable in vivo synergistic therapeutic effect is achieved through the combined chemo-photodynamic therapy, which triggers a series of antitumor immune responses at the same time. Similarly, MnO 2 as an intelligent “gatekeeper” shield can achieve the controlled release of carbide-glucose oxidase from Fe 5 C 2 nanoparticle in the acidic tumor microenvironment. Mn 2+ could serve as a magnetic resonance imaging (MRI) contrast agent for the real-time monitoring of the treatment process. The generated O 2 and released glucose oxidase in nanocatalysts could effectively exhaust glucose in tumor cells, generating plenty of H 2 O 2 simultaneously, which may accelerate the subsequent Fenton reaction catalyzed by the Fe 5 C 2 magnetic core in mildly acidic tumor microenvironments. [ 58 ] Comparing with Mn 2+ , Fe 3+ is considered much safer. However, designing a Fe 3+ based activatable agent is problematic, given that the Fe-O-Fe structure of FeIII oxide is hardly breakable at the mildly acidic pH of the tumor microenvironment. Zhang et al. developed a FeIII based activatable probe suitable for releasing FeIII in the tumor microenvironment to enhance MRI and simultaneously improve the treatment of tumors. [ 59 ] The UCL nanoparticles were used as a core, and the coordinatively unsaturated FeIII-containing Fe 3+ /gallic acid complex serves as a shell to construct the probe ( Figure 3 ). At the mildly acidic pH of the tumor microenvironment, FeIII in the nanoprobe can be released due to the unsaturated coordination structure and serves as a photothermal agent. This process can be quantitatively monitored in vivo by multiple UCL nanoparticles. Another iron-based pH-sensitive probe is reported by Li et al. [ 60 ] Iron oxide nanoparticle assemblies (IONAs) are crosslinked by the strong small-molecular aldehyde derivative ligands at neutral pH. The cleavage of these ligands in acidic environments lead to the rapidly disassembled IONAs with a significantly enhanced T1 MR contrast. Yi et al. reported self-assembled NaGdF 4 and CaCO 3 nanoconjugates. [ 61 ] In a mildly acidic tumor microenvironment, the embedded CaCO 3 nanoparticles generate CO 2 bubbles and subsequently disconnect with the nanoconjugate, releasing the spatial confined Gd 3+ ions, which shows more than 60-fold contrast enhancement in tumor visualization relative to the commercially used contrast agent Magnevist. The hydrophobic-to-hydrophilic conversion of the pH-responsive polymer is another strategy to develop an activatable probe. At physiological pH, both gadolinium metallofullerene (GMF) and drug molecules are encapsulated into the hydrophobic core of nanoparticles formed by the pH-responsive polymer and shielded from the aqueous environment, resulting in relatively low longitudinal relativity and slow drug release. However, in the acidic tumor microenvironment, the hydrophobic-to-hydrophilic conversion of the pH-responsive polymer leads to amplified MR signal and rapid drug release simultaneously. This prepared activatable MRI contrast agent can not only detect tumors but also monitor drug release. [ 62 ] Wang et al. developed size-switchable nanocapsules that respond to NIR light and the acidic TME. The nanocapsules are stable in the blood with a large initial size. In an acidic microenvironment, the capsules shrink and decompose into smaller ones induced by irradiation of the NIR laser, and DOX will be released. The capsules can also react with endogenous H 2 O 2 in tumors and overproduce ROS to overcome the tumor hypoxia-related drug resistance. These active nanocapsules provide dual-modal MRI and fluorescence imaging information with enhanced tumor accumulation. [ 63 ] In summary, there are several strategies to design pH-responsive probes. Currently, most pH-responsive probes are focusing on the tumor due to its acidic microenvironment. It is foreseeable that the same principle could be extrapolated to diagnose and treat other diseases that involve pH disruptions. Enzymes play various critical roles in different biological processes. Disease-associated enzyme dysregulations have recently become an emerging target for nanomedicine. Here, we describe several typical enzymatic triggers that could be integrated into the nanoprobe design. MMPs are closely associated with tumor invasion and metastasis. [ 64 ] The upregulated expression of MMPs within the tumor microenvironment can serve as site-specific biological cues for activating bio sensitive materials. This strategy has been applied for the quantitative detection of MMP-2 expression in vivo. [ 65 ] A NIR dye and a quencher were covalently linked through a peptide substrate of MMP-2 ( Figure 4 ). Upon cleavage with activated MMP-2, this probe presented a dramatic MMP-2 concentration-dependent absorption at around 680 nm, and an MMP-2 concentration-independent absorption at around 730 nm. Using the ratio between the PA signal at 680 nm and 730 nm, this probe can detect MMP-2 expression in breast cancer in vivo quantitatively. [ 66 ] Enzyme-activated degradation of the gelatin scaffold was also used to release drug and monitor this process simultaneously. The NIR fluorescence was first shielded by satellite CuS NPs and then recovered depending on the amount of chemo-drug doxorubicin released in MMP overexpressed tumor environment. [ 66 ] Another article describes a nanoplatform based on MMP-responsive gold nanoparticles (AuNPs). The complementary DNA strands were first attached to the AuNPs’ surface. The DOX and PEG were then coated on the particles via a thermal-labile linker and an MMP-cleavable peptide, respectively. Under the MMP-abundant microenvironment, the PEG layer was rapidly cleaved to expose the complementary DNA strands result in aggregation. This process improved the accumulation of NPs in tumors and induced the redshift of their absorption, which enhanced PA imaging signal and the DOX could be released due to the localized elevation of temperature, serving as photothermal chemotherapy (PTT). [ 67 ] Furin is a member of the proprotein convertase family, which has a crucial role in tumor progression, metastasis, and angiogenesis. [ 68 ] In one study, furin cleavage of a precursor peptide induces self-assembly to form intracellular NPs to enhance tumor microPET imaging, achieving a tumor/liver ratio of 9.1-fold compared with the control. [ 69 ] In another study, furin mediated self-assembled NPs not only has a 6.5-fold olsalazine CEST signal but also increase the antitumor therapeutic effect by 5.2-fold compared to olsalazine without furin responsiveness, which showed an excellent “theranostic correlation” (R 2 = 0.97) between the imaging signal and therapeutic response (normalized tumor size). [ 70 ] Phospholipases also function in the extracellular microenvironment when selected as therapeutic targets. [ 71 ] An in situ self-assembled nanoparticle system response to alkaline phosphatase (ALP) was designed for simultaneous enhancements in NIR fluorescence (>70-fold at 710 nm) and r1 relaxivity (~2.3-fold), which achieved real-time, high-sensitivity, high-spatial-resolution imaging and localization of the ALP activity in live tumor cells and mice. [ 72 ] Another NIR probe can efficiently be converted to IR775-Phe-Phe-Tyr-OH from IR775-Phe-Phe-Tyr(H 2 PO 3 )-OH under the catalysis of ALP and enhanced 2.3 folds of the 795 nm PA signal in vivo at four hours after NP injection. [ 73 ] Caspase-3 and −7 are hallmarks of apoptosis, and they play essential roles in the regulation of cell number and maintenance of tissue homeostasis. [ 74 , 75 ] The cleavage of peptides by caspase-3/7 promotes the dynamic response of the nanoprobe to the caspase-3/7 activity, which allowed 19 F MRI of caspase-3/7 activity in living mice for the first time using a PFC-encapsulated 19 F MRI nanoprobe with off/on-switching ability. [ 76 ] Intracellular hyaluronidase can be used as a tumor target. A core-shell nanostructure was designed with UCNPs as a core and fluorophore-doped degradable microporous silica as the shell for protein delivery. On top of that, a hyaluronic acid (HA) shell was synthesized on the surface. The HA shell of this core-shell structure could be hydrolyzed by hyaluronidase in tumor cells. Without the protection of the HA shell, the silica shell then began degradation and caused the release of the proteins. The spectral overlaps between UCNPs and silica shell can be used to track protein delivery in vivo via NIR luminescence. The probe successfully helped us to understand where, when, and how therapeutic proteins take effect in vivo. [ 77 ] The condensation reaction between 6-hydroxy-2-cyanobenzothiazole (CBT) and cysteine has been shown for various applications such as site-specific protein labeling and in vivo cancer imaging. Chen et al. further expands the substrate scope of this reaction by varying the substituents on aromatic nitriles and amino thiols, thus develop a general system for imaging hydrolytic enzymes. They identified the minimum structural requirement to form nanoparticles through the macrocyclization and assembly process. Also, they designed fluorescent probes consist of 2-pyrimidinecarbonitrile and cysteine joined by a benzyl linker to image caspase-3/7 and b-galactosidase activity in live cells. [ 78 ] Hypoxia, an absence of adequate oxygen, is associated with various diseases, including cardiomyopathy, ischemia, rheumatoid arthritis, acute and chronic vascular disease, pulmonary disease, and cancer. [ 79 – 81 ] Specifically, hypoxia is common in most solid tumors resulting from the imbalance between the high oxygen consumption and the poor oxygen supply, which because of the rapid proliferation of tumor cells and enhanced angiogenesis, respectively. Compared with normal tissue molecular oxygen (O 2 ) level (2% ~ 9%), “hypoxia” tumor microenvironment only has a O 2 levels of 0.02% ~ 2% (<2.5 mmHg pO 2 ). [ 79 , 82 ] Tumor hypoxia is generally considered negative for prognosis because of its central role in tumor progression and resistance to therapy. There are two main strategies to prepare hypoxia activable nanoprobes. One is based on hypoxia or oxygen responsive groups, including nitroaromatic derivatives, azobenzene, polypyridyl, pyrenyl units, and transition metal complexes. Another one is the indirect imaging based on the biomarkers induced by hypoxia. Hypoxia-inducible factor-1α (HIF-1α) is a crucial transcription factor that is induced by hypoxia, which can be used as the target for indirect hypoxia imaging. [ 83 , 84 ] Here, we only focus our discussion on hypoxia-responsive groups in nanoprobe design. Nitroaromatic derivatives that can be converted to hydrophilic 2‑aminoimidazole under hypoxic conditions with relatively high sensitivity are among the most widely exploited functional motifs. By incorporating a nitrobenzene-substituted BODIPY, which can be activated by reductive enzymes, especially nitroreductase (NTR), the probe obtained 55-fold fluorescence enhancement with the limit of detection as low as 7.08 ng/mL. In vivo imaging of hypoxia in a murine model of myocardial ischemia was achieved by the nanoprobe with high sensitivity and excellent biocompatibility. [ 85 ] Similarly, azobenzene, another well-established hypoxia sensitive motif previously used as an imaging probe, has been incorporated in a ratiometric surface-enhanced Raman scattering (SERS) nanoprobe for imaging hypoxic living cells or tissues. [ 86 ] The azoalkynes, which have alkyne Raman bands at 2207 cm −1 , were assembled on a single-walled carbon nanotube (SWCNT) surface-functionalized with Ag/Au alloy nanoparticles for the first time. In the hypoxia tumor microenvironment, azoalkynes would be reduced and removed from the surface. While SWCNT shows a strong Raman signal at 2578 cm −1 , which can be used as the internal standard. Thus, the hypoxia level can be determined by the ratio of two peak intensities (I 2578 /I 2207 ). [ 86 ] Li et al. developed NIR excited nanosensors, in which the donor and acceptor pairs within a biological metal-organic framework (bio-MOF) matrix is precisely controlled to enable upconversion fluorescence resonance energy transfer (FRET). It is suggested for detecting the O 2 concentration inside tumors with reduced signal disturbance and health detriment. Under NIR excitation, as-fabricated core/satellite nanosensors exhibit improved FRET efficiency and reversible hypoxic response with high sensitivity, which are effective both in vitro and in vivo (zebrafish) for cycling normoxia-hypoxia imaging. The FRET core/satellite nanostructures, in which tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride ([Ru(dpp) 3 ] 2+ Cl 2 ) as the quenchable indicator for O 2 with a linear Stern-Volmer relationship, UCNPs as antennas convert NIR light into the visible light absorbed by the [Ru(dpp) 3 ] 2+ Cl 2 , have been used for in vivo non-small-cell lung lesion tracking in a genetically engineered murine model. It allowed optical hypoxia monitoring without apparent long-term biotoxicity. [ 87 ] Zheng et al. reported a new ratiometric hypoxia luminescent imaging (RHyLI) nanoprobe based on semiconducting polymers and phosphor that can exhibit dual NIR emissions (685 and 795 nm) under the red light (up to 650 nm) excited. The phosphorescence emission at 685 nm is inert to oxygen level. While the emission at 795 nm would increase to 13.5-fold when the oxygen level dropped to 0 from 21%. Thus, the ratio of the emission intensity between 795 and 685 nm (R795/685) increased nonlinearly as the oxygen level decreased, with the sharpest response in the low oxygen range. These results showed the nanoprobe could efficiently penetrate the hypoxic regions of the tumor tissues after systemic administration and allow for quantitative imaging of tumor hypoxia dynamics during radiotherapy over one week. [ 88 ] Hydrogen sulfide (H 2 S) is a highly reactive endogenous signaling molecule, playing a critical role in diverse physiological functions. Aberrant concentration and tissue distribution of H 2 S are associated with many diseases (e.g., liver inflammation, hypertension, diabetes, and cancers). Therefore, precise spatiotemporal detection of H 2 S in living subjects is essential to the study of the biological functions and accurate diagnosis of H 2 S related diseases. In the presence of H 2 S, the boron-dipyrromethene dye could generate the NIR-II emission. Combined with another dye, which is inert to H 2 S, the probe can precisely detect H 2 S by ratiometric imaging. [ 89 ] The boron-dipyrromethene dye can also be used to construct H 2 S-activatable NIR photothermal agents for imaging-guided and photo controlled drug release. [ 90 ] H 2 S may serve as a strong reductant capable of fast donating electrons to certain organic π-electron structures and induced color changes during this electron-transfer process. For example, the strong absorption (between 500 and 850 nm) of dicationic 1,1,4,4tetraarylbutadiene would dramatically decrease due to the reduction. By doping this dye into semiconducting polymer nanoparticles, sensitive, non-invasive, and real-time detection of hepatic H2S levels in mice was achieved. [ 91 ] The researchers further optimized the structure of this dye to improve reaction kinetics toward H 2 S with longer absorption wavelength ( Figure 5 ). The final probe displays a fast reaction rate (1563 ± 141 M −1 s −1 ) and a large afterglow turn-on ratio (~122-fold) toward H 2 S, enabling high-sensitivity and specificity measurement of H 2 S concentration in blood from healthy persons, hepatic or colorectal cancer patients. [ 92 ] Adenosine 5’-triphosphate (ATP), referred to as the “molecular unit of currency” of intracellular energy transfer, was found to accumulate at high levels in inflammatory and tumor sites. [ 93 – 96 ] . Thus, ATP can serve as an attractive trigger for activatable imaging. Li group designed a NIR light-activated DNA nanodevice for sensing ATP in living cells. [ 97 ] In this nanodevice, oleic acid-capped NaGdF 4 :70%Yb,1%Tm@NaGdF 4 UCNPs (40 nm) were synthesized with a thermal decomposition approach to achieve high efficiency of NIR-to-UV conversion. The ATP aptamer strand modified with a fluorophore Cy3 and the quencher-bearing a photocleavable (PC) group was initially formed a complementary DNA. FRET occurred between Cy3 (or Cy5 for in vivo imaging) and quencher, resulting in the “off” state of fluorescence. After UCNPs convert NIR light to UV light, the photolysis of the PC group generates short DNA fragments. In this condition, the aptamer will restore its capability to switch its structure to bind ATP, leading to the dissociation of the DNA probe and the recovery of the Cy3 fluorescent signal. By introducing the pH (low) insertion peptide (pHLIP) into this DNA nanodevices, the spatially controlled imaging of ATP in the acid extracellular milieu of tumors with ultrahigh signal-to-background ratio was achieved. [ 98 ] Combining with gold nanoparticle (AuNP) with polyethylene glycol (PEG), this DNA nanodevice can further be used for bioimaging and photodynamic therapy. [ 99 ] . Chen group engineer the self-assembled quantum dots (QDs)-phenolic nanoclusters for ATP-responsive imaging and drug release. The hydrophobic core-shell structured CdSe@ZnS QDs (~9.8 nm) was assembled into nanoclusters linked by tannic acid (TA), with diameters of 55 nm. ATP could compete with TA and partly displace TA to bind on the QDs surface, leading to the disassembly of the nanoclusters and the enhancement of the fluorescence intensity. [ 100 ] We have summarized many smart probes that respond to a certain stimulus or condition. While in many cases, there is more than one condition can be used as the trigger. For example, redox, hypoxia, low pH, and some overexpressed enzymes are common in most solid tumor microenvironment, which can all be used to stimulate the smart probes. [ 46 , 79 , 101 ] A deliberately designed probe can achieve dramatically signal increase based on multi-stimulation signals. For example, the release of metal ions in the acidic environment can be combined with other strategies. A core-shell-structured iron carbide (Fe 5 C 2 @Fe 3 O 4 ) nanoparticles (NPs) can respond to acidity and overproduction of H 2 O 2 in the tumor environment, which effectively inhibits the proliferation of tumor cells through the catalysis of the Fenton reaction. [ 102 ] In acidic environments, the released ferrous ions decrease the T2 signal and enhance the T1 signal in MRI, and this T2/T1 switching process provides the visualization of ferrous ions release and ROS generation for the monitoring of tumor treatment process. For another example, a nanoparticle sensor is composed of acetylcholine catalyzing enzymes and pH-sensitive gadolinium contrast agents modified onto the surface of the particles, which can be used to specifically detect acetylcholine in the living brain. The enzymatic hydrolysis of acetylcholine leads to a localized decrease in pH, which is detected by the pH-sensitive gadolinium chelate. The concomitant change in 1/T1 in vitro saw a 20% increase from 0 to 10 μM acetylcholine concentration. [ 103 ] In addition, the thermo- and pH-responsive copolymers of poly(N-isopropylacrylamide) and poly(acrylic acid) were used to design core/shell hydrogel NPs. In the range from 10 to 80°C, the photoluminescence intensities of the NPs show linear response depend on temperature. In the range from pH6.5 to pH7.6, the blue emission of the NP’s shell is linear depend on pH with a resolution of 0.1 unit, while the red emission is inert to pH changes. [ 104 ] The weak electron acceptor (benzo[c][ 1 , 2 , 5 ]thiadiazole-5,6-diamine) can be oxidized by NO in acidic conditions to form a stronger acceptor (5H-[ 1 , 2 , 3 ]triazolo[4,5-f]-2,1,3-benzothiadiazole), which significantly increased the molecule absorption in the NIR region. Based on this transformation, a dual-stimuli-responsive theranostic nanoprobe was developed for simultaneously activatable cancer imaging and photothermal therapy. Under NO and acidity dual stimuli, the PA signal is 9.8 times and 132 times higher than that of NO and acidity alone, respectively. [ 105 ] Moreover, the poly(ethylene glycol)-conjugated iridium (iii) complex, a NIR probe, was developed to respond to both tumor acidity and hypoxia, which is akin to the cascade amplification of signals in electronics. In the presence of the first targeted physiological stimulus, the probe gets converted to a secondary form (the reporter). In the presence of a second targeted stimulus coupled to the first stimulus, the signal of the reporter is amplified and can be observed in a different readout channel. Such signal amplification involves the propagation of signals from the precursor to the reporter, which will lead to improved detection sensitivity. [ 106 ] Based on a multispectral upconverting nanophosphor (NaYF 4 : Yb, Er, Tm) as the luminescence resonance energy transfer donor, a GSH-sensitive dye and an H 2 O 2 -sensitive dye as the acceptors, a ratiometric nanoprobe was developed for real-time and synchronous monitoring of the variation of GSH and H 2 O 2 in vitro and in vivo . [ 107 ] The multi-stimulation design not only can magnify the signals but also can be used to research the correlation between these abnormal characteristics. Gao group design a probe based on a FRET system comprised of Fe 3 O 4 nanoparticles, pH-sensitive ratiometric fluorescent dye, NIR dye, MMP-9 specific peptide substrate linker, and folic acid as a tumor active targeting moiety ( Figure 6 ). The fluorescence of a ratiometric pH dye, N-carboxyhexyl derivative of 3-amino-1,2,4-triazole fused with 1,8-naphthalimide (ANNA), is quenched while attached to the surface of a Fe 3 O 4 nanoparticle, representing the “off” state. Upon cleavage of the peptide linker by MMP-9, the fluorescence of ANNA is activated, representing an “on” state, which has been previously demonstrated for in vivo pH mapping of tumor xenografts after intratumoral injection. [ 108 ] By labeling this probe with the NIR fluorescent dye Cy5.5, which is always in an “on” state, the constant Cy5.5 emission and MMP-dependent fluorescence from ANNA can be combined to map MMP-9 activity across the entire tumor quantitatively. The probe can serve as a non-invasive tool to map multiple tumor-associated signatures simultaneously to determine tumor heterogeneity, correlate abnormal characteristics with metastatic potential, and potentially help predict tumor progression and design optimal treatment strategies. [ 109 ]

Molecular imaging, which focuses on imaging molecules in living organisms in a non-invasive manner, attracted substantial attention in the past two decades. [ 1 , 2 ] Beyond structural images, molecular imaging can provide functional and molecular information to understand the mechanisms involved in physiology and disease by non-invasive imaging at the cellular or subcellular level in vivo . [ 3 ] To achieve these functions, the techniques require two essential elements: (1) the probes whose properties or concentrations can be altered by the specific biological process of interest and (2) a means by which to monitor these probes. Though most clinical molecular imaging agents are small molecular probes, nanoprobe is a promising candidate to develop a new generation of molecular imaging probes. Compared to the rapid renal clearance of small molecule probes, nanoprobes with proper surface modification usually have a longer blood circulation time. [ 4 , 5 ] Especially, nanoparticles are preferably accumulated in tumor sites due to their small size and leaky vasculature. [ 6 , 7 ] Nanoparticles also provide a platform to load functional groups or drugs with a high surface-volume ratio to achieve active targeting and/or drug delivery. In addition, nanoprobes can be designed to have many unique optical and magnetic properties for multi-modal imaging. The recent advancements in imaging nanoprobes and devices open up additional possibilities in novel imaging techniques such as fluorescence imaging, photoacoustic (PA) imaging, and near-infrared (NIR) II imaging. Optical imaging is emerging as a promising modality for real-time molecular imaging in vivo because of its high spatial resolution and high sensitivity. Although attenuation and scattering of light by tissue limit the clinical application, it has been used in small animal models to probe mechanisms involved in many disease processes. Photoacoustic (PA) imaging is an imaging technique that is based on the detection of ultrasonic waves generated by photothermal expansion of light-absorbing tissues or contrast probes under pulsed laser irradiation. [ 8 ] Combining the advantages of both ultrasound and optical imaging, PA imaging has the benefits of high spatial resolution, high contrast, and deep tissue penetration (several centimeters). [ 8 ] As a fantastic imaging modality, multispectral optoacoustic tomography (MSOT), which can supply reliable anatomy information to the disease theranostics in preclinical trials, has recently attracted considerable attention in biomedical sciences. [ 9 ] Computer X-ray tomography (CT), Single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasound scanners (US) are already powerful tools in clinical settings and are widely used to monitor the molecular probes. Recently, these techniques also have made substantial advancements. Heteronuclear MRI, especially fluorine MRI, is one of the most promising MR techniques owing to the desirable MR properties of the 19 F nucleus, lack of endogenous 19 F, and good bio-inertness of C-F bonds. The 19 F nucleus has a nuclear spin of 1/2, and a gyromagnetic ratio that is 94% of 1 H, accounting for an 83% overall signal sensitivity compared to 1 H. [ 10 ] Chemical exchange saturation transfer (CEST) MRI is another imaging technique that can indirectly detect non-labeled, native molecules by manipulating the water proton signal through selective saturation of exchangeable protons. [ 11 ] CEST MRI has now undergone clinical translation for the imaging of dilute biomarkers associated with the microenvironment as well as administered diamagnetic chemical exchange saturation transfer (diaCEST) contrast agents. [ 12 , 13 ] For imaging, the conventional probes typically emit signals continuously under all conditions and develop contrast signals through accumulation at the targeted region, named “always-on” probes. In contrast, the new generation of activatable probes are those in the “off” state at the beginning and switch to the “on” state in the “right” microenvironment of the target tissues or organs. Thereby, activatable probes have a higher signal-to-noise ratio, which can improve sensitivity and specificity for detecting biomarkers or conditions with an imaging modality. In this review, we will provide a summary of novel strategies on how the activatable probes respond to redox status ( Table 1 ), pH ( Table 2 ), enzymes ( Table 3 ), and other stimuli developed in recent years. The imaging modality includes fluorescence, PA, PET, and MRI. The responsive mechanisms of the activatable nanoprobes were illustrated in Figure 1 , which includes redox, pH, hypoxia, enzymes, etc. that will be discussed in detail below. In addition, we will discuss the challenges facing this field and the path forward.