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Tumor microenvironment-responsive contrast agents for specific cancer imaging: a narrative review

Wang, Xianwena,∗; Zhong, Xiaoyanb; Lei, Hualia; Yang, Nailina; Gao, Xiangc; Cheng, Lianga,∗

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doi: 10.1097/JBR.0000000000000075
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Cancer is currently one of the main diseases that seriously affects human health and threatens human life.[1,2] Malignant tumors are secretive, have a long incubation period, and are difficult to detect at an early stage; thus, most patients are diagnosed at a later stage when they have lost the best chance of a radical cure, indicating that the diagnosis of cancer is very important and meaningful.[3,4] Effective early diagnosis can detect cancer in its germination, which makes treatment more facile and effective, and greatly reduces economic burden of cancer patients.[5] Imaging plays an important role in the diagnosis and efficacy evaluation of malignant tumors.[6] Imaging techniques, such as magnetic resonance imaging (MRI) and photoacoustic (PA) imaging, often have high sensitivity and spatial resolution, good soft-tissue resolution, and can noninvasively monitor the occurrence and development of tumors.[7] Although contrast agents significantly improve imaging effects, the low signal-to-noise ratio and high background interference of contrast agents weakens the performance of imaging, making it difficult to obtain satisfactory efficiency for the diagnosis of early tumors.[8]

The microenvironment of tumor tissues is significantly different from that of normal tissues.[9] During tumorigenesis and development, tumor tissues are in a weakly acidic environment due to the growth and vigorous metabolism of tumor tissues, as well as their consumption of large amounts of glucose and oxygen, resulting in excessive lactic acid and H2O2.[10] In addition, as tumor cells produce excessive glutathione (GSH) and other reducing substances during the growth process, they exhibit reducing characteristics compared with normal tissues.[11,12] Thus, the tumor microenvironment (TME) presents key characteristics of weak acidity and high expression of H2O2 and GSH.[13] With rapid development of nanomedicine and the deepening of TME-related research, TME-responsive contrast agents have shown broad application prospects for tumor-specific imaging.[14] Contrast agents activated by TME play an important role in precision nanomedicine, and they have advantages of high signal-to-noise ratio and low background interference.[15] After TME-responsive contrast agents are efficiently transported to tumor tissues in the body, their structure or absorption is altered by TME stimulation, which significantly enhances the imaging effect, thereby realizing specific tumor imaging.[16,17]

Although previous reviews have summarized advances in imaging applications of nanomaterials or contrast agents,[6,18] few have described the use of TME-responsive contrast agents for specific tumor imaging based on TME characteristics.[8,17] Herein, this review systematically summarizes the use of TME-responsive contrast agents for specific tumor imaging (eg, MRI, PA imaging, and fluorescence imaging). TME-responsive agents include acidic pH, H2O2, GSH, other characteristic-responsive agents [eg, hypoxia, hydrogen sulfide (H2S), enzymes], and multiple stimuli-responsive agents (Fig. 1). After TME-responsive contrast agents reach tumor sites, changes in their structure, fluorescence, or absorption lead to significantly enhanced imaging, thus improving diagnostic efficacy. In addition, current challenges and future prospects of TME-responsive contrast agents are briefly discussed, which may provide guidance for design of TME-responsive contrast agents for specific cancer imaging.

Figure 1
Figure 1:
TME-responsive contrast agents for specific cancer imaging. TME-responsive agents including acidic pH,[27] H2O2,[37] GSH,[46], hypoxia,[58] other characteristic (eg, H2S, enzyme)-responsive agents,[50] and multiple stimuli-responsive agents.[63] Adapted from reference [22] with permission from Elsevier, copyright 2016; adapted from reference [26] with permission from American Chemical Society, copyright 2019; adapted from reference [30] with permission from Wiley-VCH, copyright 2018; adapted from reference [31] with permission from Wiley-VCH, copyright 2018; adapted from reference [37] with permission from American Chemical Society, copyright 2015; adapted from reference [39] with permission from American Chemical Society, copyright 2017. GSH = glutathione.

Database search strategy

The articles cited in this review of TME-responsive contrast agents for specific cancer imaging were retrieved from multiple search engines and databases, including: RSC, ACS, Wiley, ScienceDirect, Web of Science, and Google Scholar. References were searched using keywords including: contrast agents, cancer imaging, TME, and multimodal imaging. Specifically, we searched literature describing relevant research using the following conditions: (contrast agents) AND (cancer imaging) AND (TME) OR (multimodal imaging). The results were further screened by titles and abstracts to only present studies for TME-responsive contrast agents in cancer imaging.

TME-responsive contrast agents for cancer imaging

The primary significance of molecular imaging is early diagnosis and timely therapy of cancer. However, contrast agents used in the clinic continuously emit signal (ie, “always-on”) and lack specific recognition of tumors, leading to considerable background signal.[17] In contrast to healthy tissues, tumor cells exhibit hypermetabolism, mitochondrial dysfunction, and a non-sophisticated antioxidant system; collectively, these features represent the hallmarks of TME, namely high expression of enzymes, weak acidity, hypoxia, and high concentrations of H2O2 and GSH. Thus, developing TME-activatable contrast agents can effectively reduce background signal and maximize the target signal to improve the specificity of contrast agents and enhance molecular imaging (Table 1).

Table 1 - TME-responsive agents for specific cancer imaging
TME Agents Imaging type References
pH Smart AuNPs PA imaging Song et al[22]
c(RGDyK)-MHDA/[email protected] PA imaging Li et al[26]
HSA-Croc nanoparticles PA imaging Chen et al[27]
pRF-GQDs Fluorescence imaging Fan et al[23]
HSA-PDPA/ICG nanoprobes Fluorescence imaging Li et al[28]
[email protected] Fluorescence imaging Feng et al[24]
BODIPY nanoparticles Fluorescence/PA imaging Lin et al[25]
FNPs-PEG MRI Guo et al[29]
WS2-IO/[email protected] MRI Yang et al[30]
MnO2 nanosheets MRI Chen et al[31]
H2O2 [email protected]&ABTS PA imaging Chen et al[36]
[email protected]/PVP PA imaging Liu et al[32]
FARM:GQDzyme/ABTS PA imaging Ding et al[33]
Tyr-containing TPE derivative Fluorescence imaging Cheng et al[34]
PAAO-UCNPs-DCM-H2O2 Fluorescence imaging Wang et al[37]
CeO2: Gd nanoparticles MRI Shao et al[38]
PEG-NaxGdWO3 MRI Ni et al[35]
GSH MnO2 nanotubes PA imaging Liu et al[39]
MnMoOx nanorods PA imaging Gong et al[42]
Pyrrolopyrrolidone derivatives Fluorescence imaging Zou et al[40]
ss-diNH800CW/ss-diCy5 Fluorescence imaging Mo et al[41]
HES-SS-PTX-DiR (DHP) Fluorescence imaging Li et al[43]
99mTc-labeled Fe3O4 nanoparticles MRI/PET imaging Gao et al[46]
ICNs-RGD MRI Cao et al[44]
USPIO MRI Liu et al[45]
Hypoxia Cy7-1 Fluorescence imaging Li et al[58]
rHyP-1 PA imaging Knox et al[57]
HyP-1 Fluorescence/PA imaging Knox et al[56]
H2S Cu2O PA imaging An et al[50]
Cu-MOFs PA imaging Zhang et al[47]
CyCl-1/CyCl-2 Fluorescence imaging Li et al[48]
FTNpd Fluorescence imaging Xu et al[49]
Enzyme HACD–AdaCPT Fluorescence imaging Zhang et al[53]
BODIPY Fluorescence imaging Wang et al[55]
FLAME-DEVD 2 MR imaging Akazawa et al[65]
pH/H2O2 [email protected]2 MRI/ PA imaging Hu et al[70]
UCNPs-MnO2 Fluorescence imaging Fan et al[66]
hMUC MRI/CT imaging Sun et al[67]
pH/GSH Mo-POM PA imaging Ni et al[63]
[email protected] PA/PET imaging Yang et al[60]
2D MnO2 MRI Liu et al[61]
Manganese silicate (MnSNs) MR imaging Li et al[62]
GSH = glutathione, MRI = magnetic resonance imaging, nCT = computed tomography, PA = photoacoustic, PET = positron emission tomography, TME = tumor microenvironment.

Acidic pH-responsive contrast agents for cancer imaging

Acidic extracellular fluid is caused by significant enhancement of aerobic glycolysis, which is a common phenomenon in solid tumors and important sign of tumorigenesis.[19,20] Compared with healthy tissues and blood, the TME has slightly acidic properties, which has been widely used to design activatable imaging and therapeutic nanosystems.[21] Several strategies have been proposed to generate contrast agents with pH-responsive multimodal imaging properties. In general, pH-activated imaging is achieved by designing degraded nanomaterials, such as amine-based protonation changes, or combining fragments that are cleaved or released under slightly acidic conditions.[8] Various types of nanomaterials have been employed in this strategy and often determine the mode of activation. Therefore, design of pH-sensitive contrast agents has broad application prospects for tumor imaging and TME monitoring.[19,22–25]

Acidic pH-responsive contrast agents are useful for PA imaging.[26] To achieve the best therapeutic effect, real-time pH imaging of tumors and design of TME-responsive therapeutic strategies have become major research hotspots in nanomedicine. To this end, Chen et al[27] successfully prepared albumin-near-infrared (NIR) dye self-assembled nanoparticles for PA pH imaging and pH-responsive photothermal therapy of large tumors (Fig. 2A). Croconine (Croc) dyes with proper pH-sensitive NIR absorption could effectively adsorb to human serum albumin (HSA) through hydrophobic interactions and induce the self-assembly of albumin proteins to form HAS-Croc nanoparticles. The absorption peak of Croc could be switched between zwitterionic acid (∼790 nm) and basic anionic (∼680 nm) forms. Therefore, using the unique pH-responsive absorption of Croc, two-wavelength ratio PA imaging successfully revealed timely pH changes after intravenous injection of HSA-Croc. The resulting pH map further showed that the pH value of the inner area of large tumors was further reduced compared with its surrounding parts, which significantly enhanced NIR absorption of HSA-Croc. In addition, using HSA-Croc as a pH-responsive photothermal agent could achieve effective photothermal ablation of large tumors, which may result from more uniform distribution of intratumoral heating compared with traditional pH-insensitive photothermal agents. Therefore, this work proposed novel biocompatible pH-responsive albumin-based nanoparticles capable of realizing both real-time PA detection of pH value mapping and effective pH-responsive phototherapies, which showed obvious advantages for the treatment of large tumors and avoided nonspecific heating of normal tissues.

Figure 2
Figure 2:
Acidic pH-responsive contrast agents for cancer imaging. (A) HSA-Croc nanoparticles were prepared for real-time pH-responsive PA imaging of tumors;[27] (B) preparation process of HSA-PDPA/ICG (HDI) nanoprobes and use in acidic pH-triggered fluorescence imaging;[28] (C) two-dimensional MnO2 nanosheets were synthesized for ultrasensitive pH-triggered T1-weighted MR imaging of tumors.[31] Adapted from reference [22] with permission from Elsevier, copyright 2016; adapted from reference [23] with permission from American Chemical Society, copyright 2018; adapted from reference [24] with permission from Wiley-VCH, copyright 2018. ATRP = atom transfer radical polymerization, DPA = (2-(diisopropylamino)ethyl methacrylate, HDI = HSA-PDPA/ICG, HSA = human serum albumin, MRI = magnetic resonance imaging, NIR = near-infrared, PA = photoacoustic, PDPA = poly(2-(diisopropylamino)ethyl methacrylate, PTT = photothermal therapy.

Acidic pH-responsive contrast agents can also be used for fluorescence imaging. Due to their ability to self-assemble into stimulus-responsive micelles, site-selective protein polymer conjugates have a wide array of potential biomedical applications (from molecular imaging to drug delivery). Recently, Li et al[28] reported pH-responsive micelles with adjustable morphology for pH-triggered fluorescence imaging of tumors; preparation involved the use of a site-selective in situ growth-induced self-assembly (SIGS) method to synthesize site-specific human serum albumin-poly(2-(diisopropylamino)ethyl methacrylate) (HSA-PDPA) conjugates that formed spherical micelles, which were packed with indocyanine green (ICG) to yield pH-responsive fluorescent HSA-PDPA/ICG nanoprobes (Fig. 2B). Above the transition pH value (∼6.5), the nanoprobe remained in a silent state due to the aggregation-induced quenching (ACQ) effect. However, nanoprobes rapidly dissociated into a protonated single monomer at the transition pH (ie, the extracellular pH of the tumor), which led to a sharp increase in fluorescence intensity and significantly enhanced cell uptake. Compared with ICG alone and a non-pH-responsive nanoprobe, HSA-PDPA/ICG nanoprobes showed greatly enhanced tumor fluorescence imaging in a tumor-bearing mouse model. These findings showed pH-responsive and site-selective protein polymer-conjugated micelles synthesized by site-selective in situ growth-induced self-assembly as a new class of TME-responsive contrast agents for enhanced cancer imaging.

Acidic pH-responsive contrast agents can also be used for MRI.[29,30] For example, Chen and colleagues successfully prepared highly dispersed two-dimensional (2D) PEG-MnO2 nanosheets for ultrasensitive pH-responsive MRI of tumors (Fig. 2C).[31] The solution of PEG-MnO2 nanosheets changed from brown to colorless in a mildly acidic environment, and the pyrolysis process could be clearly observed, but there was no color change under neutral conditions. Dynamic in situ clinical MRI (3T) scanning directly showed the correlation between the break-up process and corresponding enhancement of T1-weighted MRI. After immersion in an acidic buffer for 60 minutes, the quantitative signal intensity increased to nearly 3.6 times the value observed for neutral buffer. This may have resulted because of the degradation of PEG-MnO2 nanosheets caused by exposure to an acidic pH, thus leading to the enhancement of MRI signal. The relaxation rate (r1 value) of initial PEG-MnO2 nanosheets was low ∼0.007 mM−1s−1. Such a low r1 value resulted from the high price (IV) of manganese (Mn) and shielded paramagnetic center that could not contact water molecules. Importantly, the r1 value greatly increased to 3.4 mM−1s−1 after immersion in an acid buffer (pH 6.0) for 2 hours. Such pH-responsiveness resulted in a 486-fold increase of the r1 value after changing the pH value from 7.4 to 6.0. For MRI of tumor-bearing mice, the tumor tissue at injection sites showed a time-dependent positive signal enhancement of T1-weighted MRI, while the subcutaneous injection site exhibited no obvious signal enhancement during the entire evaluation process. This interesting phenomenon not only provided direct evidence that tumor tissues were more acidic than normal tissues, but also demonstrated the utility of 2D PEG-MnO2 nanosheets for pH-responsive MRI of tumors. In summary, pH-responsive contrast agents have good performance in PA imaging, fluorescence imaging, and MRI of tumors, and significantly improve the efficiency and ability of diagnosis.

H2O2-responsive contrast agents for cancer imaging

High concentrations of H2O2 are one of the typical TME hallmarks that distinguish tumor tissues from normal tissues. Therefore, H2O2-responsive imaging has significant clinical value for disease diagnosis by improving signal-to-noise ratios.[32–35] At present, the development of imaging modalities with H2O2 responsiveness, such as fluorescence imaging, has two remaining challenges that hamper accurate detection of H2O2 in deep tissues: low sensitivity and the limited tissue-penetration depth of conventional optical imaging techniques. To solve these problems, Chen and colleagues developed liposomal nanoprobes, [email protected]&ABTS, to conduct PA imaging of inflammatory processes and tumors (Fig. 3A).[36] In this study, horseradish peroxidase (HRP) could catalyze the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) by H2O2 into its oxidized form, which has a greenish color and strong optical absorption in the NIR region (700–900 nm). With this probe, PA imaging could be used to image lipopolysaccharide- or bacterial infection-induced inflammatory processes, as well as early-stage small tumors and metastatic lymph nodes, with an elevated limit of detection to less than ∼0.8 μM, thus greatly improving the sensitivity of PA imaging compared with other imaging modalities. Moreover, PA imaging also performed well in terms of deep penetration, illustrating its promise for the diagnosis of deep-seated pathologies with great specificity.

Figure 3
Figure 3:
H2O2-responsive contrast agents for cancer imaging. (A) Preparation process of [email protected]&ABTS nanoparticles and application in H2O2-responsive PA imaging of inflammation and tumors by in vivo chromogenic assay;[36] (B) Nb3+-doped UCNPs were reported for H2O2-responsive up-conversion luminescence imaging of tumors;[37] (C) CeO2: Gd nanoparticles yielded successful H2O2-responsive MR imaging.[38] Adapted from reference [25] with permission from PNAS, copyright 2017; adapted from reference [26] with permission from American Chemical Society, copyright 2019; adapted from reference [27] with permission from American Chemical Society, copyright 2018. ABTS = 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), DCE-PWI = dynamic contrast-enhanced perfusion weighted imaging, DCM = dicyanomethylene-4H-pyran, DPPC = 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, DSPE-PEG = 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(amino-(polyethyleneglycol), DWI = diffusion-weighted magnetic resonance imaging, FRET = Förster resonance energy transfer, HRP = horseradish peroxidase, NIR = near-infrared, PAAO = PAA-n-octylamine, PEG = polyethylene glycol, PTT = photothermal therapy, UCL = upconversion luminescence.

Fluorescence imaging has high sensitivity, and H2O2-responsive fluorescent probes have achieved good results for tumor diagnosis. For example, Wang and colleagues further improved the limit of detection for H2O2 to ∼0.168 M using a PAA-n-octylamine (PAAO)-upconversion nanoparticles (UCNPs)-dicyanomethylene-4H-pyran (DCM)-H2O2 system based on up-conversion luminescence imaging (Fig. 3B).[37] In this design, core-shell UCNPs (NaYF4:Yb, Nd, [email protected]4:Nd) were synthesized and covered with PAA-n-octylamine (PAAO) to render UCNPs hydrophilic. Under 808 nm light irradiation, PAAO-UCNPs could be excited to emit two peaks located at 540 nm (515–560 nm) and 660 nm (640–675 nm). The other component of DCM-H2O2 with the donor-π-acceptor structure had high absorbance around 420 nm and 440 nm. Initially, there was no fluorescence resonance energy transfer between the donor (UCNPs) and acceptor (DCM-H2O2) due to their mismatched spectra. Ingeniously, the boronic ester group in DCM-H2O2 was transformed to phenolic hydroxyl when it came across H2O2, generating DCM-OH, which has a typical absorption at ∼559 nm (520–650 nm) that perfectly overlaps with the green emission of UCNPs. This emission change of the nanosystem in response to the addition of H2O2 indicated that it could serve as a detection signal. Moreover, the red emission intensity of UCNPs between 640 nm and 675 nm could also serve as a reference candidate for the ratiometric response of H2O2. Both in vitro and in vivo studies verified that the PAAO-UCNPs-DCM-H2O2 system is capable of detecting H2O2 with ultra-sensitivity, making it a promising new model to detect H2O2.

When evaluating the malignancy of tumors, water diffusion and microvascular permeability are the main 2 factors that need to be considered. Based on improvements of MRI, diffusion-weighted MRI (DWI) and dynamic contrast-enhanced perfusion weighted imaging (DCE-PWI) have been recently used to obtain precise information about water molecule movement, hemodynamics, and microvascular permeability of tumors.[38] To collect this information using DCE-PWI, commercial small-molecule gadolinium chelates such as Gd-DTPA (Magnevist) have been applied in clinical settings. However, Magnevist cannot enhance the sensitivity of DWI, which restricts its applications in combination DWI/DCE-PWI to fully image the tumor vascular microenvironment. Therefore, designing biofunctional contrast agents with good DWI and DCE-PWI sequences is significant for tumor malignancy detection. To this end, Shao and colleagues synthesized gadolinium (Gd3+)-doped CeO2 (CeO2:Gd) nanoparticles to act as DWI/DCE-PWI nano-contrast agents (Fig. 3C).[38] Thanks to surface oxygen vacancies, water molecules could be bonded onto the surface to accelerate the proton relaxation of water molecules with a high r1 value (19.89 mM−1s−1) of contrast agents, which significantly elevated the signal-to-noise ratio of DCE-PWI and guaranteed higher resolution DCE-PWI images compared with Magnevist at the same concentration of Gd3+. Moreover, CeO2:Gd nanoparticles also increased the relative DWI signal strength with lower magnetic susceptibility artifacts than Magnevist. To examine the assumption that water molecules fixed by oxygen vacancies enhanced the MRI signal, Ce3+-formed oxygen vacancies were depleted by H2O2 to form Ce4+. As expected, with a decreasing concentration of oxygen vacancies, the r1 value decreased rapidly. In vivo imaging of blood vessels in addition to tumor tissue further revealed the enhanced DWI signal, which indicated that CeO2:Gd nanoparticles could reduce local water diffusion in tumor tissue. Although the authors did not claim that CeO2:Gd nanoparticles were H2O2-responsive CAs to distinguish tumor tissues from normal tissues, the changes of r1 value induced by H2O2 would likely afford CeO2:Gd nanoparticles with TME responsiveness. This defect-adjusted based MRI strategy may brighten prospects for more sensitive tumor detection. In short, H2O2-responsive contrast agents can improve imaging performance and detect changes in H2O2 in the TME, thereby effectively improving diagnostic capabilities.

GSH-responsive contrast agents for cancer imaging

Among various TME hallmarks, GSH has attracted much attention because of the significant difference in its concentrations between tumors and healthy tissues. Accurate imaging of GSH in vivo can reveal physiological and pathological conditions in a real-time manner. Therefore, the synthesis of GSH-responsive PA contrast agents for specific tumor imaging is of great significance.[39–41] For instance, Gong and colleagues successfully synthesized bimetallic oxide MnMoOx nanorods as intelligent nanoprobes to detect GSH in vivo through PA imaging (Fig. 4A).[42] The obtained MnMoOx nanoprobes themselves had no NIR absorption, but possessed a strong NIR absorption in the presence of GSH, thus enabling PA imaging to detect GSH. Due to the upregulation of GSH concentrations in the TME, the as-prepared MnMoOx nanorods could be used for in vivo tumor-specific PA imaging. Importantly, MnMoOx nanorods exhibited inherent biodegradability and could be quickly removed from the body, thereby minimizing long-term retention and potential toxicity. This new type of GSH-responsive nanoprobe based on bimetallic oxide nanostructures is expected to be used for TME-specific imaging and treatments.

Figure 4
Figure 4:
GSH-responsive contrast agents for cancer imaging. (A) Bimetallic oxide MnMoOx nanorods were synthesized for GSH-responsive PA imaging of tumors;[42] (B) DHP nanoparticles containing HES-SS-PTX and DiR were prepared for GSH-responsive turn-on fluorescence imaging of cancer;[43] (C) 99mTc-labeled Fe3O4 nanoparticles were fabricated for GSH-triggering enhanced dual-model MR/PET imaging of tumors.[46] Adapted from reference [28] with permission from American Chemical Society, copyright 2018; adapted from reference [29] with permission from American Chemical Society, copyright 2019; adapted from reference [30] with permission from Wiley-VCH, copyright 2018. BSO = L-buthionine-sulfoximine, DHP = DiR and hydroxyethyl starch (HES)-SS-PTX-based nanoparticles, DiR = 1,1-Dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide, OTZ = L-2-Oxothiazolidine-4-carboxylate, PA = photoacoustic.

Numerous GSH triggered “turn-on” imaging systems, such as disulfide-bond based fluorescence imaging, have been designed with outstanding performance in lowering background interference and increasing signal-to-noise ratios. However, these designs suffer from instabilities in blood circulation and complicated preparation processes. To construct a GSH-activatable imaging system with a simple preparation procedure, Li and colleagues reported DiR and hydroxyethyl starch (HES)-SS-PTX-based DHP for dual-modal imaging and combination therapy (Fig. 4B).[43] DHP nanoparticles were prepared via a simple one-step dialysis method. Originally, DiR was encapsulated within the hydrophobic core of HES-SS-PTX, which did not allow DHP to function in fluorescence imaging due to the ACQ effect. However, upon internalization by cancer cells, -SS- could be cleaved by GSH to synchronously release PTX and DiR, making the fluorescence of free DiR recovery dependent on the concentration of GSH. With each component performing its duty, DHP could integrate dual-model (fluorescent and PA) imaging and chemo-photothermal combination therapy into a single entity with great clinical translation potential.

GSH-responsive MRI probes have also achieved satisfactory results in tumor imaging.[44,45] To solve problems associated with accurate tumor diagnosis, Gao and colleagues developed GSH-responsive anti-phagocytosis 99mTc-labeled Fe3O4 nanoprobes with active targeting and dual-modal imaging functions (Fig. 4C).[46]99mTc-labeled Fe3O4 nanoprobes were developed for GSH-responsive tumor imaging by in situ cross-linking of Fe3O4 nanoparticles modified with a responsive peptide sequence, in which tumor-targeted Arg-Gly-Asp peptides and self-labeled self-peptides were connected by disulfide bonds. Positioning of the self-peptide in the outermost layer delayed the clearance of nanoparticles from the bloodstream. After the self-peptide was cleaved by GSH in the TME, the exposed thiol group reacted with the remaining maleimide moiety in adjacent particles to cross-link the particles. Both in vitro and in vivo experiments showed that the polymer significantly improved the MRI performance of Fe3O4 nanoparticles. Using 99mTc-labeled responsive nanoprobes, single-photon emission computed tomography not only verified the enhanced imaging capabilities of cross-linked Fe3O4 nanoparticles, but also achieved sensitive dual-modal imaging of tumors in vivo. The novelty of this probe is that it combines the TME-triggered aggregation of Fe3O4 nanoparticles to enhance T2-weighted MR imaging effects with an anti-phagocytic surface coating, active targeting, and dual-mode imaging. In summary, GSH-responsive contrast agents can be used to detect changes at tumor sites, which significantly improves tumor imaging capabilities.

Other TME-responsive contrast agents for cancer imaging

In addition to the acid pH, H2O2, and GSH-activated contrast agents described above for tumor-specific imaging, other TEM-related features (eg, H2S, enzymes, and hypoxia) can activate relevant contrast agents for tumor-specific diagnosis. High expression of cystathionine-β synthase, a type of H2S-producing enzyme, results in the generation of large amounts of H2S, which plays an important role in the progression and metastasis of colon cancer. Indeed, a high concentration of endogenous H2S has become a unique hallmark of colon cancer. Although several H2S-activated fluorescence probes have been developed, they have rarely been applied to in vivo diagnostics because of the limited tissue penetration of fluorescence.[47–49] Therefore, developing H2S-activated PA imaging agents with “turn-on” properties remains a great challenge for the diagnosis of colon cancer. For example, An and colleagues developed H2S-responsive cuprous oxide (Cu2O) nanoparticles as a PA imaging agent for specific diagnosis of colon cancer (Fig. 5A).[50] Utilizing the differential expression of endogenous H2S in normal and colon cancer tissues, the in situ reaction between H2S and Cu2O that occurred at colon tumor sites forms copper sulfide (Cu9S8) nanoparticles with high absorbance in the NIR region, thus acting as both PA imaging agent and photothermal agent for PA imaging-guided photothermal therapy. Endogenous H2S was used as the switch to turn on intelligent diagnosis, which effectively improved the treatment efficacy of colon cancer.

Figure 5
Figure 5:
Other TME-responsive contrast agents for cancer imaging. (A) Cu2O nanoparticles were synthesized as H2S-responsive PA imaging agents for the specific diagnosis of colon cancer;[50] (B) BODIPY platforms with benzyl thioether as the self-immolative linker could be coupled to enzymic substrates to access diverse enzyme-activated NIR fluorescent probes with desired optical properties;[55] (C) Cy7-1 was successfully prepared for hypoxia-activated fluorescence imaging of cancer.[58] Adapted from reference [31] with permission from Wiley-VCH, copyright 2018; adapted from reference [36] with permission from Royal Society of Chemistry, copyright 2019; adapted from reference [37] with permission from American Chemical Society, copyright 2015. AOAA = aminooxyacetic acid, BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, NIR = near-infrared, NTR = nitroreductase, PA = photoacoustic, PBS = phosphate buffered saline, PTT = photothermal therapy, SAM = S-adenosyl-L-methionine, TME = tumor microenvironment.

Another notable feature of TME is the presence of overexpressed enzymes in tumors, such as matrix metalloproteinases.[51,52] Therefore, the preparation of enzyme-responsive contrast agents to achieve tumor-specific imaging also has broad prospects.[53] Indeed, activatable contrast agents capable of responding to enzyme biomarkers of cancers have already been successfully applied in cancer imaging. Current development of enzyme-activatable NIR probes are employing cyanine and rhodamine scaffolds for enzyme detection. However, they suffer from small Stokes shifts, which generally lead to severe overlap between the excitation and emission spectra.[54] Thus, innovations are required to establish other fluorescent scaffolds with large Stokes shifts to emit in the NIR region, and to further improve the resolution of detecting enzymatic cancer biomarkers. To achieve this goal, Wang and colleagues reported a new design strategy: using benzyl thioether as the self-immolative linker, BODIPY platforms could be coupled to enzymic substrates to access diverse enzyme-activated NIR fluorescent probes with desired optical properties (Fig. 5B).[55] In this study, nitroreductase (NTR)-, NAD(P)H: quinone oxidoreductase isozyme 1-, and alkaline phosphatase-activated NIR probes for enzyme-ImI and enzyme-InD were designed to explore the feasibilities of evaluating enzyme activities. Due to the diminished electron-donating ability of the sulfur atom, BODIPY platforms showed typical absorption and emission spectra in the visible region. In the presence of enzymes, self-immolation could be initiated to liberate thiol-substituted BODIPY with the enhanced electron-donating ability of the sulfur atom, eventually leading to dyes capable of emissions in the NIR region with large Stokes shifts. Both in vitro and in vivo studies demonstrated that these biomarker-activated molecular probes provided a powerful tool for accurate identification of cancer from healthy tissue. It was appreciated that this design could be extended to include a wide range of optimized optical probes, thus facilitating targeted cancer diagnostics.

The aforementioned enzyme NTR is overexpressed in hypoxic tumors; thus, selective and efficient detection of NTR is of great importance to evaluate the hypoxic degree of tumors.[56,57] Although BODIPY-based NIR fluorescent probes have been discovered, enzyme-activated probes with the aforementioned advantages and favorable responsiveness are still very limited, which motivated researchers to further increase the variety of NIR fluorescent probes. Inspired by the structure and spatial arrangement of enzymes, Li and colleagues fabricated five NIR cyanine dyes, Cy7-1 to Cy7-5, to explore rapid detection of NTR (Fig. 5C).[58] Considering the interaction between NTR and the substance, substituent group of the substance, and structural and spatial match-mismatch of the two, in vitro and in vivo studies revealed that only Cy7-1 could act as a remarkable NIR fluorescent “off-on” probe for NTR detection, with excellent selectivity, fast response, and ultra-sensitivity. This work enriched the principle for designing NTR-responsive NIR probes, and provided a new design strategy for further studies in NTR detection to monitor hypoxia of tumors. In conclusion, although TME-activated contrast agents have achieved satisfactory results for specific imaging, problems such as poor specificity and low stability still remain. However, current research in this area is in the primary stage, and there is still a broad space for development in the future.

Multiple stimuli-responsive contrast agents for cancer imaging

Compared with a single response to one of the hallmarks of TME, multiple-stimuli responses to TME can provide more accurate identification and imaging of tumor tissues.[59] pH/GSH dual-responsive contrast agents have been widely used for imaging of specific cancers.[60–62] For example, Ni and colleagues developed molybdenum (Mo)-based, polyoxometalate (POM) nanocluster-based, pH/GSH-sensitive contrast agents for pH/GSH-activated PA imaging-guided photothermal therapy (Fig. 6A).[63]Ox-POMs exhibited sequential responsiveness to TME. First, when administrated intravenously, ultrasmall Ox-POMs escaped recognition and capture by liver and spleen, and reached tumors. Once they arrived at tumors, the low pH of the TME catalyzed self-assembly of Ox-POMs into larger nanoclusters that, once formed, were retained and could not be cleared from tumors. Second, Mo(VI) elements in the retained nanoclusters were further reduced by GSH, yielding Mo(V)-containing nanoclusters with high absorbance in the NIR region due to the charge transfer between Mo(VI) and Mo(V), which is regarded as the origin of the typical NIR absorption of POM clusters. As a proof-of-concept, this finding realized sequential TME responsiveness, by lower pH-induced aggregation and GSH-induced reduction, thus establishing a new method for redox-activated bioimaging.

Figure 6
Figure 6:
Multiple stimuli-responsive contrast agents for cancer imaging. (A) Molybdenum (Mo)-based polyoxometalate (POM) nanoclusters were developed for GSH/pH dual-responsive PA imaging of tumors;[63] (B) [email protected]2 nanoparticles were fabricated as pH/H2O2 dually-activated nanoprobes for PA/MR dual-model images of cancer.[70] Adapted from reference [39] with permission from American Chemical Society, copyright 2017; adapted from reference [43] with permission from Royal Society of Chemistry, copyright 2019. MRI = magnetic resonance imaging, NPs = nanoparticles, PA = photoacoustic, PTT = photothermal therapy.

In addition, use of pH/H2O2 dual-activatable contrast agents in cancer imaging has been extensively reported.[64–67] Rational integration of two or more imaging techniques can provide complementary information and achieve higher sensitivity for cancer diagnosis.[68,69] Moreover, synchronous multimodal imaging of different TME hallmarks can realize “turn-on” imaging, further improving tumor specificity.[43] To obtain complementary information from MRI/PA imaging, Hu and colleagues reported a strategy using MnO2-based nanoparticles for pH/H2O2 dual-activatable MRI/PA imaging (Fig. 6B).[70] In the [email protected]2 system, an amphiphilic NIR absorption polymer (termed PBP) conjugated to BODIPY exhibited high absorbance in a wide range of the NIR region, with an absorption peak at of 825 nm assigned to the Aza-BODIPY molecule. This afforded [email protected]2 good capability to conduct PA imaging. In addition, MnO2 nanoparticles, a well-known MRI contrast agent, were scanned in a 7.0T Bruker MR scanner under different conditions. Under a weak acidic condition (pH 6.5) in the presence of H2O2, caged Mn ions in MnO2 nanoparticles were released and easily accessible to the water environment, resulting in an enhanced r1 value compared with the neutral pH condition (pH 7.4) without H2O2. This significance in T1-weighted MRI indicated that MnO2 nanoparticles were suitable H2O2/pH-sensitive contrast agents for tumor-specific imaging. This [email protected]2-based dual-activatable imaging was believed to enable “turn-on” performance for tumor identification. In conclusion, the design of multiple stimuli-responsive contrast agents for imaging of specific tumors can significantly improve imaging performance and sensitivity, reduce inaccurate diagnoses, and thus have broad application prospects.

Conclusions and perspectives

Although challenging, it is significant to develop intelligent contrast agents or probes for efficient tumor diagnosis because they are conducive to the early detection of cancer, which can significantly improve therapeutic efficiency and reduce treatment costs.[8,71] Based on this notion, this minireview comprehensively summarized the recent progress of TME-responsive intelligent contrast agents for achieving specific tumor imaging. TME-responsive contrast agents significantly increase the efficacy of imaging owing to changes in their structure (fluorescence or absorbance) that occur after reaching tumor sites, leading to remarkable enhancement of diagnostic efficiency. Acidic pH, H2O2, GSH, other TME-related characteristics (including H2S, enzymes, and hypoxia), and multiple stimuli-responsive agents for PA imaging, fluorescence imaging, and MRI were included in this minireview. Although considerable efforts have been made to develop new TME-responsive contrast agents or probes for tumor diagnosis, there are still some challenges and key problems that need to be resolved, including complex preparation techniques, poor/insensitive imaging effects, and side effects on healthy tissues.

Firstly, it is necessary to clarify the pharmacokinetics of TME-responsive contrast agents and solve their biocompatibility problems, including biodegradability, cytotoxicity, biodistribution, and excretion pathways. For TME-responsive contrast agents to have a chance of eventually being used in the clinic, it is necessary to thoroughly and systematically evaluate their safety, including demonstrations in appropriate animal models.[72] Secondly, it is necessary to further improve the specificity of TME-responsive contrast agents for different tumor models. A series of active tumor-targeting ligands, such as peptides and antibodies, can be combined with contrast agents to significantly increase their specific binding to tumors,[18] thereby significantly enhancing imaging performance. Thirdly, in vivo imaging performance of contrast agents needs to be further improved, including reducing the background signal and increasing the signal-to-noise ratio, which is supported by the progress of nanomaterials and imaging technology. Lastly, the combination of TME-responsive contrast agents and therapeutic reagents can achieve precise targeting and efficient destruction of malignant tumors, which is a trend for future biomedicine development.[73]

Although there are still some problems with TME-responsive contrast agents, all of these issues are expected to be solved gradually with the continuous advancement of biomedical technology. In summary, TME-responsive contrast agents skillfully use the characteristics of TME to achieve tumor-specific imaging. Compared with traditional contrast agents, they can effectively solve the problem of low contrast agent sensitivity. Thus, continuing development of new TME-responsive contrast agents is expected to provide new methods for early diagnosis and treatment of tumors.


The authors thank all the staff of the advanced biomaterials and nanomedicine Laboratory of Soochow University of China for their kind help.

Author contributions

XW and LC conceived, designed the manuscript, and revised the manuscript. XW, XZ, HL, NY and XG wrote the manuscript. All authors read and approved the final manuscript.

Financial support

LC was supported by the National Natural Science Foundation of China (No. 52072253); Collaborative Innovation Center of Suzhou Nano Science and Technology, and the State Key Laboratory of Radiation Medicine and Protection (No. GZK1201810). XW was supported a Project Funded for Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. 2019 SJKY19_1923).

Conflicts of interest

The authors declare that they have no conflicts of interest.


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cancer imaging; cancer treatment; contrast agents; diagnosis; tumor microenvironment

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