English

• 0.   Introduction

  Magnetic resonance (MR) imaging represents one of the most widely used noninvasive clinical diagnostic techniques. It provides three-dimensional images with high spatial resolution and penetration depth in the absence of ionizing radiation^[1-3]. According to the basic theory of MR imaging, the relaxation times of protons in water are monitored under an external magnetic field to generate MR signals. Specifically, a short longitudinal relaxation time (T₁) tends to improve MR signal intensity, while a fast transverse relaxation time (T₂) results in a darker MR image^{[1, 4-8]}. To visualize pathological details with brighter MR signals, T₁-weighted MR imaging has dominated in practical MR imaging scans. Accordingly, paramagnetic contrast agents have been used to alter the relaxation times of protons for contrast-enhanced T₁-weighted MR imaging. These agents include Gd(Ⅲ), Mn(Ⅱ), and Fe(Ⅲ) complexes^[9-13]. However, only a category of Gd(Ⅲ)-based contrast agents has obtained approval and is widely used in clinics. Approximately nine “always-on” Gd(Ⅲ)-based contrast agents have been approved for clinical use. To increase the specificity of MR imaging, considerable attention has been drawn to fabricating bio-responsive Gd(Ⅲ) agents since the late 1990s^[14-19].

  The paramagnetic Gd(Ⅲ) ion has seven unpaired electrons, leading to strong interaction with surrounding protons in water molecules to facilitate their T₁ relaxation and enhance MR imaging signal^[20-22]. However, the free Gd(Ⅲ) ion is toxic in biological systems^[23-26]. Both linear and macrocyclic multi-carboxylated ligands have been employed to construct stable Gd(Ⅲ) complexes for MR imaging^{[19, 27-29]}. The change of coordination environment of Gd(Ⅲ) complexes results in the alteration of longitudinal relaxivity (r₁) with distinct MR signal intensity^{[2, 30-32]}. In general, there are three parameters that are closely related to the r₁ values of Gd(Ⅲ) agents: inner-sphere bound water molecules (q), the tumbling time (τ_r), and the residence time of water molecules (τ_m). A large q value and a slow tumbling rate contribute to a higher r₁ and a brighter MR image. The modulation of q and τ_r values has dominated the molecular designs of bio-responsive Gd(Ⅲ) agents^{[15, 18, 33-37]}.

  β-galactosidase is a glycoside hydrolase lysosomal enzyme^[38-40]. It can catalyze the hydrolysis reaction of the β-galactosidic bonds in diverse biological species, such as gangliosides, lactose, galactosylceramide, and polysaccharides^[41-44]. The dysfunction of β-galactosidase activity has been associated with many diseases^[45-48]. For instance, it has been indicated that the deficiency of β-galactosidase activity is associated with GM1 gangliosidosis and Morquio B syndrome, while elevated β-galactosidase activity has been found in cell senescence, primary ovarian and glioma cancer cells^{[45, 49-55]}. Hence, β-galactosidase has been widely used as a vital biomarker reporter for the evaluation of gene expression, gene therapy, cell senescence, and cancers^{[39, 56-57]}. Among these efforts, many β-galactosidase-activated Gd(Ⅲ) agents have been reported for bio-responsive MR imaging during the last several decades^[19]. It′s worth noting that in vivo MR signal enhancement is related to many aspects, including activity-based Gd(Ⅲ) molecular-structural changes, nonspecific biodistributions and effects, and distinct physiological conditions^[19,58]. It remains challenging to track the specific q values of Gd(Ⅲ) chelates in vivo. Previous work usually employed specific control animal models such as high-or low-enzyme-activity mice to support the activity-based molecular-structural changes that can result in the observed MR signal enhancement^[59-61]. However, several τ_r-modulated multi-modal agents were successfully fabricated to coordinate β-galactosidase-responsive MR signal enhancement with ¹⁹F MR imaging or near-infrared (NIR) fluorescence changes^[62-64]. Herein, we provide a comprehensive summary of the recently developed β-galactosidase-responsive Gd(Ⅲ) agents, highlighting their structural design strategies and diverse biological applications. Based on the specific modulation parameters of the Gd(Ⅲ) coordination environment, two major classes of β-galactosidase-responsive Gd(Ⅲ) agents have been discussed, including q-modulated and τ_r-modulated probes (Fig.1).

  Figure 1

  

  Figure 1.  Schematic view of the q-modulated and τ_r-modulated pathways for the construction of β-galactosidase-responsive Gd(Ⅲ) complexes for MR imaging

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  1.   q-modulated Gd(Ⅲ) complexes for β-galactosidase-activated MR imaging

  According to the Solomon-Bloembergen-Morgan (SBM) MR theory, the parameter q for Gd(Ⅲ) complexes represents the number of water molecules that bind to the inner coordination sphere of the Gd(Ⅲ) metal ion^{[9, 20, 65-66]}. The increase in q results in more efficient shortening of the relaxation time of water protons, which contributes to the enhancement of the MR signal intensity^{[2-3, 10, 67]}. The introduction of functional groups into the inner coordination sphere of Gd(Ⅲ) can block water molecules from binding to the inner coordination sphere of Gd(Ⅲ) and result in a decrease in the q values. The removal of blocking units opens the coordination site of Gd(Ⅲ) for water molecules and leads to an increase in q. The combination of the Gd(Ⅲ) moiety with bioresponsive blocking groups displays q value differences before and after bioactivation, providing a versatile q-modulation strategy to construct bioresponsive Gd(Ⅲ) agents for functional MR imaging^{[3, 15, 17-19, 68]}. Many q-modulated bioresponsive Gd(Ⅲ) probes have recently been developed to visualize various biological events, such as enzyme activities, gene expression, senescence, metal ions, and pH for diverse bioimaging applications, including real-time visualization of gene therapy, osteoarthritis, and tumors^{[14, 16, 19, 61, 69-78]}.

  Since 1997, the Meade group has pioneered advancements in the fabrication of β-galactosidase-activated q-modulation of Gd(Ⅲ) complexes for real-time tracking of gene expression and gene therapy^{[19, 34, 59-60, 79-81]}. Generally, these agents consist of β-galactosyl or other coordinating bridging ligands to saturate the coordination sphere of Gd(Ⅲ) with a low q value to generate “dark” MR images before enzyme activation. A selective enzyme-activation reaction triggers the release of blocking ligands to afford the coordination sites in Gd(Ⅲ) for water molecules, resulting in increased q values and brighter MR signals.

  As shown in Fig.2, the first β-galactosidase-activated Gd(Ⅲ) complex (EGad, 1) contains a β-galactosidase-responsive galactosyl unit and a DO3A-Gd MR unit bridged with an ethoxy linker^[79]. It showed a low MR relaxivity (slow T₁ relaxation) at the pristine MR “off-state”. The β-galactosidase-activated cleavage of the galactosyl unit afforded the MR “on-state” of the agent, displaying a 20% enhancement of MR relaxivity (fast T₁ relaxation). The corresponding terbium complexes were prepared to determine the q values of 1 and the on-state of the agent as 0.7 and 1.2, which is consistent with the MR relaxivity enhancement upon β-galactosidase-activation. This work demonstrated the possibility of using a q-modulation strategy for the construction of bioresponsive Gd(Ⅲ) MR contrast agents. The introduction of an α-methyl group to the ethoxy linker in EGad afforded the complex EGadMe (2), which displayed a threefold increase in r₁ from 0.903 to 2.72 mmol^-1·L·s^{-1 [59]}. The optimized structure of 2 suggested that the methyl group restricts free rotation of the galactosyl blocking unit. It further limited water access to the coordination sphere of the Gd(Ⅲ) ion, resulting in a smaller q value of 2. In vivo MR imaging of β-galactosidase mRNA expression in X. laevis embryos was also demonstrated. It showed a significant 57% enhancement in MR signal intensity on the right side of X. laevis embryos with β-galactosidase gene expression compared to the left side, in the absence of β-galactosidase gene expression.

  Figure 2

  

  Figure 2.  Representative q-modulated Gd(Ⅲ) complexes

  The blue color shows the β-galactosidase recognition site. The purple color indicates the bridging linker. The black color reveals the Gd(Ⅲ) MR unit. Probe 1 is the first β-galactosidase-activated Gd(Ⅲ) complex (EGad) using an ethoxy linkage to combine a galactosyl group with a DO3A-Gd MR moiety^[79]. Complex 2 introduces an α-methyl group to the ethoxy linker in EGad with an enhanced MR relaxivity^[59]. Probe 3 employs a carbamate--based β-galactosidase-activated self-immolative unit between a galactosyl moiety and a DO3A-Gd MR group^[81]. Complex 4 attaches a pendant coordinating carboxylate ligand to the self-immolative carbamate-based chelates for in vivo detection of β-galactosidase activity^[60]. Complex 5 is a pyridyl-carbamate unit based Gd(Ⅲ) with large β-galactosidase-responsive “on-off” ratio of MR relaxivity changes^[34].

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  Another carbamate-based self-immolative linker was used to connect a galactosyl group and a DO3A-Gd MR moiety in Gd(Ⅲ) chelate 3, which incorporated a distal biotin unit to allow for noninvasive detection of both enzyme activity and receptor gene expression^[81]. It showed a distinct β-galactosidase-activated increase (120%) in r₁ relaxivity from 7.8 to 17.2 mmol^-1·L·s^-1 in the presence of avidin, while the measured q values changed from 0.14 to 1. An adduct of 3, avidin, and transferrin (Tf) in a 3∶1∶1 ratio was prepared for in vitro MR imaging of 9L cells with overexpressed transferrin receptor (TfR). A significant preferential cellular uptake of this targeting adduct was observed at different concentrations. However, it′s worth noting that a shorter linker length of biotin hindered the enzymatic reaction under similar conditions due to the interactions between avidin and the glycoside.

  Recently, Lilley et al. reported an intramolecular pathway of q-modulated self-immolative MR chelate 4 by introducing a pendant coordinating carboxylate ligand to saturate the coordination sphere of the Gd(Ⅲ) ion (Fig.3A)^[60]. It was found that a 6-carbon pendant carboxylate ligand displayed a higher enzymatic MR relaxivity response compared to the Gd(Ⅲ) chelates with 2-, 4-, or 8-carbon pendant linkers. The q value of this probe was measured as 0.1 through the luminescent decay results of the Tb(Ⅲ) analog. It showed a positive r₁ enhancement of 20% after enzyme activation. In vivo MR imaging of transgenic LacZ mice with β-galactosidase enzyme expression was then performed via an intraperitoneal injection method. As shown in Fig.3B, a global MR signal enhancement was observed in the LacZ mice, while no obvious MR signal increase was found in the native non-transgenic control mice. Gray-Edwards and coworkers extended the application of this molecular scaffold for real-time MR detection of adeno-associated virus (AAV) gene therapy in a GM1-gangliosidosis murine model^[73]. Basically, GM1 mice are an animal model for a progressive neurological disease in humans with deficient lysosomal β-galactosidase activity. The AAV gene therapy treatment of GM1 mice increased β-galactosidase activity, which can be effectively visualized by MR imaging upon the administration of agent 4.

  Figure 3

  

  Figure 3.  β-galactosidase-activated Gd(Ⅲ) probe 4 for in vivo detection of enzyme activity: (A) molecular structure changes post β-galactosidase enzyme activation^[60,74]; (B) in vivo MR imaging of LacZ transgenic mice upon intraperitoneal injection of 4 at pre-injection, 1 h, 4 h, and 24 h^[60] (Copyright 2020, Wiley-VCH GmbH); (C) senescent cells (green) and viable cells (orange) implanted in the distal femur of a live pig; (D) representative T₁-weighted MR image of the pig knee joint upon intra-articular injection of 4; (E) corresponding T₁ color-mapping in D; (F) corresponding R₁ relaxation rates of senescent and viable cell implants; (G) signal-to-noise ratios (SNR) analysis of senescent and viable cell implants; (H) senescent cells (green) and viable cells (orange) implanted in the excised distal femur with cartilage defects; (I) representative T₁-weighted MR image of the explant; (J) corresponding T₁ color-mapping in I; (K) R₁ relaxation rates of the implants; (L) SNR analysis of the implants (*p<0.05, **p<0.005)^[74] (Copyright 2025, Springer Nature)

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  MR visualization of β-galactosidase activity in large animals, such as in a pig knee joint osteoarthritis model, was further investigated by Daldrup-Link and coworkers^[74]. Recent studies have demonstrated that osteoarthritis development is usually associated with the senescence processes in cartilage and synovium. The senescence-associated β-galactosidase activity was found to be upregulated in osteoarthritis. This poses the potential of using β-galactosidase-activated MR probe 4 for real-time evaluation of osteoarthritis. As depicted in Fig.3C-3L, senescent cells and viable control cells were implanted in porcine joints (cartilage defects and explants). They showed brighter MR signals with higher r₁ relaxation rates in the senescent cells implanted areas compared to the viable controls. This work demonstrated in vitro and in vivo MR tracking of senescence-associated β-galactosidase activity in pig knee joints by the use of q-modulated Gd(Ⅲ) chelate 4. It suggested promise for real-time and noninvasive detection of osteoarthritis in patients.

  It should be noted that a pyridyl-carbamate-based self-immolative Gd(Ⅲ) complex 5 was also developed by the Meade group^[34]. The incorporation of a pyridyl coordination unit resulted in a lower r₁ before the β-galactosidase activation. The increase in MR relaxivity (Δr₁) was measured as 106% before and after the enzymatic reaction, which is significantly higher than that of probe 4 (20%). However, it displayed a slow enzymatic self-immolation reaction rate due to the coordination of the pyridyl group, limiting its further in vivo MR detection of β-galactosidase activity.

  2.   τ_r-modulated Gd(Ⅲ) complexes for β-galactosidase-activated MR imaging

  The τ_r of Gd(Ⅲ) complexes is another parameter that has often been modulated by the β-galactosidase-activated reaction to alter T₁ relaxation time and MR signal intensity^{[15, 18, 20, 82-83]}. A slower τ_r of Gd(Ⅲ) chelates leads to a decrease in T₁ relaxation time and a higher r₁ relaxivity and MR signal intensity^{[2-3, 14, 84-86]}. A general approach to slow τ_r is to increase the molecular size of the Gd(Ⅲ) agents^[87-91]. Several β-galactosidase-catalyzed molecular transformations have been developed for enhancing r₁ via τ_r-modulation, such as an enzymatic self-immobilizing strategy to bind large-sized proteins in situ, enzyme-triggered polymerization of monomeric Gd(Ⅲ) chelates, and enzyme-initiated formation of large aggregates. As shown in Fig.4, many τ_r-modulated Gd(Ⅲ) complexes (6-13) for β-galactosidase-activated MR imaging of diverse disease animal models have been reported^{[61-63, 92-95]}. However, this τ_r-modulated strategy usually generates a distinct impact on r₁ at relatively low magnetic fields (20-60 MHz)^[96]. It indicates significant translational potential of these τ_r-modulated Gd(Ⅲ) chelates. Moreover, several τ_r-modulated β-galactosidase-responsive multi-modal Gd(Ⅲ) chelates have also been developed^[62-64]. However, all current clinically approved Gd(Ⅲ) chelates are non-activity-based “always on” agents. Additional efforts are needed to move forward the translational research of these chelates, such as the large-scale production of these agents, the long-term biocompatibility and biosafety studies, and the clinical investigations.

  Figure 4

  

  Figure 4.  Representative τ_r-modulated Gd(Ⅲ) complexes

  The blue color shows the β-galactosidase recognition site. The purple color indicates the bridging linker. The black color reveals the Gd(Ⅲ) MR unit. Complex 6 is a difluoromethylphenyl-β-galactopyranoside conjugated Gd(Ⅲ) agent for β-galactosidase-expressed CT26 tumor MR imaging^[92]. Complex 7 contains a fluoromethyl pendant group to generate a protein-binding electrophilic quinone methide intermediate for MR imaging of AAV gene therapy^[61]. Complex 8 is a β-galactosidase-responsive self-immobilizing NIR fluorescence and MR imaging bimodal agent^[62]. Complex 9 represents a β-galactosidase-activated NIR fluorescence and ¹H/¹⁹F MR imaging trimodal chelate for in vivo detection of ovarian cancer^[63]. Probe 10 is a tyrosine-β-galactopyranoside functionalized Gd(Ⅲ), generating Gd(Ⅲ)-containing melanin oligomers or polymers upon the β-galactosidase and tyrosinase enzymatic reactions^[94]. Probe 11 employs a receptor-induced magnetization enhancement (RIME) mechanism to increase protein binding affinity by changing the hydrophobicity of the agent post-enzymatic activation^[93]. Complexes 12 and 13 are the β-galactopyranoside-TPE-Gd(Ⅲ) based probes to form large-sized aggregates after enzyme activation^[95].

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  β-galactosidase-catalyzed self-immobilizing strategy was first reported by Wang et al.^[92]. As shown in complex 6, a difluoromethylphenyl bridging-ligand was used to combine β-galactopyranoside and the Gd-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) MR unit. The β-galactosidase-activated cleavage of β-galactopyranoside and a fluoride unit generated an active species to bind the nucleophilic groups of human serum albumin (HSA) and β-galactosidase in situ. It resulted in a 60% decrease in T₁ relaxation time, significantly enhancing MR signal intensity. In vivo MR detection of β-galactosidase gene expression in the CT26/β-gal tumor was also demonstrated.

  Recently, we prepared a monofluoromethylphenyl-based β-galactosidase-responsive Gd(Ⅲ) chelate 7 for noninvasive evaluation of AAV gene therapy in GM1 gangliosidosis mice^[61]. As shown in Fig.5A, it generates an electrophilic quinone methide intermediate post β-galactosidase-activation, which can covalently bind to proximal nucleophilic residues on large biomolecules, such as HSA and other proteins. An obvious increase in MR relaxation rate (1/T₁) was observed upon incubation of 7 with β-galactosidase in 4.5% HSA-containing phosphate-buffered saline (PBS), suggesting the enzymatic protein-binding τ_r-modulated MR enhancement. The 7-PBS-only solution and the control agent without a fluoride substitute showed a gradual decrease in 1/T₁ under similar conditions. An intravenous injection of this probe into AAV-treated GM1 mice resulted in a distinct and persistent MR signal enhancement. Comparatively, a fast MR signal decay was observed in the untreated β-galactosidase-deficient GM1 controls.

  Figure 5

  

  Figure 5.  β-galactosidase-catalyzed self-immobilizing strategy enabled τ_r-modulated MR agent for real-time tracking of AAV gene therapy in GM1 gangliosidosis mice: (A) schematic review of β-galactosidase-activated covalent binding of Gd(Ⅲ) chelate to large bio-nucleophiles, leading to a slower molecular tumbling rate and a higher MR relaxivity; (B) MR imaging of GM1 mice (Glb1) and AAV-treated GM1 gangliosidosis mice (AAV-treated Glb1) upon intravenousⅣ injection of Gd(Ⅲ) probe 7; (C) kinetic MR signal changes in the liver slices^[61] (Copyright 2026, American Chemical Society)

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  In addition to these fluoride-based enzymatic self-immobilizing agents, Chen and coworkers used a carbamate group to generate a quinone methide electrophilic intermediate for constructing β-galactosidase-activated multimodal imaging Gd(Ⅲ) probes (8, 9)^[62-63]. Upon β-galactosidase activation, these intermediates can react with nucleophilic residues on external proteins to form large-sized structures with enhanced signals. For instance, a NIR fluorescence/MR imaging bimodal agent 8 consists of four functional components: a β-galactosidase recognition galactosyl unit, an ethyl carbamate self-immobilizing linker, a NIR fluorescence group, and a DOTA-Gd MR chelate (Fig.6A). It not only underwent a β-galactosidase-activated fluorogenic reaction to show a 42-fold increase in NIR fluorescence at 717 nm, but also displayed an approximately 1.9 times enhancement in longitudinal r₁ post-enzymatic reaction in the presence of bovine serum albumin (BSA). A control agent, 8-C without an ethyl carbamate unit, was also prepared. However, this control agent only showed similar enzymatic fluorescence enhancement. No obvious r₁ change was observed during incubation with β-galactosidase with/without BSA. In vivo fluorescence/MR imaging bimodal imaging of SKOV3 ovarian cancer cells-based tumor-bearing mice was also demonstrated. As shown in Fig.6B-6C, there was a gradual increase in fluorescence intensity with a peak at 24 h upon intravenous injection of 8, while the control agent 8-C-injected mice displayed a fast increase in fluorescence with the signal peak at 1 h followed by a rapid fluorescence decrease. T₁-weighted MR images also indicated a stronger signal enhancement in the 8 intravenously injected group (Fig.6D, 6E). Moreover, this fluorescence/MR imaging bimodal agent was also employed for real-time fluorescence-guided surgical removal of orthotopic ovarian tumors.

  Figure 6

  

  Figure 6.  β-galactosidase-catalyzed self-immobilizing strategy-enabled τ_r-modulated MR agent for NIR fluorescence and MR bimodal imaging: (A) chemical structural changes of 8 regarding β-galactosidase-catalyzed fluorogenic reaction and self-immobilizing pathways; (B) fluorescence imaging of β-galactosidase-expressing SKOV3 tumor-bearing mice via intravenous injection of 8 or 8-C; (C) corresponding fluorescence intensity changes in B; (D) T₁-weighted MR imaging of β-galactosidase-expressing SKOV3 tumor-bearing mice using intravenous injection of 8 or 8-C under a 9.4 T MR imaging scanner; (E) MR signal enhancements of the tumor area in D^[62] (Copyright 2023, Wiley-VCH GmbH)

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  Recently, the same group developed a β-galactosidase-activated NIR fluorescence/¹H/¹⁹F MR imaging trimodal probe 9 for real-time detection of ovarian cancer^[63]. This agent contains five functional moieties, including a β-galactosidase recognition unit, a carbamate self-immobilizing linker, a hemocyanin-based NIR fluorescence group, a DOTA-Gd-based MR chelate, and a trifluoromethylbenzene-based ¹⁹F signal reporter. Upon incubating this probe with β-galactosidase, it directly induced a gradual increase in fluorescence at 720 nm and the appearance of a ¹⁹F signal at δ=-62.30. The r₁ increased from (8.67±0.5) mmol^-1·L·s^-1 to (13.85±0.6) mmol^-1·L·s^-1 at 20 MHz in the presence of β-galactosidase and BSA, corresponding to an enzymatically triggered self-immobilizing enhancement pathway. In vivo fluorescence/MR imaging of β-Gal-expressing SKOV3 tumor-bearing mice was investigated using the intravenous administration method. The tumor-specific fluorescence signal was observed at 2 h after the injection, while a 33% increase in ¹H MR imaging signal intensity was obtained. Moreover, the intratumoral injection of the probe also displayed a distinct ¹⁹F MR imaging signal recovery, suggesting potential trimodal imaging applications in precise tumor detection.

  In addition to the enzymatic self-immobilization-enhanced strategy, several other mechanisms were also demonstrated for the fabrication of τ_r-modulated Gd(Ⅲ) agents, such as β-galactosidase-triggered polymerization, β-galactosidase-activated noncovalent interaction with large-sized biomolecules, and β-galactosidase-triggered aggregation^[93-94]. As shown in Fig.7A, the Aime group introduced a tyrosine bridge ligand between a Gd-DOTA MR unit and a galactose-pyranose enzyme-responsive group in probe 10^[94]. The β-galactosidase-activated cleavage of the sugar group deprotects the hydroxyl group of tyrosine, which can further form melanin oligomeric or polymeric structures in response to tyrosinase. This β-galactosidase-triggered polymerization process produces large-sized Gd-DOTA-containing oligomers or polymers with enhanced MR relaxivities via a τ_r-modulation pathway. In vitro and in vivo MR imaging investigations showed a higher MR contrast enhancement in β-galactosidase expression B16-F10LacZ cells and B16-F10LacZ tumors.

  Figure 7

  

  Figure 7.  Several other enzymatic-triggered τ_r-modulation strategies: (A) β-galactosidase-triggered polymerization^[94]; (B) β-galactosidase-activated noncovalent interaction with large-sized biomolecules^[93]

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  A receptor-induced magnetization enhancement (RIME) mechanism was proposed by Nagano et al. to construct a hydrophobic biaryl-group-linked Gd(Ⅲ) complex 11 protected with a galactosyl enzymatic unit (Fig.7B)^[93]. The β-galactosidase-activated deprotection of the galactosyl group resulted in an increase in the hydrophobicity of the biaryl moieties. It showed enhanced noncovalent binding affinity toward HSA. Specifically, a 57% increase in the MR r₁ relaxivity was observed in 4.5% HSA-containing PBS post β-galactosidase activation.

  Recently, Meade and coworkers reported a series of β-galactose-tetraphenyl ethylene (TPE)-Gd(Ⅲ)-DOTA conjugated FL/MR bimodal chelates (12, 13)^[95]. These agents displayed structure-dependent aggregation behaviors. β-galactose-substituted agents showed increased aqueous solubility, while the negatively charged derivatives were more soluble than the neutral analogues. However, these agents were unable to be cleaved by β-galactosidase due to the steric inhibition effects of the TPE unit. The negatively charged TPE-Gd(Ⅲ)-DOTA chelate 12 showed the MR r₁ increased from 7.19 to 15.57 mmol^-1·L·s^-1 during the formation of large-sized aggregates. Comparatively, the neutral TPE-Gd(Ⅲ)-DOTA complex 13 displayed the highest fluorescence enhancement with a quantum yield (Φ_F) of 4.0%. This work demonstrated the aggregation-enhanced FL/MR enhancement properties of these TPE-Gd(Ⅲ) conjugates. Further structural introduction of a space linker between β-galactose and TPE is necessary to develop β-galactosidase-responsive TPE-Gd(Ⅲ) analogues.

  3.   Conclusions and outlook

  This review provides a comprehensive summary of β-galactosidase-activated Gd(Ⅲ) agents in the broad field of developing bioresponsive MR imaging probes for diverse potential applications such as gene expression, gene therapy, senescence, osteoarthritis, and tumors (Table 1). These agents showed enzymatically responsive structural changes to effectively modulate the q or τ_r parameter of Gd(Ⅲ), corresponding to different MR imaging signals. Several probes have been successfully employed for in vivo evaluation of gene therapy and detection of tumors.

  Table 1

  

  Table 1.  Summary of β-galactosidase-activated Gd(Ⅲ) probes for MR imaging

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  Gd(Ⅲ) chelate		Responsive mechanism	q (off/on)	r1 / (mmol-1·L·s-1) (off/on)	Magnetic field strength / T	Model	Ref.
  q-modulated agents	1	Steric blocking effect	0.7/1.2	n.d.*/n.d.	n.d.	Solution	[79]
  	2	Steric blocking effect	0.65/1.02	0.903/2.72	1.41	living X. laevis embryos	[59]
  	3	Decrease in molecular weight	0.95/1.3	7.8/4.2	1.41	HeLa cells	[81]
  	4	Coordinating effect	0.1/n.d.	3.3/4.9	1.41	LacZ mice	[60]
  		Coordinating effect	0.1/n.d.	3.3/4.9	1.41	Pig knee specimens	[74]
  	5	Coordinating effect	n.d./n.d.	1.8/3.8	1.41	Normal mice	[34]
  τr-modulated agents	6	Enzymatic self-immobilizing effect	1.10/n.d.	3.96/3.77 in PBS, 7.58/15.5 in the presence of HSA	0.5	CT26 tumor-bearing mice	[92]
  	7	Enzymatic self-immobilizing effect	n.d./n.d.	6.2/5.6 in PBS, 6.5/9.0 in the presence of HSA	1.41	GM1 gangliosidosis mice	[61]
  	8	Enzymatic self-immobilizing effect	n.d./n.d.	8.3/14.2	0.5	SKOV3 tumor-bearing mice	[62]
  	9	Enzymatic self-immobilizing effect	n.d./n.d.	8.67/13.85	0.5	SKOV3 tumor-bearing mice	[63]
  	10	Enzymatic polymerization	n.d./n.d.	n.d./n.d.	n.d.	Murine melanoma tumor-bearing mice	[94]
  	11	Enzymatic receptor-induced magnetization enhancement	n.d./n.d.	5.35/3.87 in PBS, 6.06/9.51 in the presence of HSA	0.5	Solution	[93]
  	12	Aggregation-induced enhancement	n.d./n.d.	7.19/n.d.	1.41	Solution	[95]
  	13	Aggregation-induced enhancement	n.d./n.d.	n.d./n.d.	n.d.	Solution	[95]
  *n.d.: not described.

  Despite these advances, there are significant challenges to practical translation. For instance, some agents showed delivery-method-dependent enzymatic MR imaging behaviors, which showed promising results under non-clinical-use injection methods such as intrathecal or intraperitoneal administration but displayed undesirable imaging performance upon intravenous injection. Further structural optimization strategies are desired for the development of translatable Gd(Ⅲ) agents. For example, the introduction of a multivalent effect into current β-galactosidase-activated Gd(Ⅲ) platforms by combining multiple responsive or targeting moieties into one scaffold provides additional potential in enhancing the specificity of MR imaging. The stability of the Gd(Ⅲ) chelates is closely related to the biosafety profiles and the potential for translation. The linear clinically approved Gd(Ⅲ) chelates such as Gd-DTPA (Magnevist) have been suspended by the European Medicines Agency (EMA). Comparatively, the macrocyclic clinically approved Gd(Ⅲ) chelates such as Gd-DOTA (Dotarem) show better stability with reduced biosafety concerns. Hence, most reported bioresponsive Gd(Ⅲ) chelates are macrocyclic agents. The renal clearance pathway has usually been observed for these highly water-soluble Gd(Ⅲ) probes. The dose for clinical MR scans is dependent on the MR relaxivities of the Gd(Ⅲ) agent. Most clinically approved Gd(Ⅲ) chelates display MR relaxivities of about 4-5 mmol^-1·L·s^-1 per Gd, requiring a clinical dose of 0.1 mmol·kg^-1. However, the newly US Food and Drug Administration (FDA)-approved Gd(Ⅲ) agent (gadoquatrane, 2026) employs a reduced dose of 0.04 mmol·kg^-1 due to the increased MR relaxivity of 11.8 mmol^-1·L·s^-1 per Gd. It′s highly desirable to develop new Gd(Ⅲ) agents with further enhanced MR relaxivity and to reduce the dose for clinical MR scans. Another aspect is the response kinetics of the Gd(Ⅲ) agents. It usually shows faster bioresponses in vivo compared to the solutions. However, in vivo monitoring of the details of the response kinetics is still challenging. Besides, the specificity of the agents is determined by the unique bioresponsive units, which can be modulated according to the specific targeting biomarkers in diseases. Hence, the β-galactosidase-activated Gd(Ⅲ) complexes summarized in this work can also be extended to detect other biomarkers via the replacement of the β-galactosidase-specific galactosyl group.

  
  Acknowledgements: The authors are grateful for the support from the Fundamental Research Funds for the Central Universities (Grants No.E4EQ4001, E5ER4001), the National Natural Science Foundation of China (Grants No.22504147, E4144001, E5244001), and the Key R&D Program of Shandong Province, China (Grants No.2025CXPT139). Declaration of competing interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
  Data availability: No data were used for the research described in the article.
  
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  Figure 1  Schematic view of the q-modulated and τ_r-modulated pathways for the construction of β-galactosidase-responsive Gd(Ⅲ) complexes for MR imaging

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  Figure 2  Representative q-modulated Gd(Ⅲ) complexes

  The blue color shows the β-galactosidase recognition site. The purple color indicates the bridging linker. The black color reveals the Gd(Ⅲ) MR unit. Probe 1 is the first β-galactosidase-activated Gd(Ⅲ) complex (EGad) using an ethoxy linkage to combine a galactosyl group with a DO3A-Gd MR moiety^[79]. Complex 2 introduces an α-methyl group to the ethoxy linker in EGad with an enhanced MR relaxivity^[59]. Probe 3 employs a carbamate--based β-galactosidase-activated self-immolative unit between a galactosyl moiety and a DO3A-Gd MR group^[81]. Complex 4 attaches a pendant coordinating carboxylate ligand to the self-immolative carbamate-based chelates for in vivo detection of β-galactosidase activity^[60]. Complex 5 is a pyridyl-carbamate unit based Gd(Ⅲ) with large β-galactosidase-responsive “on-off” ratio of MR relaxivity changes^[34].

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  Figure 3  β-galactosidase-activated Gd(Ⅲ) probe 4 for in vivo detection of enzyme activity: (A) molecular structure changes post β-galactosidase enzyme activation^[60,74]; (B) in vivo MR imaging of LacZ transgenic mice upon intraperitoneal injection of 4 at pre-injection, 1 h, 4 h, and 24 h^[60] (Copyright 2020, Wiley-VCH GmbH); (C) senescent cells (green) and viable cells (orange) implanted in the distal femur of a live pig; (D) representative T₁-weighted MR image of the pig knee joint upon intra-articular injection of 4; (E) corresponding T₁ color-mapping in D; (F) corresponding R₁ relaxation rates of senescent and viable cell implants; (G) signal-to-noise ratios (SNR) analysis of senescent and viable cell implants; (H) senescent cells (green) and viable cells (orange) implanted in the excised distal femur with cartilage defects; (I) representative T₁-weighted MR image of the explant; (J) corresponding T₁ color-mapping in I; (K) R₁ relaxation rates of the implants; (L) SNR analysis of the implants (*p<0.05, **p<0.005)^[74] (Copyright 2025, Springer Nature)

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  Figure 4  Representative τ_r-modulated Gd(Ⅲ) complexes

  The blue color shows the β-galactosidase recognition site. The purple color indicates the bridging linker. The black color reveals the Gd(Ⅲ) MR unit. Complex 6 is a difluoromethylphenyl-β-galactopyranoside conjugated Gd(Ⅲ) agent for β-galactosidase-expressed CT26 tumor MR imaging^[92]. Complex 7 contains a fluoromethyl pendant group to generate a protein-binding electrophilic quinone methide intermediate for MR imaging of AAV gene therapy^[61]. Complex 8 is a β-galactosidase-responsive self-immobilizing NIR fluorescence and MR imaging bimodal agent^[62]. Complex 9 represents a β-galactosidase-activated NIR fluorescence and ¹H/¹⁹F MR imaging trimodal chelate for in vivo detection of ovarian cancer^[63]. Probe 10 is a tyrosine-β-galactopyranoside functionalized Gd(Ⅲ), generating Gd(Ⅲ)-containing melanin oligomers or polymers upon the β-galactosidase and tyrosinase enzymatic reactions^[94]. Probe 11 employs a receptor-induced magnetization enhancement (RIME) mechanism to increase protein binding affinity by changing the hydrophobicity of the agent post-enzymatic activation^[93]. Complexes 12 and 13 are the β-galactopyranoside-TPE-Gd(Ⅲ) based probes to form large-sized aggregates after enzyme activation^[95].

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  Figure 5  β-galactosidase-catalyzed self-immobilizing strategy enabled τ_r-modulated MR agent for real-time tracking of AAV gene therapy in GM1 gangliosidosis mice: (A) schematic review of β-galactosidase-activated covalent binding of Gd(Ⅲ) chelate to large bio-nucleophiles, leading to a slower molecular tumbling rate and a higher MR relaxivity; (B) MR imaging of GM1 mice (Glb1) and AAV-treated GM1 gangliosidosis mice (AAV-treated Glb1) upon intravenousⅣ injection of Gd(Ⅲ) probe 7; (C) kinetic MR signal changes in the liver slices^[61] (Copyright 2026, American Chemical Society)

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  Figure 6  β-galactosidase-catalyzed self-immobilizing strategy-enabled τ_r-modulated MR agent for NIR fluorescence and MR bimodal imaging: (A) chemical structural changes of 8 regarding β-galactosidase-catalyzed fluorogenic reaction and self-immobilizing pathways; (B) fluorescence imaging of β-galactosidase-expressing SKOV3 tumor-bearing mice via intravenous injection of 8 or 8-C; (C) corresponding fluorescence intensity changes in B; (D) T₁-weighted MR imaging of β-galactosidase-expressing SKOV3 tumor-bearing mice using intravenous injection of 8 or 8-C under a 9.4 T MR imaging scanner; (E) MR signal enhancements of the tumor area in D^[62] (Copyright 2023, Wiley-VCH GmbH)

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  Figure 7  Several other enzymatic-triggered τ_r-modulation strategies: (A) β-galactosidase-triggered polymerization^[94]; (B) β-galactosidase-activated noncovalent interaction with large-sized biomolecules^[93]

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  Table 1.  Summary of β-galactosidase-activated Gd(Ⅲ) probes for MR imaging

  Gd(Ⅲ) chelate		Responsive mechanism	q (off/on)	r1 / (mmol-1·L·s-1) (off/on)	Magnetic field strength / T	Model	Ref.
  q-modulated agents	1	Steric blocking effect	0.7/1.2	n.d.*/n.d.	n.d.	Solution	[79]
  	2	Steric blocking effect	0.65/1.02	0.903/2.72	1.41	living X. laevis embryos	[59]
  	3	Decrease in molecular weight	0.95/1.3	7.8/4.2	1.41	HeLa cells	[81]
  	4	Coordinating effect	0.1/n.d.	3.3/4.9	1.41	LacZ mice	[60]
  		Coordinating effect	0.1/n.d.	3.3/4.9	1.41	Pig knee specimens	[74]
  	5	Coordinating effect	n.d./n.d.	1.8/3.8	1.41	Normal mice	[34]
  τr-modulated agents	6	Enzymatic self-immobilizing effect	1.10/n.d.	3.96/3.77 in PBS, 7.58/15.5 in the presence of HSA	0.5	CT26 tumor-bearing mice	[92]
  	7	Enzymatic self-immobilizing effect	n.d./n.d.	6.2/5.6 in PBS, 6.5/9.0 in the presence of HSA	1.41	GM1 gangliosidosis mice	[61]
  	8	Enzymatic self-immobilizing effect	n.d./n.d.	8.3/14.2	0.5	SKOV3 tumor-bearing mice	[62]
  	9	Enzymatic self-immobilizing effect	n.d./n.d.	8.67/13.85	0.5	SKOV3 tumor-bearing mice	[63]
  	10	Enzymatic polymerization	n.d./n.d.	n.d./n.d.	n.d.	Murine melanoma tumor-bearing mice	[94]
  	11	Enzymatic receptor-induced magnetization enhancement	n.d./n.d.	5.35/3.87 in PBS, 6.06/9.51 in the presence of HSA	0.5	Solution	[93]
  	12	Aggregation-induced enhancement	n.d./n.d.	7.19/n.d.	1.41	Solution	[95]
  	13	Aggregation-induced enhancement	n.d./n.d.	n.d./n.d.	n.d.	Solution	[95]
  *n.d.: not described.

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