High-stability spherical lanthanide nanoclusters for magnetic resonance imaging

Abstract High-nuclear lanthanide clusters have shown great potential for the administration of high-dose mononuclear gadolinium chelates in magnetic resonance imaging (MRI). The development of high-nuclear lanthanide clusters with excellent solubility and high stability in water or solution has been challenging and is very important for expanding the performance of MRI. We used N-methylbenzimidazole-2-methanol (HL) and LnCl3·6H2O to synthesize two spherical lanthanide clusters, Ln32 (Ln = Ho, Ho32; and Ln = Gd, Gd32), which are highly stable in solution. The 24 ligands L− are all distributed on the periphery of Ln32 and tightly wrap the cluster core, ensuring that the cluster is stable. Notably, Ho32 can remain highly stable when bombarded with different ion source energies in HRESI-MS or immersed in an aqueous solution of different pH values for 24 h. The possible formation mechanism of Ho32 was proposed to be Ho(III), (L)− and H2O → Ho3(L)3/Ho3(L)4 → Ho4(L)4/Ho4(L)5 → Ho6(L)6/Ho6(L)7 → Ho16(L)19 → Ho28(L)15 → Ho32(L)24/Ho32(L)21/Ho32(L)23. To the best of our knowledge, this is the first study of the assembly mechanism of spherical high-nuclear lanthanide clusters. Spherical cluster Gd32, a form of highly aggregated Gd(III), exhibits a high longitudinal relaxation rate (1 T, r1 = 265.87 mM−1·s−1). More notably, compared with the clinically used commercial material Gd-DTPA, Gd32 has a clearer and higher-contrast T1-weighted MRI effect in mice bearing 4T1 tumors. This is the first time that high-nuclear lanthanide clusters with high water stability have been utilized for MRI. High-nuclear Gd clusters containing highly aggregated Gd(III) at the molecular level have higher imaging contrast than traditional Gd chelates; thus, using large doses of traditional gadolinium contrast agents can be avoided.


INTRODUCTION
In magnetic resonance imaging (MRI) or spin imaging, the electromagnetic waves generated by energy attenuation differences in different structural environments inside tissue are monitored by an external gradient magnetic field to form an image of the structure inside the tissue [1,2]. Because complex lesions, such as tumors, can be difficult to find and clearly visualize, it is usually necessary to use contrast agents (CAs) to enhance the MRI signal and improve diagnostic capability. The CAs indirectly change the signal intensity of the tissue through internal and external relaxation effects and the magnetic susceptibility effect to increase the difference in pixel intensity between the diseased tissue and the normal tissue [3][4][5].
Discrete mononuclear Gd(III) complexes (Gd-DTPA, Gd-DOTA, etc.) formed by Gd(III), which has a large magnetic moment and a long electron relaxation time, and poly(aminocarboxylate) chelators have been used as clinical MRI CAs [6]. These Gd(III) complexes mainly enhance tissue contrast in MRI by influencing and regulating the relaxation time of internal and external water protons [7][8][9]. Due to the low content of Gd(III), the CAs currently used in clinical practice usually require high doses to achieve effective contrast and resolution. However, the use of high-dose Gd(III)-based CAs has high toxicity that may induce many diseases, such as nephrogenic systemic fibrosis [1]. Effective solutions can be proposed based on the Solomon-Bloembergen-Morgan (SBM) paramagnetic C The Author(s) 2023. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. relaxation theory: multiple single-nuclear or lownuclear gadolinium complexes are connected through multicomponent integration to obtain CAs with high relaxation rate, high resolution and high contrast [10][11][12]. Although some progress has been made, the process is still limited to porous systems such as coordination molecular cages and metal-organic frameworks [13,14]. These systems have limited solubility, and it is difficult to keep them stabilized in body fluids for a long time. In addition, in recent years, iron-based and manganese-based complexes have been developed as CAs to replace gadolinium-based chelates, but their relaxation rate still needs to be improved, and the in vivo toxicity of these complexes is not clear [15][16][17][18]. Coordination-driven self-assembly provides an efficient method for the development of polynuclear Ln-assemblies with rigid structures, high molecular weight, intermediate sizes, and good solubilities, which all facilitate high relaxivity [13,14]. High-nuclear gadolinium clusters with superior solubility and biocompatibility can highly aggregate Gd(III) at the molecular level, which has attractive potential for MRI CAs [13]. Therefore, it is both challenging and important to develop high-nuclear gadolinium cluster aggregates to have water solubility and water stability for MRI uses.
The design and synthesis of high-nuclear lanthanide clusters with specific connections, attractive structures and rich functions have always received extensive attention [19]. To date, many different shapes and types of high-nuclear lanthanide clusters have been constructed by ligand hydrolysis and template methods [20][21][22][23][24][25], such as cage Gd 140 [20], hamburger Dy 76 [21], and tubular Dy 72 [22], and they have been applied to single-molecule magnets, magnetocaloric effects and proton conduction fields [26][27][28]. Although considerable progress has been made, high-nuclear lanthanide clusters are still largely limited to the solid-state [29,30]. Although these solid-state properties are important, the solubility and stability of these clusters in solution are also important factors for the development of other functions, such as bioimaging, therapy and catalysis [31,32]. However, it is not easy to design and synthesize high-nuclear lanthanide clusters with high solubility and stability in solution [33]. Choosing appropriate ligands, wrapping the cluster cores during the self-assembly process and forming a protective effect opens up a new way for the design and synthesis of high-nuclear lanthanide clusters with high stability in solution.
Herein, we reacted N-methylbenzimidazole-2-methanol (HL) with LnCl 3 ·6H 2 O under solvothermal conditions to obtain two spherical lanthanide nanoclusters, Ln 32 (Ln = Ho, Ho 32 ; and Ln = Gd, Gd 32 ). In the Ln 32 structure, the metal centers Ln(III) are all on the spherical surface and are connected with 6 μ 4 -O 2− and 48 μ 3 -OH − , and the ligands L − are located on the outside of the sphere and tightly wrap the cluster core, thereby ensuring the stability of the cluster. HRESI-MS and PXRD jointly verified the stability of spherical cluster Ln 32 in organic solvents and aqueous solutions. The assembly mechanism of Ho 32 is proposed to be Ho(III), (L) − and H 2 O → Ho 3 (L) 3 /Ho 3 (L) 4 → Ho 4 (L) 4 /Ho 4 (L) 5 → Ho 6 (L) 6 /Ho 6 (L) 7 → Ho 16 (L) 19 → Ho 28 (L) 15 → Ho 32 (L) 24 /Ho 32 (L) 21 /Ho 32 (L) 23 . Cluster Gd 32 , which has a high longitudinal relaxation rate and low cytotoxicity, exhibits better MRI imaging contrast than Gd-DTPA at both the solution and cell levels (Scheme 1). The same doses (100 μL) of Gd 32 and Gd-DTPA containing the same Gd(III) ion concentration (0.5 mM) were injected through the tail vein into BALB/c mice carrying the 4T1 tumor model. Notably, compared with Gd-DTPA, Gd 32 results in clearer MRI imaging contrasts and has a greater ability to mark tumors (Scheme 1). In addition, Gd 32 can be cleared from the body in a short time through the kidneys and liver. To the best of our knowledge, this is the first development of high-nuclear gadolinium nanoclusters with highly aggregated Gd(III) as MRI CAs, which effectively avoids the use of high-dose low-nuclear gadolinium chelates.

Synthesis and structure analysis of Ln 32 clusters (Ln = Ho and Gd)
The ligands HL and HoCl 3 ·6H 2 O were allowed to react for 48 h in a closed reaction vessel under solvothermal conditions at 100 • C and then placed in an open glass bottle at room temperature to volatilize for 12 h. Then, block orange crystals of Ho 32 were obtained (Fig. S1). Single crystal X-ray diffraction (SCXRD) structure analysis shows that Ho 32 crystallizes in the C2/c space group of the monoclinic crystal system, and it is a high-nuclear spherical cluster. Ho 32 is composed of a +4 valent cation cluster and four free Cl − ions on the periphery, and its molecular formula is [Ho 32 (L) 24 (μ 3 -OH) 48 (μ 4 -O) 6 Cl 8 ](Cl) 4 ·45H 2 O·5CH 3 OH·2CH 3 CN (Table S1). The cationic cluster contains 32 Ho(III) ions, 24 deprotonated ligands L − , 48 μ 3 -OH − ions formed by the removal of a proton from a water molecule, six μ 4 -O 2− ions formed by the removal of two protons from water molecules, and eight Cl − ions coordinated with end groups (Fig. 1a). Ligand L − is similar to amphiphilic surfactants. The hydrophilic terminal hydroxyl groups coordinate with the metal ions to form a cluster core, while the hydrophobic terminal benzimidazoles are located at the outermost periphery of the cluster, which ensures that the cluster has high stability and good solubility in water. Four eight-coordinated Ho1 and one seven-coordinated Ho2 together constitute the independent unit of Ho 32 , and the eight abovementioned independent units with shared vertices constitute Ho 32 (Fig. 1e). In addition, we only changed the metal salt to GdCl 3 ·6H 2 O and obtained the Gd 32 homolog of Ho 32 under the same conditions.

Stability of Ln 32 (Ln = Ho and Gd)
To explore the functions of a compound, the structure of that compound must be stable [33]. In recent years, HRESI-MS has been widely used to characterize the composition and changes of species in solution, and it has been used to detect the structural stability, fragmentation mechanism, and degree of protonation of clusters [25,33]. The fragment peaks of the Ho 32 single crystal mass spectrum mainly appear in the range of m/z = 1500-3500, and the valence states shown are +3, +4 and +5 (Figs 2a,  S8 and S9 and Table S6). Notably, the above molecular ion peaks with different valences are all generated by the main frame Ho 32 , which can be attributed to [Ho 32 Table S7). Overall, the HRESI-MS test with the Ho 32 crystal under different energies showed that the crystal has very high stability in solution. Likewise, HRESI-MS indicated that Gd 32 has high stability in solution (Figs S11 and S12 and Table S8). To verify the water stability of the giant spherical clusters of Ho 32 , they were immersed in aqueous solutions of different pH values (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) for 24 h and underwent PXRD testing. It is worth noting that Ho 32 remains stable in aqueous solutions with different pH values (Fig. 2b). In the Ho 32 structure, 24 ligands L − wrapped the cluster core and formed a dense protective layer. In addition, 48 μ 3 -OH − and 6 μ 4 -O 2− are tightly connected to the metal center through bridging, leading to a highly stable cluster core. When Ho 32 is attacked by solvent molecules such as H 2 O, the amphiphilic ligand L − (which is similar to surfactants) can effectively resist the attack of solvent molecules through weak supramolecular effects such as hydrogen bonds (Fig. 2c and S13).

Assembly mechanism of Ho 32
HRESI-MS was used to quickly detect the types of molecular ion peaks and their abundance changes in the reaction solution at different time periods to speculate on the most likely self-assembly mechanism of the high-nuclear spherical cluster Ho 32 (Figs 3a, b and S14, Table S9). Figure 3b 23 (Fig. 3c).

In vitro relaxivity and MRI performance of Gd 32
As one of the most established medical imaging techniques, MRI has received great attention [1][2][3][4][5][6][7]. The design and synthesis of CAs is essential for obtaining high-resolution and high imaging contrasts. At present, the most commonly used T 1 -weighted MRI CAs in clinical practice are gadolinium-based organic chelates, which usually require higher doses to obtain excellent imaging contrasts [13,14]. Highnuclear gadolinium clusters can be highly enriched in Gd(III) in the molecule, so they have great potential for T 1 -weighted MRI CAs. To explore the feasibility of Gd 32 as a T 1 -weighted MRI CA, the relaxation time with Gd(III) concentration changes was tested at magnetic field strengths of 1 T and 3 T, and the longitudinal (r 1 ) and transverse (r 2 ) relaxation efficiencies were obtained. The results show that the r 1 and r 2 values of Gd 32 are 265.87 and 324.96 mM −1 ·s −1 at a 1 T magnetic field strength and 250.40 and 306.90 mM −1 ·s −1 at a 3 T magnetic field strength, respectively (Fig. 4a). Cluster Gd 32 with highly aggregated Gd(III) shows a higher relaxation value than traditional Gd chelates; r 2 /r 1 = 1.22 (r 2 /r 1 < 2) indicates that Gd 32 is a potential candidate for T 1 -weighted MRI CAs [16]. As the Gd(III) concentration of Gd 32 in the aqueous solution gradually increases, the T 1 -weighted image gradually becomes brighter with a 1 T magnetic field, and the brightening effect is more obvious with a 3 T magnetic field. In addition, the T 1 -weighted pseudo color images displayed by different concentrations of Gd 32 at 1 T and 3 T both indicate that it has encouraging potential as an MRI CA for biomedical diagnosis ( Fig. 4b and c). Cluster Gd 32 has a good T 1 imaging effect not only in solution but also in cells. Figure 4d shows that as the concentration increased from 0 μM to 23 μM, the T 1 -weighted MRI contrast of Gd 32 and Gd-DTPA on 4T1 cells both increased. However, when the coincubation time was 12 h, 24 h and 48 h, Gd 32 showed a better T 1 -weighted MR imaging effect than Gd-DTPA. Similar results have also been confirmed at a 3 T magnetic field (Fig. 4e).
The above data all illustrate the excellent T 1 imaging ability of the high-nuclear gadolinium cluster (Gd 32 ) because the molecule can become highly enriched in Gd(III). The large cavity and strong hydrogen bonding with H 2 O lead to the ultrahigh T 1 relaxivity of Gd 32 (Fig. S15). The parameters obtained from the NMRD fitting results support the high relaxivity of Gd 32 (Fig. S16 and Table S10). UV−Vis absorption spectroscopy demonstrated that Gd 32 maintained high stability in PBS, serum (FBS), cell culture medium (DMEM) and PBS solution containing endogenous metal ions (Ca 2+ , Mg 2+ , Fe 3+ , Zn 2+ , etc.) (Figs S17 and S18). In addition, Gd 32 exhibits very low cytotoxicity compared with that of cisplatin, and Gd 32 can be cleared by the kidney and liver in a short time in mice, which indicates its potential for application in the field of biomedical imaging (Figs S19-S22).

In vivo tumor MRI performance and biodistribution of Gd 32
To evaluate the effects of Gd 32 on the MRI of CAs in animal models, we constructed BALB/c mice carrying 4T1 tumors for in vivo MRI experiments (Fig. 5). The cluster Gd 32 was injected into the experimental mice through the tail vein (100 μL, 0.5 mM Gd(III) ions). As shown in Fig. 5a, from 0 h to 12 h, continuous enhancement of T 1 imaging contrast at the tumor site was observed, indicating that Gd 32 can be effectively enriched at the tumor site. It can be seen from the relative MR signal value of the tumor that at 4 h, Gd 32 had high enrichment at the tumor site, exhibiting a good T 1 imaging effect. Unlike other small-particle contrast agents that are easily metabolized, Gd 32 can still achieve good contrast effects 8 h after injection, and the best contrast effects are achieved at 12 h. The signal of the tumor site at 12 h reached 1.49 times the tumor signal of the blank (Fig. 5d)  of Gd-DTPA at the tumor site gradually increased, and the best T 1 imaging effect was achieved at 2 h (Fig. 5c); however, at 2 h, the signal at the tumor site was only 1.17 times the tumor signal in the blank (Fig. 5d). The above data show that under the condition of the same extremely small dose of CAs, Gd 32 shows a far better T 1 -weighted MRI imaging effect than Gd-DTPA. Similar results appeared in 3 T MRI (Fig. S23). Excellent MRI CAs can be effectively and rapidly metabolized, which prevents the toxicity and enrichment of heavy metal ions in living bodies. Therefore, we explored the metabolism and biodistribution of Gd 32 in mice. After injecting Gd 32 through the tail vein, T 1 -weighted MRI images of the mouse kidney and liver were collected at different time points (0, 4, 8, 12 and 24 h) ( Fig. 5a and b). As shown in Fig. 5a, as the injection time increased from 0 h to 12 h, the enrichment of Gd 32 in the kidney gradually increased, and the yellow−green color gradually increased also. It is worth noting that the T 1 -weighted MRI image of Gd 32 on the kidney gradually weakened after the injection time was further increased to 24 h, indicating that it was gradually metabolized. In addition, monitoring the intensity changes in MRI images of the liver of mice after injection of Gd 32 at different time points obtained similar results, which shows that the liver can also metabolize part of Gd 32 . In general, the content of Gd 32 in the kidneys of mice is higher than that in the liver, indicating that ultrasmall Gd 32 is mainly eliminated by the kidney (Fig. 5e) [34]. Although Gd 32 with high positive charge that is diluted by mouse body fluids and easily combined with negatively charged biological macromolecules in the body, resulting in in vivo imaging effects that are not as good as those of solutions, there are still obvious T 1 imaging effects in vivo. The above results indicate that Gd 32 is an ideal candidate for MRI CAs and has great application prospects in clinical tumor diagnosis. In addition, high-nuclear Gd clusters with highly aggregated Gd(III) at the molecular level have higher imaging contrast than traditional Gd chelates, which effectively prevents the need to use large doses of traditional CAs.

CONCLUSIONS
In summary, we report two spherical high-nucleus lanthanide nanoclusters with high stability in solution. Crystallography, HRESI-MS and PXRD jointly confirmed the high stability of Ho 32 under solution conditions. Time-dependent HRESI-MS tracked the formation process of Ho 32 , a variety of different types of intermediates were screened, and the gradual assembly formation mechanism of spherical clusters was proposed for the first time. The excellent water solubility and water stability of Gd 32 prompted us to explore its potential applications in the field of biomedicine. Notably, Gd 32 , a nanocluster with low toxicity, high biocompatibility, and a high relaxation rate, shows excellent T 1 -weighted MRI effects at the cell and animal levels. The gadolinium-based nanocluster Gd 32 with highly aggregated Gd(III) has significantly better MRI imaging contrast than the clinically used commercial CA Gd-DTPA, which effectively prevents the need to use a large dose of traditional gadolinium contrast agents. To the best of our knowledge, this is the first study to explore the application of high-nuclear lanthanide clusters in the field of MRI. This work provides a detailed example of the construction of lanthanide clusters with high stability and high water solubility. In addition, this work also opens a door to study the performance of lanthanide clusters in solution.

Synthesis of Ln 32
A mixture of HL (0.1 mmol, 148 mg), HoCl 3 ·6H 2 O (0.5 mmol, 189.7 mg) and 250 μL TEA was dissolved in 5 mL MeOH and 5 mL MeCN. Then, the solution was stirred for 0.5 h. Next, the solution was transferred to a Teflon container in a stainless-steel bomb and kept at 100 • C in the oven for 48 h. The solution was then filtered and allowed to stand until evaporation.