Ligand design and synthesis

In order to weaken the intramolecular hydrogen bond formed between the amide and hydroxide groups found in many tetradentate HOPO ligands, a ligand {N,N′-[oxybis(ethane-2,1-diyl)]bis[2-(3-hydroxy-2-oxopyridin-1(2H)-yl) acetamide], denoted as 5LIO-1-Cm-3,2-HOPO, was designed by introducing the carboxylic group on the N site of pyridine ring. The distance between the amide and carbonyl group is increased by the addition of a methyl group. 5LIO-1-Cm-3,2-HOPO was obtained from a 4-step synthesis as illustrated in Fig. 125,26,27. Commercially available 1,2-dihydro-2,3-pyridinediol was first alkylated at the nitrogen position of the pyridinone ring by reacting it with excess amounts of ethyl bromoacetate at 150 °C to yield the ester (product A). Next, the hydroxyl group in A was protected with a benzyl moiety, and the ester group was activated via hydrolysis to afford product B. 5LIO-1-Cm-3,2-HOPOBn was then obtained via amidation of the carboxylate group with the amine backbone. Benzyl protection was finally removed by 5% Pd/C to obtain 5LIO-1-Cm-3,2-HOPO with a total yield of 43%. The chemical shifts observed in the spectra of 1H NMR and 13C NMR all corroborate with the chemical structure of the molecules, and the results of elemental analysis and LC-MS are consistent with the formula of 5LIO-1-Cm-3,2-HOPO (Supplementary Figure 1). In the FTIR spectra, the pattern of 5LIO-1-Cm-3,2-HOPO contains peaks at 2947 cm−1 and 1080 cm−1, corresponding to -CH 2 group and the symmetric stretch of C–O-C, respectively (Supplementary Figure 2a).

Fig. 1 Synthesis procedure for 5LIO-1-Cm-3,2-HOPO. i: Br-CH 2 COOC 2 H 5 , 150 oC, 24 h; ii: BnCl, NaOH, 80 oC, 12 h; iii: NHS/EDC, 1,5-diamino-3-oxapentane, 30 oC, 12 h; iv: Pd/C, H 2 , 30 oC, 4 h Full size image

Solution thermodynamic studies

The solution thermodynamic data of 5LIO-1-Cm-3,2-HOPO was first measured to evaluate its complexation behavior with U(VI). The protonation constants of the free ligand, 5LIO-1-Cm-3,2-HOPO (denoted as LH 2 in this part), were determined by potentiometric titrations. The formation constants could be calculated with equation (1) in the experimental section (Table 1). The two protonation constants, pK a1 and pK a2, were measured to be 8.3(5) and 9.3(4), respectively.

Table 1 Protonation Constants of Ligands and Uranyl Chelation Stability Constants Full size table

The formation constants of UO 2 -L were measured by competition titration with EDTA28. Uranyl hydrolysis was taken into consideration for the refinement of the cumulative constants of the UO 2 -L complexes, logβ mlh calculated by equation (2). The stability constants of the UO 2 -L complexes, logβ mlh , were established as logβ 111 = 24.8(7), logβ 110 = 18.6(7), and logβ 11–1 = 7.5(7) (Table 1). These formation constants are noticeably higher than those of the previously reported and most optimal ligand, 5LIO-(Me-3,2-HOPO) (denoted as LcH 2 )29. Only one mixed TAM-HOPO ligand has been reported with higher stability constants (logβ 111 = 19.75 and logβ 11–1 = 11.92), probably owing to the high affinity of TAM moiety for uranyl30. Additionally, the formation constants of 5LIO-1-Cm-3,2-HOPO complexes containing biological trace elements were also measured, including Zn–L (logβ 111 = 15.9(6), logβ 110 = 9.8(7)), Cu–L (logβ 111 = 16.6(5), logβ 110 = 9.7(4)), Ca–L (logβ 111 = 13.9(3), logβ 110 = 5.7(2)), and Mg–L (logβ 110 = 4.6(4)) (Supplementary Table 1). These values are significantly lower than the formation constant of UO 2 -L, indicating that 5LIO-1-Cm-3,2-HOPO is a highly selective sequestration ligand for uranyl. Furthermore, the species distribution of the M–L system based on their formation constants was calculated with Hyss at the defined condition of 10−3 M (L) and 10−4 M (metal ion) from pH 3.0 to 11.0 (Supplementary Figure 3). Taking the formation of uranyl-hydroxide and uranyl-carbonate complexes into consideration, the speciation diagram of the M–L system illustrates that at physiological pH (7.4), UO 2 L (94.0%) and UO 2 LH+ (5.9%) are the only uranyl complexes in solution. In comparison, only 2.5% MgL, 56.3% CaLH+, and 7.6% CaL are present in solution, suggesting that 5LIO-1-Cm-3,2-HOPO barely bind to Ca2+ or Mg2+ at physiological pH (7.4). Furthermore, the low formation constants of ZnL and CuL demonstrate that it is unlikely that ZnL and CuL complexes can form in the presence of uranyl ion, despite that 94.0% ZnL, 24.0% CuLH+, and 75.9% CuL exist in solution at pH 7.4 in the absence of uranyl ion. The high solubility of UO 2 L and UO 2 LH+ suggests that they can be easily transported and rapidly excreted from the body. Moreover, the pUO 2 value of L, which is an assessment of the ligand affinity, was found to be 16.6(5). This value is obviously larger than those of other tetradentate HOPO ligands including 5LIO-(Me-3,2-HOPO), and potentially assigns 5LIO-1-Cm-3,2-HOPO as one of the most efficient chelators for uranyl ions29,30,31.

Characterizations of UO 2 −5LIO-1-Cm-3,2-HOPO Complex

The 1H NMR and 13C NMR spectra of the complex in DMSO-d 6 were collected (Supplementary Figure 1f). A slight difference between the chemical shifts of the carbon atoms from UO 2 −5LIO-1-Cm-3,2-HOPO complex and from the ligand can be observed in the 13C NMR spectra collected in DMSO-d 6 . More notably, the feature at 8.97 ppm in 1H NMR spectrum, which can be observed for the raw ligand and assigned to the hydroxyl group, disappears for the UO 2 -5LIO-1-Cm-3,2-HOPO complex, initially suggesting the complexation between the ligand and U(VI). In comparison with the FTIR spectrum of 5LIO-1-Cm-3,2-HOPO, the spectrum of UO 2 -5LIO-1-Cm-3,2-HOPO exhibits an additional peak at 899 cm−1 attributed to the uranyl group (Supplementary Figure 2b). A significant intensity reduction of the peak at 3282 cm−1 assigned to the hydroxyl group was observed for the spectrum of UO 2 −5LIO-1-Cm-3,2-HOPO, when compared with that of 5LIO-1-Cm-3,2-HOPO. Nevertheless, much more powerful evidence comes from the elemental analysis and LC-MS analysis, confirming the formation of UO 2 -5LIO-1-Cm-3,2-HOPO complex with a metal to ligand molar ratio of 1:1 (Supplementary Figure 1f).

Extended X-ray adsorption fine structure (EXAFS)

To characterize the local coordination environment of the uranyl ion in these complexes, a solid sample of UO 2 -5LIO-1-Cm-3,2-HOPO was precipitated from a mixture of methanol and water, and was analyzed using synchrotron radiation EXAFS technique. The EXAFS spectra for the solid samples of UO 2 (NO 3 ) 2 and UO 2 -5LIO-1-Cm-3,2-HOPO contain two distinct oxygen coordination shells: axial oxygen, O ax , and equatorial oxygen, O eq (Supplementary Figure 4). The refinement results are provided in Supplementary Table 2, listing all structural parameters, such as coordination number (CN), bonds distance (R), and the Debye/Waller factor (σ2). Within the experimental error, the coordination environment of uranium contains 2.0–2.2 O ax atoms at bond distances ranging from 1.77–1.82 Å, and 4.70 ± 0.60 O eq atoms at distances ranging from 2.41–2.48 Å. Notably, the average U–O eq bond distance in UO 2 -5LIO-1-Cm-3,2-HOPO (2.41 Å) is shorter than that of UO 2 (NO 3 ) 2 (2.48 Å), whereas the U–O ax bond distance in UO 2 -5LIO-1-Cm-3,2-HOPO (1.82 Å) is longer than that of UO 2 (NO 3 ) 2 (1.77 Å). These come as a result of the change in coordination numbers between UO 2 -5LIO-1-Cm-3,2-HOPO (4.7 ± 0.6) and UO 2 (NO 3 ) 2 (5.6 ± 0.6). The calculated bond distances of UO 2 -5LIO-1-Cm-3,2-HOPO are in good agreement with the structural data reported of the uranyl hydroxypyridinone compounds25,32,33.

Density functional theory (DFT)

We performed DFT calculations to reveal the effect of intramolecular hydrogen bonds on the interaction between the ligand and U(VI). In 5LIO-(Me-3,2-HOPO), the strong intramolecular hydrogen bond (approximately 1.96 Å) between the amide group and the hydroxyl group of the pyridinone ring promotes the formation of a planar local structure between the two bonding components (Fig. 2a). This strong hydrogen bond and local planar structure remain intact when chelating a UO 2 2+ cation (Fig. 2b). On the other hand, for 5LIO-1-Cm-3,2-HOPO, we obtained two different stable structures, one containing–NH···N (pyridine) intramolecular hydrogen bonds and the other containing –NH···O (pyridinone) intramolecular hydrogen bonds [Fig. 2c State I and State II]. Stable structure I is more energetically favorable than II by a relatively small energy difference of approximately 0.96 kcal mol−1. The calculated transition state (Fig. 2c) between the two structures shows a 1.37 kcal mol−1 energy barrier for this structural transformation. Such a low energy barrier and small energy difference imply that the C–N and C–C bonds on either side of the methylene group can freely rotate and that the two types of intramolecular hydrogen bonds in 5LIO-1-Cm-3,2-HOPO can spontaneously transform at room temperature. Compared to 5LIO-(Me-3,2-HOPO), the increase in the local degrees of freedom of 5LIO-1-Cm-3,2-HOPO can be attributed to the cooperation of the inversion of the pyridinone ring and the addition of the methylene group, leading to the weakening of the intramolecular hydrogen bonds (Fig. 2c). We then calculated the Gibbs free energy change for the deprotonation reactions (ΔG depro ) of the hydroxyl group in the pyridinone ring within both ligands. The ΔG depro values of 5LIO-1-Cm-3,2-HOPO [36.87 and 37.26 kcal mol−1 for I and II, respectively] are both larger than that of 5LIO-(Me-3,2-HOPO) (29.44 kcal mol−1), offering a qualitative explanation for the relatively larger pK a value for 5LIO-1-Cm-3,2-HOPO determined experimentally.

Fig. 2 DFT optimized structures, geometric parameters, and relevant energy information. a 5LIO-(Me-3,2-HOPO). b The UO 2 -5LIO-(Me-3,2-HOPO) chelate. c State I and State II show two types of stable 5LIO-1-Cm-3,2-HOPO states containing –NH···N (pyridine) and –NH···O (pyridinone) intramolecular hydrogen bonds, respectively. Transition state represents the transition state between State I and State II. The inset energy diagram shows the energy relationship for all states. d Two types of UO 2 -5LIO-1-Cm-3,2-HOPO chelates. The left structure contains two –NH···N (pyridine) intramolecular hydrogen bonds, whereas the right contains one –NH···O (pyridinone) intramolecular hydrogen bond and one pyridine –NH···N intramolecular hydrogen bond. The gray, white, red, blue, and yellow spheres represent C, H, O, N, and U atoms, respectively. The pink dotted lines represent hydrogen bonds. ΔG(depro) in (a, c) denotes the Gibbs free energy changes of the deprotonated reactions. E b in (b, d) represents the binding energies between the UO 2 2+ cations and the chelating ligands. The U–O eq distances in b and d were calculated by averaging the four U–O distances between uranium and oxygen atoms from the chelating agents. Source Data are provided as Supplementary Data 1–3 Full size image

Fig. 2d displays two stable UO 2 -5LIO-1-Cm-3,2-HOPO chelates, where one contains two –NH···N (pyridine) hydrogen bonds (left) while the other contains one –NH···O (pyridinone) and one –NH···N (pyridine) hydrogen bond (right). The calculated geometric parameters of both complexes (U–O ax = 1.80 Å, U–O eq = 2.41 Å and U–O ax = 1.80 Å, U–O eq = 2.42 Å) are consistent with the EXAFS measurements (U–O ax = 1.82 Å, U–O eq = 2.41 Å; see Supplementary Table 2). The small difference (~0.02 eV) in the values of their binding energies between the UO 2 2+ cation and the chelator indicates that alteration of the hydrogen-bonding scheme would not drastically affect the complexing ability of the ligand. The intramolecular hydrogen bonds transformation and minor change of the chain folding form are not expected to significantly affect the binding energy (−9.23/−9.21 eV). In contrast, the binding energy difference between UO 2 -5LIO-(Me-3,2-HOPO) and UO 2 -5LIO-1-Cm-3,2-HOPO (−8.81 eV vs −9.23/−9.21 eV) is obvious. Therefore, this calculation result reveals the enhancement of the binding ability of the 5LIO-1-Cm-3,2-HOPO ligand, especially the oxygen denticity.

We propose that the enhanced uranyl binding in 5LIO-1-Cm-3,2-HOPO originates from its intrinsic structural and electronic features. From a classical point of view, the negatively charged oxygen of the deprotonated hydroxyl group is strongly attracted to the hydrogen atom of the nearby amide in 5LIO-(Me-3,2-HOPO), thereby generating a strong –NH···O (pyridine) hydrogen bond (Fig. 3a). In contrast, when the pyridinone ring is reversed, the negatively charged oxygen of the deprotonated hydroxyl group in 5LIO-1-Cm-3,2-HOPO can be completely exposed to the environment without any intramolecular interactions (Fig. 3b). This intrinsic structural feature is expected to endow a spatial advantage in coordinating the positively charged UO 2 2+ cation. From a quantum perspective, DFT calculations show that, for both ligands, the global electrostatic potential (ESP) minima over the electron density surface are located between the two oxygen atoms of the pyridinone ring (Fig. 3c, d). Consequently, these two oxygen atoms act as the targeted chelating sites during complexation reactions. The calculated ESP minimum of 5LIO-1-Cm-3,2-HOPO (−189.21 kcal mol−1) is lower than that of 5LIO-(Me-3,2-HOPO) (−178.25 kcal mol−1), indicating that 5LIO-1-Cm-3,2-HOPO could provide more effective long-range electrostatic attractions for UO 2 2+. Moreover, the negative ESP area originating from the two oxygen atoms is further broadened over the electron density surface in 5LIO-1-Cm-3,2-HOPO (46.07 Å2) than in 5LIO-(Me-3,2-HOPO) (38.67 Å2). This observation indicates that 5LIO-1-Cm-3,2-HOPO could provide a wider effective landing surface region for UO 2 2+ complexation. These calculation results indicate that the exposure of the deprotonated hydroxyl group and the weak and transformable intramolecular hydrogen bond synergistically contributes to the unique distribution of the negative ESP between the two oxygen atoms. To help directly visualize and compare the ESP features, we depicted the two ESP isosurfaces for the two ligands, at a same value of +/− 188.25 kcal mol−1, in Fig. 3e, f. Clearly, the negative ESP distribution space is much larger in 5LIO-1-Cm-3,2-HOPO than in 5LIO-(Me-3,2-HOPO). In addition, Morokuma scheme energy decomposition analyses (EDA) results show that the E(elstat) term in UO 2 -5LIO-1-Cm-3,2-HOPO is ca. 1.16 eV larger than that in UO 2 -5LIO-(Me-3,2-HOPO). This result confirms our perspective that the electrostatic effect of 5LIO-1-Cm-3,2-HOPO can be fully released.

Fig. 3 The structural and electrostatic features of 5LIO-(Me-3,2-HOPO) and 5LIO-1-Cm-3,2-HOPO. a, b Represent the deprotonated structures of 5LIO-(Me-3,2-HOPO) and 5LIO-1-Cm-3,2-HOPO, respectively. The red dotted boxes highlight the inverted carbonyl group and deprotonated hydroxyl group of the pyridinone ring. c, d Represent the ESP distributed on the electron density surface of a, b (isodensity = 0.001 a.u.). The arrows denote the global ESP minima. The red dotted boxes surround the major negative ESP areas. The negative ESP areas contributed by the two oxygen atoms were calculated and are shown below (c, d). e, f Represent the ESP isosurfaces at +/− 188.25 kcal mol−1. The blue dotted boxes surround the major spatial distribution regions at the specific ESP isosurface Full size image

Most surprisingly, DFT calculations further reveal an important interaction between the axial oxygen of uranyl and the side chain of 5LIO-1-Cm-3,2-HOPO in the UO 2 -5LIO-1-Cm-3,2-HOPO complex. For 5LIO-1-Cm-3,2-HOPO, the amide group could simultaneously form an intramolecular hydrogen bond with the oxygen of pyridinone ring, as well as an intermolecular hydrogen bond with the axial oxygen of the uranyl ion (Fig. 4a, b). The bond distance of the intramolecular hydrogen bond (–NH···O (pyridinone), 2.05 Å) is shorter than the intermolecular hydrogen bond (–NH···O (uranyl), 2.39 Å), showing relatively stronger intramolecular hydrogen bonding interactions in the former. This is mainly because of the electrostatic advantage of the oxygen of the pyridinone ring (Fig. 3d). Meanwhile, an intermolecular hydrogen bond between the methylene and the axial oxygen of uranyl can be found in the UO 2 -5LIO-1-Cm-3,2-HOPO complex. The longer –CH···O (uranyl) hydrogen bond distance (2.71 Å) reveals that this hydrogen bonding interaction is relatively weaker. Furthermore, we performed reduced density gradient (RDG) analyses based on the ground state electron density of the UO 2 -5LIO-1-Cm-3,2-HOPO complex. The small RDG values (blue areas, arrows point) between H and O (uranyl) clearly confirmed the two types of hydrogen bonding interactions, as shown in Fig. 4c. Formation of the two intermolecular hydrogen bonds are mainly attributed to the longer and more flexible side chain of the 5LIO-1-Cm-3,2-HOPO chelator. In contrast, for 5LIO-(Me-3,2-HOPO), the side chain of this chelator is relative shorter and more rigid, thus only intramolecular –NH···O (pyridine) hydrogen bonds can be formed (Fig. 2b).

Fig. 4 The intramolecular hydrogen bonds and RDG analysis between 5LIO-(Me-3,2-HOPO)/ 5LIO-1-Cm-3,2-HOPO and U(VI). a Illustration of the intramolecular –NH···O (pyridinone) hydrogen bonds in the UO 2 -5LIO-(Me-3,2-HOPO) complex. b Illustration of the intramolecular –NH···N (pyridine) and –NH···O (pyridinone) hydrogen bonds and intermolecular –NH···O (uranyl) hydrogen bonds in the UO 2 -5LIO-1-Cm-3,2-HOPO complex; c the RDG color-filled maps of the N–H–O(uranyl) and sections of b, respectively. The hydrogen bond distances in (a, b) are consistent with the structures of Fig. 3b (right) and d, respectively Full size image

In order to quantitatively evaluate the bonding contributions of the intermolecular hydrogen bond, we also performed topology analyses for the UO 2 -5LIO-1-Cm-3,2-HOPO complex (Fig. 2d, right). The UO 2 -5LIO-(Me-3,2-HOPO) complex (Fig. 2b) was also subjected to this analysis for comparison. Based on the quantum theory of atoms in molecules (QTAIM), bond critical points (BCP) between relevant H and O atoms were found in the ground state electron densities, and the potential energy density, V(r), of these BCPs were calculated. According to the relationship between V(r) and the hydrogen bond energy, EHB34, the EHB can be estimated as EHB = V(r BCP ) / 2. It can be seen that the EHB value of the intramolecular –NH···O (pyridinone) hydrogen bond in UO 2 -5LIO-(Me-3,2-HOPO) is about -0.36 eV, signifying a relatively stronger hydrogen bond interaction. This intramolecular –NH···O (pyridinone) hydrogen bond was significantly weakened to −0.27 eV in UO 2 -5LIO-1-Cm-3,2-HOPO. The EHB values of the intermolecular –NH···O (uranyl) hydrogen bonds is −0.11 eV, implying relatively weaker intermolecular hydrogen bond interactions (Supplementary Table 3). Although the binding contributions (−0.16 eV in total) of the intermolecular hydrogen bond is relatively small compared to the total binding energy (−9.21 eV), it can be expected that the intermolecular hydrogen bond could provide an additional driving force for the coordination of uranyl and therefore enhance the chelating ability of 5LIO-1-Cm-3,2-HOPO.

Cytotoxicity of chelating agents

Given the high affinity of 5LIO-1-Cm-3,2-HOPO for uranyl, the U(VI) sequestration performance and toxicity of 5LIO-1-Cm-3,2-HOPO were evaluated and compared with those of the clinically-used ZnNa 3 -DTPA and the previously reported most optimal tetradentate ligand 5LIO-(Me-3,2-HOPO) in vitro and in vivo. Renal injury is one of the major concerns in the case of uranium contamination. Therefore, U(VI) uptake and release assays were first conducted using renal proximal tubular epithelial cells from rat (NRK-52E cells). The toxicity assay of U(VI) with chelation therapy agent has been performed, and 12.4 μM was considered as the acceptable concentration for the following cellular assays35. Then, a comprehensive toxicity assay of U(VI) and chelating agents was performed by adding 12.4 μM U(VI) and different concentrations of chelating agents ranging from 20.0 to 320.0 μM. The results show that the comprehensive toxicity of UO 2 -5LIO-1-Cm-3,2-HOPO is slightly lower than that of UO 2 -ZnNa 3 -DTPA at low dosage, and notably lower than that of UO 2 -5LIO-(Me-3,2-HOPO) (Supplementary Table 4, Fig. 5a).

Fig. 5 The comprehensive cytotoxicity and uranium removal efficiency of NRK-52E cells. a Dosage-dependent cell growth rate of NRK-52E cells treated with U[(VI), 12.4 μM] + 5LIO-1-Cm-3,2-HOPO, U[(VI), 12.4 μM] + 5LIO-(Me-3,2-HOPO), and U[(VI), 12.4 μM] + ZnNa 3 -DTPA, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 vs control, paired-sample T test for independent-samples, n = 6 samples. Bars indicate SD, n = 6 samples. b Effects of ligand on U(VI) uptake and release when ligand and U(VI) are given together, and on U(VI) release in NRK-52E cells when U(VI) and ligand are given one after the other. Source data are provided as a Source Data file. *p < 0.05, **p < 0.01, ***p < 0.001 vs U(VI)-treated control, paired-sample T test for independent-samples, n = 3 samples. Bars indicate SD, n = 3 samples Full size image

U(VI) uptake and release

In vitro U(VI) uptake and release assays were conducted to investigate the uranyl removal efficiency of 5LIO-1-Cm-3,2-HOPO at the cellular level. In vitro assay for U(VI) uptake and release from NRK-52E cells were performed by adding 12.4 μM U(VI) solution to cells, followed by 320.0 μM solution of the chelators. Fig. 5b shows that the addition of 5LIO-1-Cm-3,2-HOPO can remove 99.5% of uranium from NRK-52E cells, whereas ZnNa 3 -DTPA can remove only 12.5% of uranium under the same condition. Clearly, the uranium removal efficiency of 5LIO-1-Cm-3,2-HOPO is much higher than that of ZnNa 3 -DTPA at an equal molar dosage (Supplementary Table 5). However, since the NRK-52E cells were cultured in medium that contain uranium throughout the experiment, question remains as whether the ligand mainly play the role of preventing the uranium uptake of the cells, or enhancing the release of the intracellular uranium. Therefore, another in vitro assay for U(VI) release from NRK-52E cells was designed by adding 12.4 μM U(VI) solution to cells first, then removing the U(VI) culture medium, followed by the addition of 320.0 μM solution containing the chelators. As shown in Fig. 5b, the treatment of 5LIO-1-Cm-3,2-HOPO resulted in a uranium removal efficiency of 75.8%, illustrating that 5LIO-1-Cm-3,2-HOPO plays a major role for enhancing the U(VI) release from cells (Supplementary Table 6).

In vivo uranyl decorporation

Considering the remarkable performance of 5LIO-1-Cm-3,2-HOPO in removing U(VI) at the cellular level, further evaluation of 5LIO-1-Cm-3,2-HOPO, 5LIO-(Me-3,2-HOPO), and ZnNa 3 -DTPA was conducted via in vivo chelation of U(VI) in mice. Fig. 6 illustrates the procedure of rounded in vivo decorporation assays with different administration methods and time. Three batches of in vivo decorporation assays were designed to study and compare the performance between the three ligands, 5LIO-1-Cm-3,2-HOPO, 5LIO-(Me-3,2-HOPO), and ZnNa 3 -DTPA, including single dosage group with intraperitoneal (ip) injection, single dosage group with oral administration, and multiple dosage and delayed multiple dosage groups with ip injection.

Fig. 6 In vivo decorporation efficiency and in vitro desorption efficiency results. a–d Removal efficiency of U(VI) deposited in the kidneys and femurs compared to the control group. For the single-dose group, mice were ip injected (193 μmol kg−1, molar ratio 92:1) or oral administered (644 μmol kg−1, molar ratio 307:1) with ligands after the iv injection of U(VI) (0.5 mg U(VI) kg−1) and then were killed 24 h later. e The urine & feces excretion percentage for the group 5LIO-1-Cm-3,2-HOPO and 5LIO-(Me-3,2-HOPO) treated with single-dose ip injection or oral administration. f Removal efficiency of U(VI) deposited in the kidneys and femurs compared to the control group, for the multiple-dose group. Mice were given the ligand by ip (97 μmol kg−1, molar ratio 46:1) injection at 3 min, 6 h, 24 h, and 48 h, or at 1 h, 7 h, 25 h, and 49 h after the iv injection of U(VI) (0.5 mg U(VI) kg−1) and were killed 72 h later. g Removal efficiency of U(VI) deposited in the kidneys and femurs compared to the control group, for the delayed multiple-dose group. Mice were given the ligand by ip (193 μmol kg−1, molar ratio 92:1) injection at 6 h, 12 h, 30 h, and 54 h (6 h delayed multiple-dose group), or at 12, 18, 36, and 60 h (12 h delayed multiple-dose group), or at 24, 30, 48, and 72 h (24 h delayed multiple-dose group) after the iv injection of U(VI) (0.5 mg U(VI) kg−1) and were killed 7 d later. *p < 0.05, **p < 0.01, ***p < 0.001 vs. U(VI)-treated control, paired-sample T-test for independent-samples, n = 5 mice. Bars indicate SD, n = 5 mice. h U(VI) desorption efficiency from HAP using 5LIO-1-Cm-3,2-HOPO, 5LIO-(Me-3,2-HOPO) or ZnNa 3 -DTPA. Source data are provided as a Source Data file Full size image

In the single-dose group with intraperitoneal (ip) injection, the first assay was performed to compare the performance of 5LIO-1-Cm-3,2-HOPO and ZnNa 3 -DTPA on the decorporation of uranium, where 5LIO-1-Cm-3,2-HOPO and ZnNa 3 -DTPA (193 μmol kg−1, molar ratio to uranium is 92:1) were injected intraperitoneally (ip) three min after an initial intravenous (iv) injection of U(VI) (0.5 mg kg−1). At 24 h after this iv injection, the kidneys and femurs from the control group were determined to contain 6.72 ± 0.72 and 2.64 ± 0.36 μg U(VI) per gram of tissue, respectively. The group receiving the ip injection of 5LIO-1-Cm-3,2-HOPO displayed a reduced accumulation of U(VI) in the kidneys and femurs, where uranium levels were decreased by 82.9 and 39.0%, respectively. The groups given ZnNa 3 -DTPA, however, showed limited removal of U(VI) under identical experimental conditions (20.5% and 9.1%, respectively) (Fig. 6a and Supplementary Table 7). Another assay was performed to directly compare the performance of 5LIO-1-Cm-3,2-HOPO and 5LIO-(Me-3,2-HOPO) (193 μmol kg−1, molar ratio to uranium is 92:1) on decorporation of uranium in a similar fashion. The group treated with 5LIO-1-Cm-3,2-HOPO yielded a 86.8% and 47.9% of U(VI) removal efficiency in the kidneys and femurs, respectively. Notably, this ligand shows a removal efficiency in the femurs six times higher than that of 5LIO-(Me-3,2-HOPO), since the group given 5LIO-(Me-3,2-HOPO) showed limited effect on removing U(VI) deposited in femurs under the identical experimental conditions (Removal percentage of 8.0%) (Supplementary Table 8 and Fig. 6b). These results are well consistent with the previous study when 5LIO-(Me-3,2-HOPO) was originally synthesized15,23. The results of uranium concentrations in urine and feces for the groups treated with 5LIO-1-Cm-3,2-HOPO and 5LIO-(Me-3,2-HOPO) via ip injection are listed and compared in Supplementary Table 9, showing a clear increase of uranium excretion in the 5LIO-1-Cm-3,2-HOPO treated group. This further supports the observation of the enhanced decorporation efficiency of 5LIO-1-Cm-3,2-HOPO, in comparison with 5LIO-(Me-3,2-HOPO). The other in vivo assays were performed to compare the decorporation ability between 5LIO-1-Cm-3,2-HOPO, 1-Hydroxyethylidene-1,1-diphosphonic acid (HEDP), and NaHCO 3 , which have been reported to be effective for U(VI) decorporation36,37. Supplementary Table 10 lists the U(VI) removal performance in kidneys and femurs following the same experimental procedure. The group treated with HEDP showed reduction of 31.2% of U(VI) deposited in kidneys and 17.4% of U(VI) deposited in femurs, respectively. The group treated with NaHCO 3 showed very limited effect on removing U(VI) from mice.

More importantly, in vivo assay with single-dose oral administration was performed to evaluate the oral efficiency of the 5LIO-1-Cm-3,2-HOPO, 5LIO-(Me-3,2-HOPO), and ZnNa 3 -DTPA. Similiarly, an assay with single-dose oral administration was first performed to compare the performance of 5LIO-1-Cm-3,2-HOPO and ZnNa 3 -DTPA. 5LIO-1-Cm-3,2-HOPO and ZnNa 3 -DTPA (644 μmol kg−1, molar ratio to uranium is 307:1) were orally administered by gastric tube three min after an initial intravenous (iv) injection of U(VI) (0.5 mg kg−1). The group received the oral administration of 5LIO-1-Cm-3,2-HOPO showed a reduction of 58.2% uranium from kidneys and 39.5% from femurs (Supplementary Table 11 and Fig. 6c). Subsequently, a comparison between 5LIO-1-Cm-3,2-HOPO and 5LIO-(Me-3,2-HOPO) was conducted in a similar fashion. As shown in Fig. 6d, the group given 5LIO-1-Cm-3,2-HOPO displayed similar U(VI) removal percentage in kidneys (68.2%) with the group given 5LIO-(Me-3,2-HOPO) (63.2%), but a much higher U(VI) removal ratio in femurs (30.5%) was observed for the group of 5LIO-1-Cm-3,2-HOPO than the group of 5LIO-(Me-3,2-HOPO) (3.5%) (Supplementary Table 12). Fig. 6e shows the excretion results of uranium concentrations in urine and feces for the groups orally treated with 5LIO-1-Cm-3,2-HOPO and 5LIO-(Me-3,2-HOPO). For the mice given ip injection, 76.8% and 86.9% of uranium were excreted in the group treated with 5LIO-(Me-3,2-HOPO) and 5LIO-1-Cm-3,2-HOPO, respectively, which correspond to enhancement of 2.2- and 2.5-fold comparing to that of the control group (35.1%). For the oral administration group, 53.8% and 78.9% of uranium was excreted in the group treated with 5LIO-(Me-3,2-HOPO) and 5LIO-1-Cm-3,2-HOPO, respectively, corresponding to an enhanced excretion of 1.5- and 2.3-fold in comparison to the excretion of the control group, respectively. In comparison with the ip injection groups, U(VI) excretion efficiency is lower in the oral administration groups of both ligands, consistent with the relatively lower U(VI) decorporation efficiency in kidneys and femurs in the orally treated groups (Supplementary Table 12).

In the multiple-dose group, 5LIO-1-Cm-3,2-HOPO and ZnNa 3 -DTPA (97 μmol kg−1; molar ratio to uranium of 46:1) were ip injected at 3 min, 6 h, 24 h, and 48 h after the initial iv injection of U(VI). At 72 h after the iv injection of 0.5 mg U(VI) kg−1, the kidneys and femurs from the control group contained 5.22 ± 1.13 and 3.50 ± 0.39 μg U(VI) per gram of tissue, respectively (Fig. 6f). Compared with the control group, 5LIO-1-Cm-3,2-HOPO significantly reduced U(VI) levels in the femur by 47.4%. Another multiple-dose assay was conducted by ip injection of 5LIO-1-Cm-3,2-HOPO and ZnNa 3 -DTPA (97 μmol kg−1; molar ratio to uranium is 46:1) at 1, 7, 25, and 49 h after the initial iv injection of U(VI). Similarly, kidneys and femur samples were obtained 72 h after the initial U(VI) injection. Notably, 70.9% of U(VI) deposited in the kidneys and 50.0% in femurs were removed in the 5LIO-1-Cm-3,2-HOPO group compared with the untreated U(VI)-injected controls (Fig. 6f). These values are almost identical with those of the multiple-dose group treated 3 min after the U(VI) injection. This achievement overcomes a large hurdle in developing actinide chelation agents and opens up the possibility of complete uranium removal from bones by increasing the dosage further (Supplementary Table 13).

In most nuclear accidents, an immediate response to radionuclide introduction into human bodies is not applicable. Therefore, decorporation agents that can still function despite delayed administration are preferred for chelation therapy with elevated significance. To fully study the relationship between the decorporation efficiency and the time of administration, 5LIO-1-Cm-3,2-HOPO (193 μmol kg−1; molar ratio to uranium is 92:1) was given via ip injection at the following three different time intervals: (1) 6, 12, 30, and 54 h; (2) 12, 18, 36, and 60 h; (3) 24, 30, 48, and 72 h, after the initial iv injection of U(VI). The three groups with 6, 12, and 24 h delayed multiple-dose administration display a similar removal level of U(VI) from kidneys of 61.4%, 62.4%, and 65.0%, respectively. For the decorporation efficiency in femurs, all three groups significantly reduced U(VI) accumulation in femurs by 39.6, 24.2, and 30.8%, respectively (Fig. 6g and Supplementary Table 14), suggesting that the decorporation efficiency is nearly independent of the time of administered treatment.