Bioconjugate Chem. 2006, 17, 571−574
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Fast Water-Exchange Gd3+-(DO3A-like) Complex Functionalized with Aza-15-Crown-5 Showing Prolonged Residence Lifetime in Vivo Cong Li,† Ying-Xia Li,‡ Ga-Lai Law,† Kwan Man,§ Wing-Tak Wong,*,† and Hao Lei*,‡ Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, Department of Surgery, The University of Hong Kong, Pokfulam Road, Hong Kong, and State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics & Mathematics, The Chinese Academy of Sciences, Wuhan, P. R. China . Received January 7, 2006; Revised Manuscript Received March 17, 2006
A bis-hydrated Gd3+ complex based on tris acetic acid-1,4,7,10-tetraazacyclododecane (DO3A) that was functionalized with aza-15-crown-5 demonstrated a nearly optimal water-exchanging rate (kex ) 3.1 × 107 s-1) and low acute cytotoxicity. Efficient magnetic resonance signal intensity enhancements and prolonged residence lifetime induced by this small molecular complex in vivo were demonstrated even with one-fifth of the standard dosage used in the clinic.
Small molecular gadolinium complexes, such as Gd-DOTA (Dotarem), and its derivatives, are widely used as magnetic resonance imaging (MRI) contrast agents (CAs) to aid the diagnosis of pathologies by enhancing the morphology and functionality of the tissue with the advantage of low inherent toxicity (1). However, their pharmacokinetic properties, especially the transient retention lifetime in the blood pool, result in their poor performance in cardiovascular and tumor imagings (2). Additionally, these small complexes demonstrate relatively low relaxivity, so a high dosage is required to achieve a satisfactory signal enhancement. To overcome the problems, Gd3+ complexes were covalently bound to macromolecules, such as proteins (3), antibodies (4), dendrimers (5), and micellar aggregates (6) because their large sizes not only lead to the confinement and a long circulation lifetime in the vasculature (7) but also improve the relaxivity by increasing their rotational correlation time τR (8). Unfortunately, the increasing toxicity induced by the slow clearance rate and the inefficient delivery of these macromolecules to the targeting sites seriously limit their applications (5, 7, 9). In response to the problems, Lu et al. designed the biodegradable macromolecular agents, in which a Gd3+ complex was grafted with poly(ethylene glycol) (PEGs) through the disulfide bond (10, 11). After the imaging, Gd3+ chelates can be released from the macromolecules and excreted in time through the cleavage of the disulfide bond by disulfidethiols exchange. However, because of the low concentration of endogenous thiols in human plasma, multiple administrations of extraneous thiols are needed to keep the efficient cleavage of the disulfide bond. For the above reasons, innovative designs for MRI contrast agents with high relaxivity and moderate excretion rates in vivo are needed. PEGs are nontoxic and nonantigenic polymers that have been widely used in the modification of biomolecules to improve their biocompatibility and pharmacokinetics (12). Covalently * To whom correspondence should be addressed. (W.-T.W.) Tel.: +852 2859 2157. Fax: +852 2547 2933. E-mail: wtwong@ hkucc.hku.hk. (H.L.) Tel. +86 27 8719 8542. Fax: +86 27 8719 9291. E-mail:
[email protected]. † Department of Chemistry, The University of Hong Kong. ‡ Wuhan Institute of Physics & Mathematics, The Chinese Academy of Sciences. § Department of Surgery, The University of Hong Kong.
bound PEGs not only promote the passive accumulation of the conjugates into the tumors (13) but also decrease their clearance rate from the circulation system (14). Crown ethers as artificial molecules have been extensively investigated in supramolecular chemistry (15). What attracts us here are their well-known amphiphilicity (16), identical building block (ethylene glycol) as PEGs, and much smaller molecular weights as compared to PEGs. Considering these properties of crown ethers, a compromise between a prolonged circulation lifetime and low inherent toxicity is expected after functionalization of the crown ether moiety to the Gd3+ chelates. In this communication, we report the synthesis and preliminary evaluation of the crown ether functionalized Gd3+ complex. Tris acetic acid-1,4,7,10-tetraazacyclodecane (DO3A) was chosen as the chelate here because its Gd3+ complexes not only demonstrated a satisfactory thermodynamic stability [log K ) 22.0, 298 K] (17), but also a maximum of two binding sites is left for water molecules, which makes their relaxivity increase substantially because the inner-sphere proton relaxivity is proportional to the q values (8, 18). Meanwhile, aza-15-crown-5 was selected because of its moderate ring size and the modification availability of the secondary amine. The synthesis of LnL1 is depicted in Scheme 1. Mono-crown ether alkylated cyclen 1a (19) was prepared straightforwardly with high yield according to our previous method. 1a reacted with excess tert-butyl bromoacetate to afford 1b. Following a deprotection step, the resulting ligands L1 were treated with Ln2(CO3)3 to give the desired Gd3+, Tb3+, and Eu3+ complexes. To clarify the effect of the pendant crown ether to relaxivity and other relaxometric parameters, Ln3+ complexes of DO3A grafted with ethylamine LnL2 as control molecules were prepared (20, 21). The luminescent lifetimes of TbL1 (τH2O ) 1.39 ms, τD2O ) 2.94 ms) and TbL2 (τH2O ) 1.25 ms, τD2O ) 3.05ms) were measured in H2O and D2O, respectively, and both of their hydration numbers q were given as 2 (22). The water proton relaxivity r1p of GdL1 and GdL2 at 20 MHz, 25 °C was measured as 9.5 and 5.9 mM-1 s-1, respectively at pH 7.0. Significantly, the r1p of GdL1 nearly doubled compared with its parent complex Gd-DO3A (5.7 mM-1 s-1) (23). A main reason that determines the lower relaxivity of small molecular complexes compared to their theoretical prediction is their water/proton residence lifetime
10.1021/bc060003m CCC: $33.50 © 2006 American Chemical Society Published on Web 04/15/2006
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Li et al.
Scheme 1. Synthesis Procedure of LnL1a
a Reagents and conditions: (a) 3.3 equiv of tert-butyl bromoacetate, Na2CO3/THF-H2O, r.t., 4 h, 84%; (b) TFA/CH2Cl2, r.t., 2 h, 96%; (c) Ln2(CO3)3, H2O, 70 °C, 10 h, Ln ) Gd, Tb, and Eu. Yields of the lanthanide complexes range from 88-95%.
Figure 1. VT-17O NMR transverse relaxation rate of GdL1 and GdL2 at 2.1 T, 25 °C, pH 7.0. GdL1, filled circle; GdL2, open circle.
τM (τM is the reciprocal of the water exchange rate, kex) is far from optimal values (8). Therefore, many efforts have been dedicated to investigating ligand systems for Gd3+ ion by optimizing the water exchange rate kex (24, 25). To clarify the unusually high r1p of GdL1, the measurement of the water 17O NMR transverse relaxation rate, R2, at variable temperature (VT), which allows a precise assessment of the water residence lifetime τM, was conducted. The VT 17O NMR curve of GdL1 (Figure 1) demonstrated that it is a typical system within the fast exchange regime. The τM value of 32 ns (kex ) 3.1 × 107 s-1) for GdL1 at 25 °C was provided by analyzing the curve in terms of the Swift-Connick equation (3). Notably, this τM value is rather rare for a neutral complex and very close to its optimal range of 20-30 ns (24). τM is generally determined by several factors including steric crowding at the water binding site, the overall charge of the complex as well as the geometry of the complex. In comparison to GdL2 (τM ) 240 ns, kex ) 4.2 × 106 s-1, Figure 1) and Gd-DO3A (τM ) 159 ns, kex ) 6.3 × 106 s-1) (23), the fast water exchange rate of GdL1 could arise from the increasing negative charge density and steric congestion around the Gd3+ ion upon the approach of the crown ether, which promotes the departure of the bound water molecules. Therefore, the fast water-exchanging rate of GdL1 should be an important reason for its high relaxivity. Besides the relaxivity, another important factor to evaluate the potential application of Gd3+ complexes as MRI contrast agent is their toxicity, which is generally caused by the free gadolinium ion. To determine the cytotoxic effect of GdL1, an MTT assay (26) (MTT ) 3-(4,5-dimethythiazol-2-yl)-2,5diphenyltetrazolium bromide) was performed on the human nontumotigenic immortalized liver cell line (MIHA) (27). In this work, the commercial available MRI contrast agent, Gd-
Figure 2. Results obtained from the MTT assay showing the effect of Gd-DOTA (white bars), Gd-DO3A (light gray bars), and GdL1 (gray bars) on MIHA cells. The viability (%) refers to the growth inhibition induced by the Gd3+ complex in comparison to the cells in the absence of Gd3+ complex. Error bars represent the standard deviation (( SD) on a triplicate analysis (n ) 3).
Figure 3. 3D maximum intensity projection T1-weighted MR images of a rat at preinjection (a), 4 (b), 30 (c), 100 (d), and 200 (e) min after administration of GdL1 (0.04 mmol/kg). In panel b, the MR signal in abdominal aorta (1), kidney (2), liver (3), and adrenal gland (4) was enhanced significantly and attained their maximum at different times.
DOTA, and Gd-DO3A with two bound water molecules were used as the control molecules under the same condition. The experimental results demonstrated that the cell’s proliferation and viability were not obviously affected after incubating from 1 to 24 h, while the concentration of GdL1 ranged from 0.5 to 10 mM (Figure 2, Figure S1, Supporting Information). Additionally, the morphology of live MIHA cells in response to 100 µM GdL1 was monitored by laser scanning confocal microscopy after 1, 2, 8, and 24 h incubation at 37 °C (Figure S2, Supporting Information). Compared to the cells in the control experiments, no obvious morphological changes of the MIHA cell were detected. The MTT assay showed that the acute cytotoxicity of GdL1 is quite low in the concentration range required for obtaining the detectable MR signal. In vivo T1-weighted images were acquired after the administration of GdL1, Gd-DOTA, and the liver-specific CA GdBOPTA (MultiHance) to anesthetized rats. Figure 3 shows the three-dimensional (3D) maximum intensity projection (MIP) abdomen images of a rat before and after intravenous injection of GdL1 at 0.04 mmol/kg. MR signals in the kidney, liver, adrenal gland, and abdominal aorta were enhanced significantly. Time dependent MR signal intensity enhancements (IEs) after administration of different CAs were demonstrated in Figure 5. GdL1 offered much higher vascular and renal contrast enhancements than commercial available Gd-DOTA under the same dosage (Figure 5A,B). The IEs in the abdominal aorta from GdL1 remained strong in the first 30 min, and still visible at 45-100 min (Figures 3 and 5A), while the enhancement in kidney cortex was even longer; obvious renal IEs lasted for more than 3 h (Figures 4A and 5B). Comparatively, the IEs induced by Gd-DOTA in the blood vessel and kidney vanished completely in less than 15 and 40 min, respectively. To clarify the prolonged resident lifetime of GdL1 in vasculature, the titration
Communications
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Figure 4. 2D axial T1-weighted renal and hepatic MR images of rats before (a) and 10 (b), 100 (c) and 200 (d) min after the administration of GdL1 (A, B) and Gd-BOPTA (C) in the dosage of 0.04 mmol/kg.
of human serum albumin (HSA) to this complex was conducted at pH 7.4 (Figure S3, Supporting Information). The r1p (20 MHz, 25 °C) of GdL1 increased substantially with the concentration of HSA, and the binding constant Ka of 3009 M-1 was measured. This result implied that the nonspecific binding between the amphiphilic crown ether moiety and serum proteins resulted in the prolonged resident lifetime of GdL1 in vasculature (28). Pronouncedly different time courses of hepatic IEs were observed between GdL1 and Gd-BOPTA (Figures 4B,C and 5C). IE induced by Gd-BOPTA reached its maximum within the first 10 min, while the peak value of GdL1 appeared in 80-100 min after administration. Notably, the significant hepatic IEs induced by GdL1 was sustained for more than 4 h. Moreover, it is worth mentioning that the vascular, renal, and hepatic enhancements of GdL1 reached their maximal values at different time windows, which implies this complex can be used to image multiple organs successively after the single injection. Even though the mechanism of extended residence lifetime of GdL1 in liver parenchyma is not very clear, the experimental results suggest that, like the pendant phenyl group in Gd-BOPTA, amphiphilic crown ether can enhance the passive accumulation of GdL1 in the reticuloendothelial system (RES). Moreover, the uptake of this complex by hepatocytes, and following biliary excretion, may be other reasons for the prolonged hepatic IE (29). Dosage-dependent MR signal IE behaviors induced by these three complexes were also investigated (Figure S4, Supporting Information). Even in 0.02 mmol/ kg, one-fifth of the standard dosage used in the clinic, bishydrated GdL1 achieved higher and longer IEs in blood vessel, kidney, and liver compared to two other commercial available CAs. Additionally, it is noteworthy that all rats recovered spontaneously from anesthesia after the MRI studies, which further indicates that GdL1 has no acute fatal toxicity at the dosage used. In conclusion, this work describes a fast water-exchange Gd3+-DO3A complex showing efficient MR signal enhancements, an extended excretion rate, and low acute cytotoxicity. The relaxometric and in vivo imaging studies demonstrate the crown ether moiety as a functional group is a potential choice
Figure 5. Time-dependent abdominal aortic (A), renal (B), and hepatic (C) IEs induced by GdL1, Gd-DOTA, and Gd-BOPTA at the dosage of 0.04 mmol/kg. Error bars represent the standard deviation (( SD) on a triplicate analysis (n ) 3).
to realize the compromise between the prolonged excretion rate and the low toxicity of MRI contrast agents.
ACKNOWLEDGMENT The authors thank the Hong Kong Research Grants Council (HKU 7119/00P and HKU 7116/02P) and Natural Science Foundation of China (10234070 and 30370419). This work was also supported by the Area of Excellence Scheme of the University Grants Committee (Hong Kong). We are grateful to Prof. M. Botta and his student S. Avedano for their assistance with the relaxometric measurements. Supporting Information Available: Details of chemical synthesis and characterization, time-dependent MR IEs at different dosages, and cytotoxicity studies. This material is available free of charge via the Internet at http://pubs.acs.org.
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