Diethylenetriaminepentaacetic Acid−Gadolinium (DTPA-Gd

Bioconjugate Chem. 2006, 17, 700−706

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Diethylenetriaminepentaacetic Acid-Gadolinium (DTPA-Gd)-Conjugated Polysuccinimide Derivatives as Magnetic Resonance Imaging Contrast Agents Ha Young Lee,†,‡ Hye Won Jee,‡ Sung Mi Seo,‡ Byung Kook Kwak,§ Gilson Khang,† and Sun Hang Cho*,‡ Department of Advanced Organic Materials Engineering, Chonbuk National University, Jeonju, Jeonbuk, Republic of Korea, Nanobiomaterials Laboratories, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea, and College of Medicine, Chung-Ang University, Seoul, Republic of Korea. Received January 23, 2006; Revised Manuscript Received March 19, 2006

Biocompatible polysuccinimide (PSI) derivatives conjugated with diethylenetriaminepentaacetic acid gadolinium (DTPA-Gd) were prepared as magnetic resonance imaging (MRI) contrast agents. In this study, we synthesized PSI derivatives incorporating methoxy-poly(ethylene glycol) (mPEG) as hydrophilic ligand, hexadecylamine as hydrophobic ligand, and DTPA-Gd as contrast agent. PSI was synthesized by the polycondensation polymerization of aspartic acid. All the synthesized materials were characterized by proton nuclear magnetic resonance (1H NMR). Critical micellization concentrations were determined using fluorescent probes (pyrene). Micelle size and shape were measured by electro-photometer light scattering (ELS) and atomic force microscopy (AFM). The formed micelle size ranged from 100 to 300 nm. The T1-weighted MR images of the phantom prepared with PSImPEG-C16-(DTPA-Gd) were obtained in a 3.0 T clinical MR imager, and the conjugates showed a great potential as MRI contrast agents.

INTRODUCTION Magnetic resonance imaging (MRI) utilizes a strong magnetic field to image the human body (1). Recently, MRI contrast agents have been used specifically to improve the image contrast between normal and pathological tissue. These MRI contrast agents must have contrast enhancing ability, high stability, and favorable pharmacokinetic properties (2). Several poly(amino acid) derivatives have been investigated as a type of delivery system (3, 4). Particularly, polysuccinimide (PSI) is one of the most promising particle carriers since it was demonstrated to possess suitable physicochemical characteristics for development of macromolecular prodrugs, due to the biocompatibility, biodegradability, and the ease of synthesis and functionalization (5-11). Micellization of biologically active substances prepared by advanced bioconjugation technology is a useful method to increase the solubility, stability, and bioavailability of lipophilic drugs and nutrients (12, 13). The rational design of polymeric bioconjugates represents a key issue to achieve derivatives that have the desired physicochemical and biopharmaceutical properties. Therefore, the introduction a of specific pendant group in the polymer backbone is often carried out to modify the carrier properties (14-16). Especially, the polymeric micelle system can offer some advantages such as wide applicability to drugs and small particle size (17-19). Polymeric micelles having hydrophilic/hydrophobic groups were prepared by conjugating a hydrophilic methoxy-poly (ethylene glycol) (mPEG) and a hydrophobic group to PSI, a biodegradable polymer, that is known to be excreted by the kidney (20, 21). The size of micelles with different molecular weights of mPEG and lengths of hydrophobic chains was measured by electro-photometer light scattering (ELS). * To whom correspondence should be addressed. Tel: + 82-42-8607222. Fax: + 82-42-860-7228. E-mail: [email protected]. † Chonbuk National University. ‡ Korea Research Institute of Chemical Technology. § Chung-Ang University.

We also attached an MRI contrast agent, Gd-DTPA, to the polymeric micelles, PSI. Diethylenetriaminepentaacetic acid (DTPA) is a very efficient chelating agent for lanthanide metal ions. Due to its contrast enhancement and high stability in vivo, a DTPA complex has been clinically used as a contrast agent in MRI (22, 23), but because this DTPA-Gd complex has a low molecular weight and is removed rapidly from the blood pool, the study has been directed toward stable DTPA-Gd complexes that can circulate for a longer time in blood (24, 25). In this study, we synthesized PSI-mPEG-C16-(DTPA-Gd) which formed a micelle. The size diameter of the micelles was 100-300 nm which is suitable for a particle carrier to be well incorporated into the internal organs (26, 27). Furthermore, we investigated the potential of PSI-mPEG-C16-(DTPA-Gd) as a MRI contrast agent by an MRI test of the phantom.

EXPERIMENTAL PROCEDURES Materials. D,L-Aspartic acid and hexadecylamine were purchased from Aldrich Co., Milwaukee, WI. Methoxy-poly(ethylene glycol)-amine [mPEG-NH2, number-average molecular weight (Mn) ≈ 5000] was purchased from Fluka, Buchs, Switzerland. Reagents for DTPA preparation were obtained from Fluka and Aldrich. Phosphoric acid, sulfolane, N,N-dimethylformamide (DMF), methyl alcohol (MeOH), and diethyl ether were commercially available and were used without further purification. Omniscan was purchased from Guerbet Co., Germany. Synthesis of Polysuccinimide (PSI). Polysuccinimide was synthesized by a polycondensation reaction. A suspension of D,L-aspartic acid (25 g, 0.188 mol) and phosphoric acid (9.4 mmol) in sulfolane (80 g) was stirred at 160 °C under a nitrogen atmosphere. After 24 h, the reaction solution was precipitated by into MeOH. PSI was filtered, washed with MeOH, and dried under vacuum. Synthesis of Poly(succinimide-ethylene glycol) (PSImPEG). A solution of mPEG-NH2 (0.1 × 10-4 mol) in DMF (5 mL) was added dropwise to a solution of PSI (2.5 × 10-4

10.1021/bc060014f CCC: $33.50 © 2006 American Chemical Society Published on Web 05/02/2006

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Figure 1. The overall synthetic scheme of PSI-mPEG-C16 from poly(aspartic acid).

mol) in DMF (50 mL). The mixture was stirred at 70 °C, under a nitrogen atmosphere. After 24 h, the reaction solution was precipitated and filtered with ethyl ether for removal of DMF and unreacted materials. The filtered PSI-mPEG was washed with ether and dried under vacuum. Synthesis of PSI-mPEG-C16. A solution of hydrophobic ligand, hexadecylamine (2 × 10-4 mol), in DMF (5 mL) was dropped to a solution of PSI-mPEG (2.5 × 10-4 mol) in DMF (30 mL). The mixture was stirred at 70 °C under a nitrogen atmosphere. After 7 h, the reaction solution was precipitated and filtered with ethyl ether. The filtered PSI-mPEG-C16 was washed with ethyl ether and dried under vacuum. The distribution of micelle size was experimentally evaluated. Synthesis of PSI-mPEG-C16-(DTPA-Gd). Lysine-DTPA tert-butyl ester was prepared according to the method reported in the literature (22). The solution of DTPA (3.75 × 10-4 mol) in DMF (5 mL) was added dropwise to a solution of PSImPEG-C16 (2.5 × 10-4 mol) in DMF (20 mL). The mixture was stirred at room temperature. After 18 h, ethyl ether was added. The precipitate was filtered, washed with ethyl ether, and dried under vacuum. The dried residue was dissolved in formic acid and stirred at 100 °C to perform the deprotection of tert-butyl group, according to the literature (1). After 3-5 h, the reaction solution was evaporated under vacuum condition. The residue was dissolved in water and stirred at room temperature for 3 h. Thereafter, the mixture was filtered and lyophilized. To support gadolinium in PSI-mPEG-C16-DTPA, a solution of GdCl3 (1.5 × 10-3 mol) in water (5 mL) was added dropwise to a solution of PSI-mPEG-C16-DTPA (1.5 × 10-3 mol) in water (30 mL), maintaining the solution mixture at pH 7-8. After 1 h, the reaction mixture containing PSI-mPEG-C16(DTPA-Gd) was precipitated by addition of acetonitrile and was filtered. The filtered solid was washed with ethyl ether and dried under vacuum.

Figure 2. 1H NMR spectra of (a) PSI, (b) PSI-mPEG, and (C) PSImPEG-C16. Table 1. Degree of Derivatization (DD) of mPEG and C16 sample

mPEG DD (mol %)

C16 DD (mol %)

PSI-mPEG PSI-mPEG- C16

13.5 14.7

1.37

Characterization Analysis. The molecular weight of PSI was confirmed by GPC ((PL-GPC220, Polymer Laboratory, England) analysis. GPC had two columns (10 µm, MIXED-B ×2. 50 nm), and DMF (Aldrich) was used as mobile phase. Polystyrene (Showa Denko, Japan) was used as the standard sample. The flow rate was 1 mL/min. The results of polymerization were confirmed by 1H NMR (Bruker 300, 500 MHz). Dimethyl sulfoxide (DMSO, Aldrich) was used as solvent. The size of micelles and shape were confirmed by ELS (ELS-8000, Otsuka Electronics, Japan) and AFM (Nanoscope IV). Also, Gd incorporation and Gd content were confirmed by EA, EDS, and ICP-AES. Fluorescence studies were carried out using a F-4500 fluorescence spectrophotometer (Hitachi Co. Ltd., Japan).

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Figure 3. The overall synthetic scheme of PSI-mPEG-C16-(DTPA-Gd) from PSI-mPEG-C16.

Figure 4. 1H NMR spectrum of PSI-mPEG-C16-DTPA tert-butyl ester.

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Polysuccinimide Derivatives as a MRI Contrast Agent Table 2. Elemental Analysis of Complex complex PSI-mPEG-C16-DTPA PSI-mPEG-C16-(DTPA-Gd)

element

elemental anal. average (%)

nitrogen carbon hydrogen nitrogen carbon hydrogen

8.911348 44.71717 6.167405 4.997967 22.12623 2.562378

In Vitro Contrast Effect Measurement. In vitro MR imaging test was performed with a 3.0T magnet at Philips Achieva 3.0T MRI (Philips Co., Netherlands). The T1-weighted MR images of the prepared compounds and a commercially available contrast agent, Omniscan, were obtained. MR images were taken with different concentrations of Gd solution (1 × 10-3 M Gd/L to 5 × 10-6 M Gd/L). The experimental condition was as follows: TR (recycle time) ) 9.6 ms, TE (echo delay time) ) 4.6 ms, flip angle: 8.0, field of view: 10 × 5 cm2.

RESULTS AND DISCUSSION Synthesis and Characterization of PSI-mPEG-C16. To prepare the micelle-formed MRI contrast agent with Gd, at first, PSI, PSI-mPEG, and PSI-mPEG-C16 were sequentially synthesized following the literature (28). The overall synthetic route of PSI-mPEG-C16 is shown in Figure 1. PSI was synthesized by the polycondensation polymerization of aspartic acid. The yield and weight average molecular weight of PSI were 80% and 21000 by GPC measurement. 1H NMR spectra of PSI, PSI-mPEG, and PSI-mPEG-C 16 are shown in Figure 2a-c. In the PSI spectra, the signals at 5.04 ppm were assigned to the methyne proton (a) of the repeating succinimide unit and the signals at 2.5-3.5 ppm were assigned to the methylene protons (b) (29). The degree of derivatization (DD) of PEG was determined by 1H NMR and was calculated by comparing the integral of the NH peak at δ 7.9 that appears during the opening of the ring when being combined with PEG to the integral of the peak at δ 5.04 assigned to protons that belong to the PSI unit. The DD of C16 was calculated by comparing the integral of the CH2 peak at δ 1.2 of hexadecylamine to the integral of the peak at δ 5.04 to protons that belong to the PSI unit (28). The DD values of PSI-mPEG and PSI-mPEG-C16 are reported in Table 1. Synthesis and Characterization of PSI-mPEG-C16(DTPA-Gd). The overall synthetic route of PSI-mPEG-C16(DTPA-Gd) from PSI-mPEG-C16 is shown in Figure 3. DTPA used as a chelating agent was combined with PSI-mPEG-C16, and Gd was incorporated into the complex. Figure 4a,b shows 1H NMR analysis data of PSI-mPEG-C -DTPA and PSI16 mPEG-C16-(DTPA-Gd). These spectra show the existence of a tert-butyl peak at 1.2-1.8 ppm before formic acid treatment. Incorporation of Gd into the polymer chain was confirmed by EDS (energy dispersive spectroscopy) analysis and EA (elemental analysis) data. Table 2 showed that the content of C, H, N decreased after incorporation of Gd. Figure 5 showed that the peak of Gd appeared after incorporation of Gd. The content of Gd was measured by ICP (Table 3). The results showed that a moderately consistent amount of Gd was incorporated in the polymer from two different batches, because the binding site of PSI involved in the conjugation of DTPA was limited. Critical Micelle Concentration (CMC). The CMC of PSImPEG, PSI-mPEG-C16, and PSI-mPEG-C16-(DTPA-Gd) were determined using pyrene emission (30, 31). The concentrations of sample solution were varied from 1 × 10-5 to 1 mg/mL. The ratio I3/I1 of the intensities of the first (λ1 ≈ 335 nm) and the third (λ3 ≈ 338 nm) vibronic band is a sensitive measure for pyrene. This ratio increases with increasing hydrophobicity in the core because pyrene is expected to be distributed in

Figure 5. The analysis of gadolinium chelating measured by EDS: (a) PSI-mPEG-C16-DTPA, (b) PSI-mPEG-C16-(DTPA-Gd). Table 3. Concentration Analysis of Gadolinium in Final Product by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES) DTPA content ratio of final product

gadolinium concentration (%)

batch 1 batch 2

29 26.9

hydrophobic regions of a self-organized system in solution due to low water solubility. Figure 6 shows the fluorescence emission spectra of pyrene incorporated into PSI-mPEG, PSImPEG-C16, and PSI-mPEG-C16-(DTPA-Gd) in water, respectively. The ratio I3/I1 increases rapidly, indicating that interactions between pyrene and the hydrophobic core are occurring and pyrene is in the micellar core. The critical micelle concentration (CMC) was determined by the interception of two straight lines. This CMC data indicate that the CMC values increase with increasing hydrophilic groups such as PEG and DTPA-Gd and that all prepared copolymers form micelles. Size Distribution. The size effect of micelles with different molecular weights of mPEG is shown in Figure 7. This result shows that the size of micelles is decreased as the molecular weight is increased. The decrease in micelle size could be due to increased hydrophilicity when more mPEG units are added. The size effect of micelle with the length of alkyl chain is shown in Figure 8. This result showed that the size of micelles increased as the length of hydrophobic chain increased. Figure

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Figure 6. Change of intensity ratio (I338/I335) versus the concentration of PSI-mPEG, PSI-mPEG-C16,and PSI-mPEG-C16-(DTPA-Gd).

Figure 8. The size distribution of micelles with different hydrophobic ligand lengths by ELS analysis; (a) PSI-mPEG5000-C8 micelles average size: 238 nm, (b) PSI-mPEG5000-C14 micelles average size: 288 nm and (c) PSI-mPEG5000-C18 micelles average size: 342 nm.

Figure 7. The size of micelles with different molecular weight of mPEG by ELS analysis: (a) PSI-mPEG2000 micelles average size: 212.3 nm, (b) PSI-mPEG5000 micelles average size: 129.3 nm, and (c) PSI-mPEG10000 micelles average size: 97 nm.

9 shows the AFM image of micelles. The formed micelles had a spherical shape, and the diameter of micelle ranged from 100 to 300 nm. In Vitro MRI Test. T1-weighted MR images of the phantom are shown in Figure 10. In the phantom image, when the concentration of Gd was above 9.4 × 10-4 M, the image contrast

showed similar patterns, but when the concentration of Gd was below this amount, the image contrast seemed better than that of Omniscan. Table 4 shows relaxivity values according to different concentrations of Gd. From these data, we can know that the signal intensity value of PSI-mPEG-C16-(DTPA-Gd) in 1.2 × 10-4 M (Gd) is similar with the signal intensity value of Omniscan in 4.7 × 10-4 M (Gd). These results showed that the prepared sample had better contrast imaging at a lower concentration than that of Omniscan.

CONCLUSION In this study, we have prepared PSI micelle conjugates by combining PSI with a hydrophilic chain, a hydrophobic chain, and a contrast agent. We have tested the compounds to determine its potential use as a carrier and a contrast agent. First, PSI with various molecular weights was synthesized through condensation polymerization with different reaction

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ACKNOWLEDGMENT This work was supported by KMOCIE(10011394).

LITERATURE CITED

Figure 9. The shape of micelles measured by AFM: (a) PSI-mPEG micelles, (b) PSI-mPEG-C16 micelles.

Figure 10. Contrast effect of the phantom measured by MRI. Table 4. Signal Intensity Value with Different Concentrations of Gd Measured by MRI Gd concn (M)

signal intensity of Omniscan

signal intensity of PSI-mPEG-C16-(DTPA-Gd)

1.9 × 10-3 M 9.4 × 10-4 M 4.7 × 10-4 M 2.3 × 10-4 M 1.2 × 10-4 M 6.0 × 10-5 M

1992.17 1724.86 1468. 42 1096 771.05 580.96

2112.12 2043.92 1912.2 1812.43 1430.12 1043.42

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