Bioconjugate Chem. 2010, 21, 175–181
175
γ-Glutamyl PAMAM Dendrimer as Versatile Precursor for Dendrimer-Based Targeting Devices Tomoya Uehara,† Daisuke Ishii,† Tomoe Uemura,† Hiroyuki Suzuki,† Tomoko Kanei,† Kyoko Takagi,† Masashi Takama,‡ Masahiro Murakami,‡ Hiromichi Akizawa,§ and Yasushi Arano*,† Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Faculty of Pharmaceutical Sciences, Osaka Ohtani University, Osaka, and Health Sciences University of Hokkaido, Sapporo, Japan. Received September 16, 2009; Revised Manuscript Received November 24, 2009
Poly(amidoamine) (PAMAM) dendrimers are highly branched spherical polymers that have a unique surface of primary amine groups and provide a versatile design for targeted delivery of pharmaceuticals and imaging agents. Acetylation or succinylation of surface amine groups of PAMAM dendrimer derivatives is frequently performed to reduce nonspecific uptake. However, since targeting molecules, drugs/imaging agents, and acylating reagents react with the amine groups on dendrimer, such modification may limit the number of targeting molecules and/or drugs or may result in insufficient charge reduction. In this study, a γ-glutamyl PAMAM dendrimer was designed and synthesized as a new precursor for targeting device. The relationship between surface electrical properties of the PAMAM dendrimer derivatives and pharmacokinetics was also determined. A PAMAM dendrimer (generation 4.0) was modified with a small number of Bolton-Hunter reagent to prepare Phe-P (pI 9.2). The amine residues of Phe-P were γ-glutamylated to prepare Glu-P (pI 7.1). The R-amine residues of Glu-P were then acetylated or succinylated to prepare Ac-Glu-P (pI 5.3) or SucGlu-P (pI 3.6). For comparison, Phe-P was acetylated or succinylated to prepare Ac-P (pI 6.0) or Suc-P (pI 5.1). All the PAMAM dendrimer derivatives exhibited similar molecular size (7.2 to 7.8 nm) except for Ac-P (5.1 nm). The biodistribution studies were performed after radioiodination of each PAMAM dendrimer derivative with Na[125I]I. When injected intravenously to mice, both [125I]Ac-P and [125I]Suc-P exhibited prolonged radioactivity levels in the blood and significantly lower hepatic and renal radioactivity levels than those of [125I]Phe-P. Both [125I]Glu-P and [125I]Ac-Glu-P showed residence times in the blood similar to those of [125I]Ac-P and [125I]Suc-P. However, [125I]GluP also registered higher radioactivity levels in the kidney. High hepatic and renal radioactivity levels were observed with highly anionic [125I]Suc-Glu-P. These results indicate that, while the manipulation of pI between 5 to 6 would be appropriate to enhance blood retention and reduce renal and hepatic uptake, the amount of primary amine residues on dendrimer surface may also play a crucial role in their renal uptake. The findings in this study show that γ-glutamyl PAMAM dendrimers would constitute versatile precursors to prepare PAMAM dendrimer-based targeting devices due to their neutral molecular charge (pI 7.1) and the presence of a large number of R-amine residues available for conjugation of targeting molecules and drugs/imaging agents.
INTRODUCTION Dendrimers are highly branched macromolecules with welldefined architecture. A large number of dendrimer derivatives of different constituent atoms and sizes have been synthesized so far (1). The unique characteristics of dendrimers such as uniform and controlled size, monodispersity, and modifiable surface group functionality make these molecules appealing for biomedical applications (2). The ability to functionalize their terminal groups with targeting molecules and drugs/ imaging agents in a specific and controllable manner renders the molecules potential carriers for targeted drug delivery (3-7). In addition, their interior can encapsulate various molecules (8). Among the dendrimer derivatives, the starburst polyamidoamine (PAMAM) dendrimers, highly branched spherical polymers, are soluble in aqueous solution and have a unique surface of primary amine groups (1). PAMAM dendrimers have been investigated as carriers in gene transfection (9), * Address correspondence to Yasushi Arano, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan. Phone: +81-43-226-2896, Fax: +81-43-2262897, E-mail:
[email protected]. † Chiba University. ‡ Osaka Ohtani University. § Health Sciences University of Hokkaido.
MRI contrast agents, boron-neutron capture therapy (10) (11), and drug delivery applications (8, 12, 13). When PAMAM dendrimers with surface amine groups were injected into mice, however, they were eliminated rapidly from circulation and accumulated in the liver, kidney, lung, and spleen (14-16). This would be attributable to the electrostatic interaction between positively charged dendrimer molecules and negative charged residues on cell surface at physiological pH (17, 18). Indeed, carboxylic acid-terminated dendrimers exhibit longer residence time in the blood and lower accumulations in the liver and kidney when compared with amineterminated counterparts (15, 19). The acetylation or succinylation of free terminal amine groups of drug-conjugated PAMAM dendrimers also improved pharmacokinetics (4, 20, 21). However, since the acylation reagents, targeting molecules, and drugs/imaging agents react with amine groups on PAMAM dendrimer surface, such chemical modification may cause insufficient charge reduction and/or may limit the number of targeting molecules or drugs/imaging agents especially when the targeting molecules and/or drugs possess positive charges. The lack of knowledge concerning the relationship between molecular charges and pharmacokinetics of PAMAM dendrimer makes the modification cut-and-try, and further understanding of the relationship would provide insight for rationale design of PAMAM dendrimer-based targeting devices.
10.1021/bc900410q 2010 American Chemical Society Published on Web 12/15/2009
176 Bioconjugate Chem., Vol. 21, No. 1, 2010
In the present study, γ-glutamylation of a PAMAM dendrimer was estimated as a new approach to manipulate the molecular charges of an amine-terminated PAMAM dendrimer (generation 4.0) without impairing the number of targeting molecules and drugs/imaging agents. The relationship between the molecular charges and pharmacokinetics a PAMAM dendrimer was also investigated.
EXPERIMENTAL PROCEDURES Materials. Na[125I]I was purchased from MP biomedicals Inc. (Irvine, CA). PAMAM dendrimer (polyamidoamine dendrimer, generation 4.0) was purchased from Aldrich (St. Louis, MO). TLC analyses were performed with silica plates (Silica gel 60 F254, Merck, Tokyo) developed with 80% methanol. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a JEOL JNM-ALPHA 400 spectrometer (JEOL Ltd., Tokyo) with tetramethylsilane as an internal standard. Fast atom bombardment mass spectra (FAB-MS) were taken on a JEOL JMS-HX-110A mass spectrometer (JEOL Ltd.). The diameter of PAMAM dendrimer derivatives was determined by dynamic light scattering using DLS-700 (Photal Otsuka Electronics, Osaka, Japan). Zeta potentials were measured using an electrophoretic light scattering spectrophotometer (ELS-6000, Photal Otsuka Electronics). N-Succimidyl-3-(4-hydroxyphenyl)propionate (compound 1) was synthesized according to the procedure of Bolton et al. (22). Other reagents were of reagent grade and used as received. Synthesis of N-r-tert-Butoxycarbonyl-L-glutamic Acid γ-N-Succinimidyl Ester r-tert-Butyl Ester (2). N-R-tertButoxycarbonyl-L-glutamic acid R-tert-butyl ester (Boc-GluOtBu; 3 g, 9.89 mmol) and N-hydroxysuccinimide (NHS; 1.25 g, 10.8 mmol) were dissolved in tetrahydrofuran (THF, 20 mL). The reaction mixture was cooled in an ice bath, and a solution of N,N′-dicyclohexylcarbodiimide (DCC, 2.2 g, 10.8 mmol) in THF (8 mL) was added dropwise while keeping the reaction temperature below 5 °C. The reaction mixture was then stirred at room temperature overnight. After removing precipitated DCC urea by filtration, the filtrate was purified by column chromatography on silica gel using a mixture of ethyl acetate/hexane (1:2) as an eluent to produce compound 2 (3.1 g, 79.3%) as a white solid. 1H NMR (CDCl3): δ 1.44-1.51 (18H, overlapped, Boc, tert-butyl), 2.02 (1H, m, CH), 2.26 (1H, m, CH), 2.70 (2H, m, CH2), 2.83 (4H, s, CH2), 4.25 (1H, s, CH), 5.14 (1H, d, CONH); FAB-MS (M+H)+: m/z 401. Found: 401. 3-(4-Hydroxyphenyl)propionate-Conjugated PAMAM Dendrimer (Phe-P). Since the PAMAM dendrimer is supplied as a methanol solution, the solvent was replaced with a mixture of 8 mL N,N-dimethylformamide (DMF) and 1 mL water to prepare the PAMAM dendrimer at a concentration of 200 mg (1.4 × 10-5 mol)/9 mL. A solution of compound 1 (37.0 mg) in DMF (1 mL) was then added dropwise to the PAMAM solution, and the reaction mixture was stirred for 2 h at room temperature. The resulting Phe-P was purified by diafiltration using an Amicon Ultra-4 (NMWL 5 kDa, Millipore Japan, Tokyo). After removing the solvent in vacuo, Phe-P was obtained as a yellow oil (140 mg, 63.4%). 1H NMR (D2O): δ 2.26-2.54 (m, PAMAM methylene), 2.66 (s, PAMAM methylene), 2.75-3.00 (overlapped, PAMAM methylene, propionate methylene), 3.02-3.18 (overlapped, PAMAM methylene, propionate methylene), 3.18-3.38 (m, PAMAM methylene), 3.43 (t, PAMAM methylene), 6.73 (d, CH), 7.02 (d, CH). Acetyl-PAMAM (Ac-P). To a solution of Phe-P (50 mg) in dimethyl sulfoxide (DMSO, 2 mL) was added a mixture of acetic anhydride (48.8 µL) and diisopropylethylamine (89.9 µL). After stirring for 2 days at room temperature, the solution was evaporated in vacuo. The residue was dissolved in 1 N NaOH
Uehara et al.
solution (5 mL) and heated at 50 °C for 1 h. The resulting Ac-P was purified by diafiltration using the Amicon Ultra-4. After removing the solvent in vacuo, Ac-P was obtained as a yellow oil (33 mg, 55.8%).1H NMR (D2O): δ 1.95 (s, CH3), 2.26-2.51 (m, PAMAM methylene), 2.62 (s, PAMAM methylene), 2.81 (overlapped, PAMAM methylene, propionate methylene), 3.04-3.38 (overlapped, PAMAM methylene, propionate methylene), 6.77 (d, CH), 7.06 (d, CH). Succinyl-PAMAM (Suc-P). A solution of succinic anhydride (330 mg) in THF (5 mL) was added dropwise to a solution of Phe-P (150 mg) dissolved in a mixture of THF (3.3 mL) and 1 N NaOH (7.7 mL) while maintaining the reaction solution at pH 9 by an addition of 1 N NaOH. The reaction mixture was then stirred for 2 h at room temperature. The solvent was removed in vacuo, and the crude Suc-P dissolved in 0.5 M ammonium acetate buffer (pH 7.4) was purified by diafiltration using the Amicon Ultra-4. After removing the solvent in vacuo, Suc-P was obtained as a yellow oil (130 mg, 61.8%). 1H NMR (D2O): δ 2.38-2.58 (m, PAMAM methylene), 2.78 (s, PAMAM methylene), 2.95 (overlapped, PAMAM methylene, propionate methylene), 3.12-3.42 (overlapped, PAMAM methylene, propionate methylene), 6.77 (d, CH), 7.07 (d, CH). γ-Glutamyl-PAMAM (Glu-P). To a solution of Phe-P (135 mg) in DMSO (1.5 mL) was added a mixed solution of compound 2 (380 mg) and diisopropylethylamine (248 µL) in DMSO (2 mL), and the reaction mixture was stirred for 2 h at room temperature. After removing the solvent in vacuo, the residue was purified by gel permeation chromatography (TOYOPEARL HW-40S, 1 cm × 25 cm) using methanol as an eluent to produce Boc-Glu(OtBu)-Phe-P. Boc-Glu(OtBu)-Phe-P was then dissolved in a mixture of trifluoroacetic acid (TFA, 4.5 mL) and anisole (0.5 mL), and the reaction mixture was stirred for 2 h at room temperature. The solution was neutralized with 1 N NaOH under cooling in an ice bath, and the conjugate (GluP) was purified by diafiltration using the Amicon Ultra-4. After removing the solvent in vacuo, Glu-P was obtained as a yellow oil (81 mg, 43.5%). 1H NMR (D2O): δ 2.08 (d, CH2), 2.21-2.46 (m, PAMAM methylene, glutamic acid), 2.59 (s, PAMAM methylene), 2.78 (overlapped, PAMAM methylene, propionate methylene), 3.04-3.35 (m, PAMAM methylene), 6.73 (d, CH), 7.02 (d, CH). Succinyl γ-Glutamyl PAMAM (Suc-Glu-P). To a solution of Glu-P (54 mg) in DMSO (2 mL) was added a mixed solution of diisopropylethylamine (123 µL) and succinic anhydride (42 mg) in DMSO (1 mL). The reaction mixture was stirred overnight at room temperature, and the resulting Suc-Glu-P was purified by diafiltration using the Amicon Ultra-4. After removing the solvent in vacuo, Suc-Glu-P was obtained as a yellow oil (26 mg, 39.3%). 1H NMR (D2O): δ 1.85 (d, CH2), 2.10 (d, glutamic acid), 2.27 (d, glutamic acid), 2.32-2.57 (m, PAMAM methylene, glutamic acid), 2.65 (s, PAMAM methylene), 2.83 (m, PAMAM methylene), 2.95 (overlapped, PAMAM methylene, propionate methylene), 3.05-3.41 (m, PAMAM methylene), 3.48 (s, PAMAM methylene), 3.73 (t, glutamic acid), 4.11 (t, glutamic acid), 7.06 (d, CH), 7.64 (d, CH). Acetyl γ-Glutamyl PAMAM (Ac-Glu-P). A mixed solution of diisopropylethylamine (190 µL) and acetic anhydride (51 µL) in DMSO (1 mL) was added to a solution of Glu-P (40 mg) in DMSO (1 mL). The reaction mixture was stirred overnight at room temperature. The resulting Ac-Glu-P was purified by diafiltration using the Amicon Ultra-4. After removing the solvent in vacuo, Ac-Glu-P was obtained as a yellow oil (32 mg, 72.7%). 1H NMR (D2O): δ 1.84 (d, CH2), 1.97-2.15 (m, glutamic acid), 2.27 (t, glutamic acid), 2.35-2.66 (m, PAMAM methylene, glutamic acid), 2.69-3.19 (m, PAMAM methylene, propionate methylene), 3.23-3.53 (m, PAMAM methylene),
γ-Glutamyl PAMAM Dendrimer-Based Targeting Devices
Bioconjugate Chem., Vol. 21, No. 1, 2010 177 Scheme 1a
Figure 1. Cumulative radioactivity levels in urine and feces for 48 h postinjection of [125I]PAMAM derivatives in normal mice.
3.61 (t, glutamic acid), 4.11 (t, glutamic acid), 6.78 (d, CH), 7.25 (2d, CH). 125 I-Labeled PAMAM Derivatives ([125I]PAMAM Derivatives). A solution of 169 µg of freshly prepared chloramine-T in 10 µL of 0.1 M phosphate buffer (pH 7.4) was added to a mixture of Na[125I]I (5 µL) in 0.01 M NaOH and a PAMAM derivative (0.22 mg/100 µL) in 0.1 M phosphate buffer (pH 7.4). The reaction mixture was incubated for 15 min before quenching the reaction with aqueous sodium bisulfite (73 µg, 10 µL). All the PAMAM derivatives were radioiodinated under similar conditions. [125I]PAMAM derivatives were then purified by diafiltration using the Microcon YM-3 (NMWL 3 kDa, Millipore Japan). The radiochemical purity was determined by TLC. Zeta Potential and Particle Size of PAMAM Derivatives. Each PAMAM derivatives were dissolved in 0.05 M phosphate buffer (pH 7.4) at a concentration of 5 mg/3-5 mL. The zeta potentials and the sizes of the PAMAM derivatives were determined by an electrophoretic light scattering spectrophotometer and by a dynamic light scattering. Isoelectric Points (pIs) of [125I]PAMAM Derivatives. Polyacrylamide isoelectric focusing (IEF) was performed to determine the pI values of each [125I[PAMAM derivative. Aliquots of each [125I]PAMAM derivative (3 µL) along with standard solution were applied onto the gel (TEFCO, Tokyo). IEF was performed at 100 V for 1 h, 200 V for 1 h, and 500 V for 30 min. The IEF gel was autoradiographed to reveal radioactive bands using a bioimaging analyzer, FUJIX BASS2000 II (Fujifilm, Tokyo). The molecular markers were stained with Coomassie Brilliant. Biodistribution of [125I]PAMAM Derivatives. Animal studies were conducted in accordance with our institutional guidelines and were approved by the Chiba University Animal Care Committee. The biodistribution of radioactivity after intravenous administration of [125I]PAMAM derivatives (70.4 fmol, 100 µL) to 6-week-old mice was determined at 10 min, 1 h, 3 h, 24 h, and 48 h postinjection. Groups of 4-5 mice were used for the experiments. Organs of interest were removed and weighed, and the radioactivity counts were determined with an auto well gamma counter. Urine and feces were collected for 48 h postinjection, and the radioactivity counts were also determined (Figure 1). Statistical Analysis. Data are expressed as the means ( SD where appropriate. Results were statistically analyzed using the unpaired Student’s t-test. Differences were considered statistically significant when p was
