Synthesis and Biological Evaluation of Diethylenetriamine Pentaacetic

Sep 2, 2005 - These properties are highly desirable for a targeted radiopharmaceutical for imaging neoplastic cells overpressing folate receptors in l...
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Bioconjugate Chem. 2005, 16, 1126−1132

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Synthesis and Biological Evaluation of Diethylenetriamine Pentaacetic acid-Polyethylene Glycol-Folate: A New Folate-Derived, 99mTc-Based Radiopharmaceutical Min Liu,† Wen Xu,† Ling-jie Xu,† Gao-ren Zhong,† Shao-liang Chen,‡ and Wei-yue Lu*,† Fudan-Pharmco Targeting Drug Research Center, Department of Pharmaceutics, School of Pharmacy, University of Fudan, Shanghai, 200032, China, and Department of Nuclear Medicine, Zhongshan Hospital, Shanghai, China. Received April 21, 2005; Revised Manuscript Received August 2, 2005

99m Technetium-labeled diethylenetriamine pentaacetic acid-polyethylene glycol-folate (DTPA-PEGfolate) was synthesized and tested as a radiopharmaceutical agent, which targeted the lymphatic system with metastatic tumor. Folic acid was reacted with H2N-PEG-NH2 to yield H2N-PEGfolate. After purification by anion-exchange chromatography, the product was reacted with cyclic DTPA. By removal of unreacted DTPA by size-exclusion chromatography, DTPA-PEG-Folate was obtained. Fluorescein-5-isothiocyanate (FITC)-labeled DTPA-PEG-folate and DTPA-PEG-OCH3 were prepared via a dicyclohexylcarbodiimide-mediated coupling. In vitro competitive binding test showed that the uptake of [125I] folic acid was inhibited by DTPA-PEG-folate and the 50% inhibitory concentration was 4.37 pmol/L (R2 ) 0.9922). The relative affinity of DTPA-PEG-FITC was 0.18 for human folate receptor comparing with folic acid. In cultured tumor cells, uptake of fluorescence-labeled DTPAPEG-folate was found to increase significantly in folate-deficient medium compared with that of untargeted DTPA-PEG-OCH3 and FITC-ethylenediamine. The competition with free folic acid blocked the cell uptake of DTPA-PEG-folate. These results confirmed the DTPA-PEG-folate entered into KB cells through the folate receptor endocytosis pathway in vitro. The radiolabeled yield of [99mTc] DTPA-PEG-folate was in excess of 98%, and specific activities of 7.4 kBq (0.2µCi/µg) were achieved. After subcutaneous injection, [99mTc] DTPA-PEG-folate exhibited an initial increase and successive decline of accumulation in popliteal nodes in normal Wistar rats. Expect for the kidney, uptake by other tissues was rather low. In a normal rabbit imagine study, the lymphatic vessels were readily visualized by single-photon-emmission computed tomography following subcutaneous injection of [99mTc] DTPA-PEG-folate. In conclusion, the [99mTc] DTPA-PEG-folate conjugate may have a potential as a lymphatic tumor-targeted radiopharmaceutical.

INTRODUCTION

The lymphatic system comprises a network of lymphatic vessels and lymph nodes throughout the body. It often serves as a primary route for dissemination of many solid tumors, including carcinomas of colon, lung, breast, renal, cervix, and prostate (1-4). Furthermore, lymph nodes could provide ideal tumor-cell “incubators” with long residence time. Assessment of lymph node status could determine the stage of numerous malignancies. Therefore, it would be great interest to develop reliable techniques for evaluating the lymph nodes stage in cancer patients. The direct lymphography (such as lymphoangiogram) and other modern indirect techniques (such as compounded tomography and magnetic resonance) are commonly used for detecting nodal metastases. However, these methods could only detect the lymph node’s abnormality by nodal enlargement, but that does not always imply malignant involvement at the initial stage (many nodes have been infiltrated or replaced by tumor without change in size) (5). Since Sherman first reported lymph * Corresponding author. School of Pharmacy, University of Fudan, 138 Yixueyuan load, 190 BOX, Shanghai, 200032, People’s Republic of China. Fax: (86 21) 64178790. E-mail: [email protected]. † University of Fudan. ‡ Zhongshan Hospital.

node accumulation of interstitially injected radioactive colloidal gold in 1953, many studies have exploited radiolabeled colloidal particles (liposomes, emulsions, or micelles) and macromolecules (dextran, polylysine, human serum albumin, monoclonal antibody and their fragments, polyethylene glycol, and other synthetic polymers) for better detection of lymph nodes (5-14). Among these radiological agents, only monoclonal antibodies could actively bind and target to tumor cells in the lymph nodes and detect occult metastases of tumor (10, 11). On the contrary, labeling with monoclonal antibodies has been rather disappointed because of its concomitant clearance by lymph node macrophages as well as the lack of a uniform expression of surface antigen specific for a designed monoclonal antibody. Folate receptor is a glycosyl-phosphatidylinositallinked membrane glycoprotein with an apparent molecular weight of ∼38-40 kDa. It preferentially expresses with enhanced binding affinity for folic acid in many types of tumors, including ovarian, lung, colorectal, renal, breast cell cancers, and non-Hodgkin’s lymphomas (15). Radioactive tracers and drug carriers utilizing folate conjugates for tumor targeting have been studied (1521). Additionally, folate receptor was associated with parameters of biological aggressiveness in cancers. With high histological grade, advanced stage, and serious histology in ovarian neoplasmas, folate receptor was overexpressed to a higher degree. A multiple comparison

10.1021/bc050122m CCC: $30.25 © 2005 American Chemical Society Published on Web 09/02/2005

Synthesis and Evaluation of DTPA−PEG−Folate

analysis showed that folate receptor expression of stage IV and omental metastases was significantly higher that that in stage I (22). Moreover, it had been reported that folate-fluorescein could be easily distinguished the tumor loci of submillimeter size, which spread through the vascular system, from adjacent normal tissues (23). Thus, folic acid owned a potential value as a targeting ligand for metastatic tumor through lymphatic system. To our knowledge, few were reported in the literatures concerning folate conjugates for lymphatic targeting. As a first step toward the design of a radiological agent for imaging the tumor metastatic lymph node, we adopted a macromolecular receptor-binding radiotracer, [99mTc] diethylenetriamine pentaacetic acid-polyethylene glycolfolate (DTPA-PEG-folate). The only study where PEG was adopted for targeting lymphatic system after subcutaneous injection was reported by Desser et al. (9). In their work, PEG conjugate provided excellent popliteal nodal enhancement. However, further study on delivering H2N-PEG-folate conjugates to tumor metastatic lymph nodes via receptor-mediated pathways has not yet been reported. In this paper, we described the synthesis of DTPA-PEG-folate conjugates, which displayed good targeting activities for tumor cells via the folate receptor in vitro, as well as lymphatic system in the normal animal.

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Figure 1. Purified H2N-PEG-folate on a DEAE-Sepharose anion-exchange column by gradient elution. Peak 1, uncharged impurities including H2N-PEG-NH2 and some small molecules; peak 2, H2N-PEG-folate; peak 3, folate-PEG-folate; peak 4, folic acid.

EXPERIMENTAL PROCEDURES

Materials. H2N-PEG-NH2 (MW 2000) and H2NPEG-OCH3 (MW 2000) were prepared as described previously (24). Cyclic DTPA dianhydride, folic acid, N,N′dicyclohexylcarbodiimide, and ethylenediamine (EDA) were purchased from Sigma (St. Louise, Mo, USA). Fluorescein-5-isothiocyanate (FITC) was obtained from Molecular Probes, Co. (Eugene, Oregon, USA). [125I] folic acid came from Naval radioimmunity center (Beijing, China, the radioactivity was 7.69 TBq/mmol). Other chemicals reagents used in the study were certified analytical reagent grade (Shanghai Chemical Reagent Company, Shanghai, China). Solvents for high-performance liqui chromatography (HPLC) were HPLC grade accordingly. KB cell, a human oral epidermoid carcinoma cell line that overexpresses folate receptor, was provided by Shanghai Institutes for Biological Sciences (Chinese Academic of Sciences). Synthesis of H2N-PEG-Folate. Two grams (1 mmol) of H2N-PEG-NH2 was dissolved in 15 mL of dry dimethyl sulfoxide containing an equimolar folic acid. Dicyclohexylcarbodiimide (0.2 g) and 40µL of pyridine were added. The mixture was stirred overnight in the dark at 25 °C. Deionized water (30 mL) was added, ss and the insoluble byproduct, dicyclohexylurea, was removed by centrifugation. The supernate was dialyzed against deionized water to remove dimethyl sulfoxide in the reaction mixture and lyophilized. A yellow powder (0.35 g) was obtained. The H2N-PEG-folate was separated from folate-PEG-folate and unreacted folic acid and H2N-PEG-NH2 by using a DEAE-sepharose anionexchange column (26 mm × 10 cm, Biosciences, Uppsala, Sweden) on a A ¨ KTA explorer 100 system (Amersham Biosciences, Uppsala, Sweden) consisting of Pump P-900, Pump P-950, Monitor UV-900, and Frac-900 at room temperature. The column was loaded with 10 mg of H2N-PEG-folate crude sample per mL bed volume. The column was then washed with a gradient (eluent A, 100 mM ammonium acetate buffer, pH 10.0; eluent B, water; gradient, 90% B for 3 column volumes, 90-75% B at 3 column volumes, keeping 75% B and 50% B at 1 column

Figure 2. Analysis for H2N-PEG-folate and impurities on a TSK GEL G4000PWXL column 4.6 mm × 30 cm. Elution, 5 mM NaCl; flow rate, 0.5 mL/min; temperature, 25 °C; detection, UV@280 nm).

volume, respectively, in the end washing the column with 0.5 M NaCl, at a flow rate of 5 mL/min). The result was seen in Figure 1. Fractions were collected, desalted, and lyophilized. Analytical chromatography was performed on an Agilent 1100 series quaternary HPLC system (Palo Alto, California, USA) equipped with a vacuum degasser, an autosampler, a thermostetted column, and a UV and refractive index detector. The chemical purity of H2NPEG-folate was examined by analytical HPLC on a 300 × 7.8 mm inside diameter TSK-GEL G4000PWXL (10 µm particle size, 300 Å pore size, Tosoh Corporation, Minato-ku, Tokyo, Japan) with a TSK-GUARDCOLUMN at 25 °C [H2N-PEG-folate, tR ) 21.76 min; folic acid, tR ) 19.97 min; folate-PEG-folate, tR ) 18.89 min; H2N-PEG-NH2, tR ) 31.84 min; eluent, 5 mM NaCl]. The results were shown in Figure 2. 1H NMR (500 MHz, D2O) δ (ppm) for H2N-PEG-folate: 8.67 (s, C7-H, 1H), 7.66 (d, 2′,6′-H, 2H), 6.63 (d, 3′,5′-H, 2H), 4.34 (minor and major RCH2 of Glu, 1H), and ∼2.0-2.4 (m, β, γ-CH2 of GLu, 4H) for folate; ∼3.1-4.0 (m, -OCH2CH2) for PEG. Synthesis of DTPA-PEG-Folate. DTPA-PEGfolate was obtained by reacting 200 mg H2N-PEG-folate

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Figure 3. Analysis for DTPA-PEG-folate and impurities on a TSK GEL G4000PWXL column 4.6 mm × 30 cm. Elution, 5 mM NaCl; flow rate, 0.5 mL/min; temperature, 25 °C; detection, UV@280 nm for both DTPA-PEG-folate and H2N-PEGfolate, UV@200 nm for DTPA).

with 1.25 times molar quantity of DTPA dianhydride in 10 mL of dimethyl sulfoxide at 25 °C. When analytical HPLC showed the absence of the H2N-PEG-folate, 10 mL of deionized water was added to the mixture. The mixture was dialyzed against deionized water to remove dimethyl sulfoxide in the reaction solution and lyophilized. The DTPA-PEG-folate was separated using the A ¨ KTA explorer 100 system equipped with a Sephadex G-15 column to remove unreacted DTPA. The eluent was 50 mM acetate buffer (pH 3.2) containing 50% alcohol with 1 mL/min flow rate. The sample was collected, desalted, and lyophilized. Analytical HPLC on the TSK GEL G4000PWXL column revealed a single peak with a retention time of 17.59 min (eluent, 5 mM NaCl). The starting material DTPA and H2N-PEG-folate was individually eluted at 18.84 min and 21.76 min. Figure 3 revealed the results. 1H NMR (500 MHz, D2O) δ (ppm) for DTPA-PEG-folate: 3.29 (t, C5, C7-2H) and 3.42 (t, C4, C8-2H) for DTPA; 8.67 (s, C7-H, 1H), 7.66 (d, 2′,6′-H, 2H), 6.63 (d, 3′,5′-H, 2H), 4.34 (minor and major RCH2 of Glu, 1H), and ∼2.0-2.4 (m, β,γ-CH2 of GLu, 4H) for folate; ∼3.6-3.8 (bs, -OCH2CH2) for PEG. Similarly, we used the above method to obtain the conjugate of DTPA-PEG-OCH3. 1H NMR (500 MHz, D2O) δ (ppm): 3.29 (t, C5, C7-2H) and 3.42 (t, C4, C82H) for DTPA; ∼3.6-3.8 (bs, -OCH2CH2) for PEG. Synthesis and Purification of the FITC-EDA. FITC (62 mg, 0.16 mmol) dispersed in 0.2 mL of N,N′dimethylformamide was reacted with 50 molar equiv of EDA at 25 °C for 30 min. The resulting EDA-FITC was mixed with the same volume of deionized water and separated from unreacted FITC and EDA by HPLC on SymmetryPrep C-18 reversed column (300 mm × 19 mm, Waters Co.) using a stepped gradient (eluent A, 10 mM CH3COONH4; eluent B, 15% acetonitrile in 10 mM CH3COONH4; gradient, 2 column volume for eluent A, and then 2 column volume for eluent B at a flow rate 10 mL/min). The eluent time for EDA, EDA-FITC, and FITC were 8.2, 30, and 39 min, respectively. Analytical chromatography was performed on an SHIMADZA HPLC equipped with a liquid chromatography, a vacuum degasser, an autosampler, and a fluorescence detector (Japan). The chemical purity and identity of EDA-FITC was examined by analytical HPLC on a Diamonsil C-18 reversed-phase column (200 mm × 4.6

Liu et al.

mm, Dikma Technologies) [EDA-FITC, TR ) 8.5 min; FITC, TR ) 13.2 min; the eluent, 15% acetonitrile in 10 mM CH3COONH4 at a flow rate of 0.8 mL/min; Fluorescence detection, Ex ) 490 nm, Em ) 518 nm]. Highresolution mass spectroscopy (MS) (Agilent, California, USA): EDA-FITC, C23H19O5N3S [M + H]+ 450.5, found 450.0. Fluorescent Labeling of PEG Derivatives. DTPAPEG-folate (5.6 mg, 2 mmol) and dicyclohexylcarbodiimide (1.5 mg, 7.3 mmol) were dissolved in 0.25 mL of dimethyl sulfoxide containing 10 µL of pyridine. The reaction mixture was stirred for 1 h before FITC-EDA (0.9 mg, 2 mmol) was added. The free FITC-EDA was monitored of by analytical HPLC on a TSK GEL G4000PWXL column (300 mm × 7.8 mm,) [FITC-labeled DTPA-PEG-folate, TR ) 26.1 min; FITC-EDA, TR ) 35.9 min; the eluent, 20% acetonitrile in 100 mM NaCl at a flow rate of 0.5 mL/min; fluorescence detection, Ex ) 490 nm, Em ) 518 nm]. As soon as the free FITCEDA disappeared, the same amount of deionized water was added to the reaction mixture and the unsolved material was filtered. Other small molecules were removed by G-15 sephadex column. The chemical purity assay of FITC-labeled DTPA-PEG-folate was conducted as described above. As a nontargeted agent, FITC-labeled DTPA-PEGOCH3 was prepared similarly. In Vitro Competitive Binding Assay. All biological experiments were carried out with KB cell line, enriched with folate receptors. The competitive binding-assay was conducted as described previously (18, 25, 26) with slight modification. Briefly, 24 h prior to experiment, KB cell were cultured in folate-deficient RPMI medium (RPMI1640, Gibcol Co., USA) supplemented with 10%v/v heatinactivated fetal calf serum (Gibcol Co., USA), and temperature was maintained at 37 °C in an atmosphere containing 5% carbon dioxide/95% air humidified atmosphere. A total of 5 × 105 KB cells in 0.5 mL of serumsupplemented folate-deficient RPMI medium was added to the plastic test tube containing 0.1 mL [125I] folic acid. Nonradiolabeled folic acid, DTPA-PEG-OCH3, and DTPA-PEG-folate with different concentrations were added to the cell suspension, respectively. After incubation for 1 h at 4 °C, the KB cells were centrifuged to remove the unbinding [125I] folic acid and washed 3 times with phosphated-buffered saline and placed in a γ counter (SN-682 Radioimmunity γ-Counter, Shanghai Institutes for Nuclear Sciences, China) to determine the amount of cell-associated [125I] folic acid. The total binding and nonspecific binding were determined at the same time. All data were recounted depending on [125I] decay, and the result was shown in Figure 4. Fluorescent Microscopy Studies. KB cells were transferred to 33-mm culture dishes at 2 × 105 cells per dish 24 h prior to the assay adopting folate-deficient RPMI-1640 medium supplemented with 10%v/v heatinactivated fetal calf serum. The cells were incubated for 24 h at 37 °C with 0.5 µM solution of FITC-labeled DTPA-PEG-folate, DTPA-PEG-OCH3, and FITCEDA diluted in folate-deficient culture medium, respectively. In free folate competition studies, 1 mM folic acid was added to incubation medium. After washing three times with PBS, the cell-associated fluorescence was determined by FITC fluorescence on an Olympus ImagePro Plus fluorescence microscope (Japan). Radiolabeling of DTPA-PEG-Folate. To label DTPA-PEG-folate with 99mTc as described previously (13, 18), first a solution of DTPA-PEG-folate (10 mg/ mL) in 10 mmol/L phosphate buffer (pH 7.2, 0.9%NaCl)

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Synthesis and Evaluation of DTPA−PEG−Folate

were euthanized and popliteal lymph nodes and other tissues were excised and assayed for radioactivity. The biodistribution of the tracer in each animal was calculated as a percentage of injected dose per gram of tissue wet weight (%ID/g), using reference counts from an accurately diluted sample of the original injection. In addition, [99mTc] DTPA-PEG-folate (0.1 mL, 10 mg/ mL) was injected into the web space between the second and third in both hind feed of the rabbits. Then the continuous imaging was performed in whole body and the radioactivity change of [99mTc] DTPA-PEG-folate was measured by single-photon-emmission computed tomography (SPECT) at 5, 15, and 120 min, respectively. The experiment results were given in Figure 7. RESULTS

Figure 4. Competition study of binding to cellular folate receptor with free folic acid (--2--), DTPA-PEG-OCH3 (--9--), and DTPA-PEG-folate (--b--). Radiolabeled folic acid was added to KB cell monolayers, which were incubated at 4 °C for 1 h with or without the competitions at the concentrations shown (n ) 3).

was mixed with 0.02 mL solution of tin chloride (10 mg SnCl2- 2H2O in 1 mL of 1 mol/L HCl followed by 8.8 mL of distilled water). Second, the mixture was incubated with freshly eluted 0.1 mL (approximately 74 MBq) of sodium pertechnetate (NaTcO4). The reaction was allowed to stand for 15 min at room temperature. The purity of radiolabeled compound was analyzed by paper chromatography (27-29) and by electrophoresis (30). The paper chromatography was used for distinguishing [99mTc] DTPA-PEG-folate from unbounded Na99mTcO4, and the electrophoresis was performed for determining the [99mTc] colloid. Biodistribution and Imaging of [99mTc]DTPAPEG-Folate. The biodistribution of Wistar rats was obtained following subcutaneous injection of [99mTc] DTPA-PEG-folate. During the test, Wistar rats (three weeks old) were fed on normal folate-rich diet. [99mTc] DTPA-PEG-folate (0.1 mL, 10 mg/ml) was injected into foodpad of Wistar rats. Three rats were studied at each time point. At different times after injection, the animals

Synthesis. The general strategy of the synthesis is shown in Figure 8. H2N-PEG-folate was synthesized as previously described (14). The conjugation of folic acid to the unprotected primary amine of PEG was a complicated process. Because of no selectivity of two end functional groups of H2N-PEG-NH2, the byproduct folate-PEG-folate was synthesized as soon as the yield ratio of H2N-PEG-folate was over 25% (determined by HPLC). Additionally, unreacted folic acid and H2NPEG-NH2 interfered with the purity of H2N-PEGfolate. Because of the ionic difference of these molecules, we decided to adopt ion-exchange chromatography for separation. Figure 1 showed the optimized gradient purification of crude H2N-PEG-folate performed on a DEAE-Sepharose anion-exchange column. As expected, the uncharged H2N-PEG-NH2 was eluted first, followed by H2N-PEG-folate, folate-PEG-folate, and folic acid successively. The purity was confirmed by analytical HPLC (Figure 2). The liquid exclusion-adsorption chromatography (LEAC), which we reported previously (17), was an important method for a qualifying HPLC analysis of PEG derivatives. On the basis of LEAC, H2N-PEGfolate could be efficiently identified from other impurities. Since H2N-PEG-NH2 could not be detected by UV spectroscope, we had adopted refractive index detection (data not shown). The DTPA-PEG-folate conjugate was obtained by reaction of DTPA dianhydride with the purified H2N-

Figure 5. Cellular accumulation of fluorescein-labeled PEG conjugates in KB cells. Cells were incubated with 0.5 µM substrates for 24 h at 37 °C and photographed in both the phase-contrast and fluorescence mode on microscope as described in methods. Left, KB cells treated with FITC-labeled DTPA-PEG-folate; middle, cells treated with DTPA-PEG-OCH3; right, cells treated with FITC-labeled DTPA-PEG-folate containing 1 mM of free folic acid. Upper panels, micrographs taken in the phase-contrast mode; lower panels, the same field viewed in the fluorescence mode.

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Figure 6. Percent injected dose vs time of lymphotrophic imaging agent [99mTc] DTPA-PEG-Folate using the Wistar rats. Data were acquired following subcutaneous administration of the radiotracers (n ) 3; DTPA-PEG-folate dose ) 1 mg per rat).

Figure 8. Synthesis of DTPA-PEG-folate and FITC-labeled PEG conjugates.

Figure 7. SPECT imagines of rabbits injected subcutaneously in the hind feet with [99mTc] DTPA-PEG-folate. (n ) 2; DTPAPEG-folate dose ) 1 mg/per rabbit).

PEG-folate. Because of higher reactive activity of cyclic DTPA, the synthesis was completed within 10 min. To control the process more easily, the amount of DTPA was a little more than that of H2N-PEG-folate. The major side product DTPA-bis-PEG-folate did not emerge when the H2N-PEG-folate had run out. Therefore, the excess of unreacted DTPA became the only impurity in the reaction mixture and was easily removed by size-exclusion chromatography. Figure 3 showed the purity of DTPA-PEG-folate. EDA was conjugated to FITC to serve as a linker between the fluorescein and DTPA-PEG-Folate. The mass spectroscope analysis showed the FITC-EDA [M + H]+ parent ion peak at m/e ) 450.5 (calculated 450.0), indicating the desired 1/1conjugation ratio. The chemical purity was confirmed by analytical HPLC. FITC-labeled PEG conjugates were synthesized in order to evaluate, by fluorescence-sensitive techniques, whether the conjugated folate altered the PEG compounds accumulation in KB cells. FITC-labeled DTPA-PEG-folate and DTPAPEG-OCH3 were prepared via a dicyclohexylcarbodiimide-mediated coupling. The chemical purity was also confirmed by analytical HPLC. In fact, the FITC labeling in the DTPA-PEG compounds was randomly placed. It is possible that the FITC-EDA moiety reacted with not the carboxylates of DTPA but also the lone remaining carboxylate group of folic acid. Competition Binding Study in Vitro. To determine whether DTPA-PEG-folate could compete as the folate receptor against [125I] folic acid, the in vitro binding tests on KB cell line were carried out. As shown in Figure 4, with the increasing concentration of the unlabeled folic acid and DTPA-PEG-folate, the uptake of [125I] folic acid by KB cell gradually decreased. As a control group,

unlabeled DTPA-PEG-OCH3 had no effect on the uptake of [125I] folic acid. Therefore, DTPA-PEG-folate, as well as folic acid, could combine with folate-receptor enriched on KB cell membrane. The data were analyzed adopting Graphpad Prim 4.0 software. Compared with free folic acid, DTPA-PEG-folate had higher IC50 values (folic acid, IC50 ) 0.7726 pmon/L; DTPA-PEG-folate, IC50 ) 4.37 pmol/L (R2 ) 0.9922). The relative affinity was adopted to evaluate a ligand’s ability of folate derivatives to directly compete with folic acid for binding to cell surface receptors. Simply, the relative affinity was defined as the inverse molar ratio of compound required to displace 50% of [125I] folic acid bound to folate receptor on KB cells, and the relative affinity of folic acid for the folate receptor was set to 1. Therefore, we calculated that DTPA-PEG-folate had an affinity of 0.18 relative to that of folic acid for human folate receptors. Thus, DTPA-PEG modification of folate lowered the vitamin’s intrinsic affinity for folate receptors. The possible reason may be the interference by PEG lineal molecule. Cell Uptake Studies of Fluorescence-Labeled PEG Derivatives. All biological experiments were carried out with the KB cell line, enriched with folate receptors. The cell uptake of FITC-labeled compounds was evaluated by the fluorescent microscopy. The results clearly indicated that the uptake of DTPA-PEG-folate increased significantly in folate-deficient medium compared with the uptake of untargeted DTPA-PEG-OCH3 and FITC-EDA. The competition with free folic acid blocked the cell uptake of DTPA-PEG-folate (shown in Figure 5). These results confirmed the DTPA-PEGfolate entered into KB cells through the folate receptor endocytosis pathway. Radiolabeling. The paper chromatography demonstrated an absence of unbounded Na99mTcO4 that would be detected by the radioactivity profile at the front (data not shown). [99mTc] Colloid was detected in the product by electrophoresis, which would be existed by radioactivity profile at the end (data not shown). The radiolabeled yield was in excess of 98%. The specific activity was 7.4 kBq (0.2µCi/µg).

Synthesis and Evaluation of DTPA−PEG−Folate

Biodistribution. Figure 6 showed the %ID/g for the popliteal nodes and other tissues at 15 min, 30 min, 1 h, 2 h, and 4 h postadministration of [99mTc] DTPA-PEGfolate. In this test, a fast accumulation and clearance was obtained throughout popliteal nodes. The amount of radioactivity in the lymph nodes was 5.913 ( 1.549 %ID/g at 15 min postinjection, increased to the maximum (13.432 ( 2.207 %ID/g) at 1 h postinjection and then decreased to 2.314 ( 0.278 %ID/g at 4 h. In addition, this radiotracer kept a high accumulation in the kidney during the whole test. Figure 7 showed continuous images obtained 2 h after subcutaneous injection of [99mTc] DTPA-PEG-folate at web space between second and third toes in both hind feet of rabbits. The lymphatic vessels were prominent as early as 5 min after injection. The lymph images continued to develop at 15 min as well as the early development of a body pool. Kidneys and bladders began to be prominent at this time. At the end of 2 h, node images faded. DISCUSSION

The lymphatic route is known to be one of the primary pathways for tumor metastasis. Tumor cells that have detached from the tissue or have invaded a lymphatic vessel became trapped in the meshwork of a lymph node. Some metastatic cancers appear to spread via lymphatics, particularly those of epithelial origin including breast, colon, lung, ovary, and prostate (1-4). The previous studies have shown that the folate receptor is overexpressed in many types of tumors derived from epithelial cancers (15). Furthermore, high dedifferentiated metastatic cancers express considerably more folate receptor than their localized, low-grade counterparts (22). Therefore, with respect to folic acid with high affinity for folate receptors (Kd ) 10-9-10-10 M) (31, 32), the vitamin was worthy of attention as a targeting ligand for metastatic tumor thought lymphatic system. To our knowledge, most studies about tumor located the lymphatic system focused on monoclonal antibodies, which could actively bind and target to tumor cells in the lymph nodes (10, 11). Compared with monoclonal antibody, folic acid has lower imunogenicity and relatively simpler chemistry. Thus, folate conjugate was made for targeting lymphatic tumor in this study. Otherwise, the widely used macromolecules targeting the lymphatic system include dextran, polylysine, human serum albumin, monoclonal antibodies, and other synthetic polymers. Our choice of PEG as a molecular backbone was based on the following reason: PEG is hydrophilic and structurally flexible, potentially evading the recognization and phagocytosis by macrophage cells in the lymphatic system. This enables folatemodified PEG to selectively bind with a metastatic tumor-cell leading to receptor-mediated endocytosis. As a first step, in this paper, we prepared the folate conjugates, determined the folate receptors mediated endocytosis in vitro, and lymphatic targeting in normal animals. Our following study was the demonstration of the tumor model with lymphatic metastases to better verify folate conjugate targeting. Our results from competition binding study and cell uptake studies in vitro demonstrated the receptor-mediated endocytosis properties of the folate conjugate with a PEG backbone. The biodistribution and imaging measurement in a normal animal revealed a lymph targeting effect of DTPA-PEG-folate. These properties are highly desirable for a targeted radiopharmaceutical for imaging neoplastic cells overpressing folate receptors in lymphatic system.

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In the biological test, the DTPA-PEG-folate showed a high uptake in the kidneys in normal rabbits, consistent with the known presence of folate receptors in the proximal tubules of the kidneys (33-36). In fact, the amount of the radiopharmaceutical in the kidneys was affected by two factors: first, the kidneys behaving as a clearance organ; second, as a tissue capable of receptorspecific trace accumulation. We once determinated the biodistribution of [99m] DTPA-PEG-folate in the athymic mice. Dates showed that the kidneys’ uptake of the agent was 3.872 ( 0.294, 5.844 ( 0.442, and 0.130 ( 0.009 %ID/g (n ) 4) at 1 h, 4 h, and 24 h, respectively, at a dose of 1 mg per rat. The agent was absolutely cleared by the kidney at 24 h. At present, the folate receptor positive tumor model in the lymphatic system is being set up. The suitable time point would be identified for not only adequate clearance of the DTPA-PEG-folate in the kidneys but keeping folate receptor mediated bound in tumor. According to previous studies (14), compounds with MW > 16 000 Da are mainly absorbed by the lymphatic vessels, hardly gaining access to blood capillaries. In our paper, the molecular weight of DTPA-PEG-folate is about 2 800 Da. We considered whether the micelles appear during subcutaneous administration of DTPAPEG-folate. To test the hypothesis, we tried to determine the hydrodynamic diameter of DTPA-PEG-folate by dynamic light scattering (Zeta Potential/Particle Sizer NICOMP 380ZLS (PSS‚NICOMP, USA). The conjugate was assayed for 20 min at a concentration of 10 mg/mL in phosphate buffer (pH 7.2, 0.9% NaCl). However, there were no nanoparticles to be detected. Additionally, we investigated the biodistribution of DTPA-PEG-folate at different concentrations (from 0.1 to 10 mg/mL) after subcutaneous injection; the data still revealed the popliteal node targeting. In addition, DTPA-PEG-OCH3 also showed lymph nodes targeting after subcutaneous injection (13.243 ( 2.345 and 23.730 ( 5.473 %ID/g at 1 and 4 h, respectively, in athymic mice after subcutaneous injection at dose of 1 mg per rat). Therefore, we supposed the possible reasons resulting in the phenomenon, which might be due to its linear structure and negatively charged free carboxyl groups in the DTPA-PEG-folate conjugate. It was reported that negatively charged particles showed a faster lymph delivery than cationic charged or neutral ones (14). Certainly, this hypothesis still needs further verification. ACKNOWLEDGMENT

This work was supported by the National Science Foundation of China (No. 39670859) and by the Research Fund for the Doctoral Program of Higher Education (No. 20030246050). LITERATURE CITED (1) Swartz, M. A. (2001) The physiology of lymphatic system. Adv. Drug Deliv. Rev. 50, 3-20. (2) Ayhan, A., Glutekin, M., Taskiran, C., Celik, N. Y., Usubutun, A., Kucukali, T., and Yuce, K. (2005) Lymphatic metastasis in epithelial ovarian carcinoma with respect to clinicopathological variables. Gynecol. Oncol. 97, 400-404. (3) Gajdos, C., Tartter, P. I., and Bleiweiss, I. J. (1999) Lymphatic invasion, tumor size, and age are independent predictors of axillary lymph node metastases in women with Ti breast cancers. Ann. Surg. 230, 692-896. (4) Gunther, K., Leier, J., Henning, G., Dimmler, A., Weissbach, R., Hohenberger, W., and Forster, R. (2005) Prediction of lymph node metastasis in coloretal carcinoma by expression of chemokine receptor CCR7. Int. J. Cancer 116, 726-733.

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