Transferrin Conjugates of Triazacyclononane-Based Bifunctional NE3TA Chelates for PET Imaging: Synthesis, Cu-64 Radiolabeling, and In vitro and In vivo evaluation
Three different polyaminocarboxylate-based bifunctional NE3TA (7-[2-[carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) chelating agents were synthesized for potential use in copper 64-PET imaging applications. The bifunctional chelates were comparatively evaluated using transferrin (Tf) as a model targeting vector that binds to the transferrin receptor overexpressed in many different cancer cells. The transferrin conjugates of the NE3TA-based bifunctional chelates were evaluated for radiolabeling with 64Cu. In vitro stability and cellular uptake of 64Cu-radiolabeled conjugates were evaluated in human serum and prostate (PC-3) cancer cells, respectively. Among the three NE3TA-Tf conjugates tested, N-NE3TA-Tf was identified as the best conjugate for radiolabeling with 64Cu. N-NE3TA-Tf rapidly bound to 64Cu (>98% radiolabeling efficiency, 1 min, RT), and 64Cu-N-NE3TA-Tf remained stable in human serum for 2 days and demonstrated high uptake in PC-3 cancer cells. 64Cu-N-NE3TA-Tf was shown to have rapid blood clearance and increasing tumor uptake in PC-3 tumor bearing mice over a 24 h period. This bifunctional chelate presents highly efficient chelation chemistry with 64Cu under mild condition that can be applied for radiolabeling of various tumor-specific biomolecules with 64Cu for potential use in PET imaging applications.
Introduction<!>Instruments and analyses<!>2-{[(4-aminophenyl)methyl]({2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]ethyl})-amino}acetic acid (2)<!>2-({2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]ethyl}[(4-isothiocyanatophenyl)-methyl]amino)acetic acid (3)<!>2-{[1-(4-aminophenyl)-3-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]propan-2-yl](benzyl)amino}acetic acid (5)<!>2-[benzyl({1-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]-3-(4-isothiocyanatophenyl)propan-2-yl})amino]acetic acid (6)<!>Conjugation of NE3TA-based bifunctional chelating agents to transferrin and Spectroscopic Determination of Ligand to Protein (L/P) Ratio for transferrin conjugates<!>Radiolabeling of NE3TA-Tf conjugates with 64Cu<!>In vitro stability of 64Cu-radiolabeled NE3TA-Tf conjugates<!>Cell uptake studies<!>Biodistribution<!>Statistical Analysis<!>Synthesis of NE3TA-based bifunctional chelates and NE3TA-transferrin (Tf) conjugates<!>Radiolabeling and specific activity of NE3TA-Tf conjugates with 64Cu<!>Serum stability of 64Cu-radiolabeled NE3TA-Tf conjugates and 64Cu-C-DOTA-Tf<!>In vitro cellular uptake assay<!>Biodistribution study<!>Conclusion<!>
<p>Positron emission tomography (PET) is a non-invasive and sensitive nuclear imaging technique that uses two highly energetic photons (511 keV) produced from interaction of positrons emitted from decay of radionuclides and electrons. [1,2] Among the clinically available positron-emitting radionuclides, 18F (t1/2 = 110 min) has been successfully applied for PET imaging, and 18F-2-Fluoro-2-deoxy-glucose (18F-FDG) is one of the most frequently used tracers for detection of tumors. [3] However, the short half-life of 18F presents a limitation for its broad application in imaging of cancers, particularly for biomolecules with slow tumor accumulation such as antibodies. Copper-64 (t1/2 = 12.7 h) with a relatively longer half-life and useful decay property (Iβ+ =17%, Iβ− = 39%, IEC = 43%, β+max = 0.656 MeV) is an attractive metallic radionuclide for PET imaging. [4] Research efforts have been made to develop clinically viable bifunctional chelating agents for radioimmuno-PET imaging applications of 64Cu using tumor-specific antibodies or peptides. [4–6]</p><p>Macrocyclic polyaminocarboxylate-based chelating agents including DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N‴-tetraacetic acid), and CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid) have been evaluated for development of 64Cu-based PET imaging agents. [4–6] However, DOTA, TETA, and CB-TE2A are not optimal chelating agents of 64Cu for PET applications due to in vivo dissociation of 64Cu-radiolabeled complex or harsh radiolabeling conditions. [7–9]</p><p>Recently, we reported NE3TA (7-[2-[carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) and bifunctional versions of NE3TA (N-NE3TA and C-NE3TA, Figure 1) as potential chelating agents of 64Cu. [10–11] The heptadentate NE3TA analogues rapidly bound to 64Cu, and 64Cu-radiolabeled complexes of the chelates remained inert in serum. [11] Copper-64-radiolabeled complexes of C-NE3TA (7-[2-[carboxymethyl)amino]-3-(4-nitrophenyl)propyl]-1, 4,7-triazacyclononane-1,4-diacetic acid) and N-NE3TA (7-[2-[(carboxymethyl) amino]-2-(4-nitrophenyl)methyl]-1,4,7-triazacyclononane-1,4-diacetic acid) were shown to have lower uptake in normal organs and more rapid blood and muscle clearance and superior in vivo stability than the 64Cu-C-DOTA complex. [10]</p><p>With the encouraging data on chelation chemistry of bifunctional NE3TA analogues with 64Cu, we proceeded with evaluation of the bifunctional NE3TA-based chelates for targeted PET imaging using transferrin as a model tumor targeting vector. Transferrin (Tf) binds to ferric iron (Fe3+) in circulation and transports the iron into cells and tissues. When the transferrin binds to transferrin receptor (TfR), the Tf-TfR complex is transported into the cell via endocytosis. TfR is highly expressed on some cancers including cervical, colon, prostate, and breast cancers. [12–13] Due to its elevated expression level in cancer cells, TfR has been used as a therapeutic target for cancer treatment and diagnosis. Particularly, holotransferrin (Holo-Tf) in the form of its iron complex has high binding affinity to TfR and has been used as a targeting moiety of therapeutics for cancers with high expression of TfR. [14]</p><p>We herein report preparation, characterization, and evaluation of transferrin conjugates of three bifunctional NE3TA analogues for targeted PET applications of 64Cu. The NE3TA-transferrin conjugates were prepared, characterized, and evaluated for radiolabeling kinetics with 64Cu. The corresponding 64Cu-radiolabeled NE3TA-Tf conjugates were further evaluated for complex stability in human serum and binding affinity to TfR using PC-3 cancer cells, as well as in vivo biodistribution in PC-3 xenograft-bearing mice.</p><!><p>1H and 13C NMR spectra were obtained using a Bruker 300 instrument, and chemical shifts are reported in ppm on the δ scale relative to TMS or solvent. Fast atom bombardment (FAB) or Electrospray iodization (ESI) high resolution mass spectra (HRMS) were obtained on JEOL double sector JMS-AX505HA mass spectrometer (University of Notre Dame, IN). Size-exclusion HPLC (SE-HPLC) chromatograms were obtained on Agilent 1200 equipped with a diode array detector and an in-line IN/US γ-Ram Model 2 radiodetector (Tampa, FL), fitted with BioSep-SEC S 3000 column (Phenomenex, Torrance, CA). All absorbance measurements for the protein concentration and ligand protein ratio were obtained on an Agilent 8453 diode array spectrophotometer equipped with an 8-cell transport system (designed for 1-cm cells). Arsenazo III (AAIII, 2,2-(1,8-dihydroxy-3,6-disulfonaphthylene-2,7-bisazo) bis-benzenearsonic acid), copper atomic absorption standard solution, and diferric (holo) transferrin were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. C-DOTA analogues were purchased from Macrocyclics (Dallas, TX).</p><!><p>4M HCl/dioxane (6 mL) was added to 1 [15] (39 mg, 62.9 µmol) in a flask was cooled with an ice bath. After the addition was complete, the reaction mixture was gradually warmed to room temperature and allowed to stir overnight at room temperature. Ethyl ether (50 mL) was added to the reaction mixture with vigorous stirring, and the resulting slurry was kept in the freezer for 2 h. The precipitate was collected and washed with ethyl ether, and immediately dissolved in water, and the resulting solution was lyophilized to provide pure salt 2 (34 mg, 85%) as a light yellow solid. 1H NMR (D2O, 300 MHz) δ 2.97 (m, 4H), 3.21 (m, 6H), 3.33 (s, 4H), 3.48 (t, J = 5.4 Hz, 2H), 3.93 (s, 4H), 3.99 (s, 2H), 4.46 (s, 2H), 7.40 (d, J = 6.4 Hz, 2H), 7.59 (d, J = 6.4 Hz, 2H). 13C NMR (D2O, 75 MHz) δ 48.4 (t), 48.7 (t), 49.8 (t), 49.9 (t), 51.1 (t), 51.3 (t), 53.9 (t), 56.1 (t), 58.5 (t), 124.1 (d), 129.5 (s), 131.8 (s), 133.2 (d), 168.7 (s), 171.2 (s); HRMS (Positive ion ESI) Calcd for C14H26N4O6 [M – BnNH2 + H]+
m/z 347.3874, Found: [M – BnNH2 + H]+
m/z 347.1958.</p><!><p>To a solution of 2 (20.1mg, 31.7 µmol) in water (0.3 mL) was added CSCl2 in CHCl3 (39.4 µL). The resulting mixture was stirred at room temperature for 4 h. The aqueous layer was transferred to a round bottom flask, and the CHCl3 layer was washed with water (1 mL × 2). The combined aqueous layers were lyophilized to provide compound 3 (N-NE3TA-NCS) as a yellow solid (20 mg, 99%). 1H NMR (D2O, 300 MHz) δ 2.97 (m, 4H), 3.02–3.36 (m, 12H), 3.44 (t, J = 5.4 Hz, 2H), 3.93 (s, 4H), 3.98 (s, 2H), 4.36 (s, 2H), 7.29 (d, J = 6.1 Hz, 2H), 7.47 (d, J = 6.1 Hz, 2H). HRMS (Positive ion ESI) Calcd for C22H32N5O6S [M + H]+
m/z 494.2073, Found: [M + H]+
m/z 494.2098.</p><!><p>To a solution of 4 [16] (14.5 mg, 20.2 µmol) in ethanol (8.0 mL) was added dry 10% Pd/C (3.9 mg) under Argon gas. The reaction mixture was subjected to hydrogenolysis (20 psi) for 14 h. at room temperature. The resulting mixture was filtered via Celite® bed and washed thoroughly with ethanol. The filtrate was concentrated in vacuo to provide compound 5 as a light yellow solid (14 mg, 96%). 1H NMR (D2O, 300 MHz) δ 2.35–2.66 (m, 1H), 2.69–3.28 (m, 18H), 3.50 (s, 2H), 3.56–3.97 (m, 8H), 4.21 (s, 1H), 7.18–7.49 (m, 9H). 13C NMR (D2O, 75 MHz, DEPT-135) δ 45.0 (t), 48.5 (t), 50.1 (t), 50.9 (t), 51.9 (t), 55.9 (t), 57.6 (t), 61.0 (d), 62.3 (t), 123.5 (d), 123.7 (d), 129.4 (d), 130.0 (d), 130.1 (d), 130.6 (d), 130.8 (d). HRMS (Negative ion ESI) Calcd for C28H38N5O6 [M - H]+
m/z 540.6312, Found: [M - H]+
m/z 540.2823.</p><!><p>To a solution of 5 (10 mg, 13.8 µmol) in water (100 µL) was added 1M CSCl2 in CHCl3 (16.6 µL). The resulting mixture was stirred at room temperature for 4 h. The aqueous layer was transferred to a round bottom flask, and the CHCl3 layer was washed with water (2 × 1 mL). The combined aqueous layers were lyophilized to provide compound 6 as a pale yellow solid (9.2 mg, 91%). 1H NMR (D2O, 300 MHz) δ 2.35–2.66 (m, 1H), 2.69–3.28 (m, 18H), 3.50 (s, 2H), 3.56–3.97 (m, 8H), 4.21 (s, 1H), 7.18–7.49 (m, 9H). HRMS (Positive ion ESI) Calcd for C29H37N5O6S [M + H]+
m/z 583.6990, Found: [M + H]+
m/z 584.2534.</p><!><p>A stock solution of holotransferrin (holo-Tf) was freshly prepared. HEPES (N-(2-hydroethyl)-piperazine-N'-ethanesulfonic acid) buffer (50 mM HEPES, 150 mM NaCl, pH 8.6) was prepared and stored at 4 °C. Each Tf conjugate was prepared by reaction of a bifunctional chelating agent in the isothiocyanate form with holo-Tf. In brief, holo-Tf (40 mg) was dissolved in HEPES buffer (2.0 mL, 50 mM HEPES, 150 mM NaCl at pH 8.6). To a sterilized test tube containing the resulting solution, 10-fold molar excess of each chelating agent was added. The resulting solution was gently agitated at room temperature overnight. The following day, this solution was placed on an Amicon C-50 membrane (50,000 MWCO) and spun down to reduce volume. PBS (3 × 2 mL) was added to the remaining solution of the Tf conjugate, followed by centrifugation in order to remove unreacted ligand. The final volume of purified transferrin conjugates was brought to ~400 µL using PBS. The protein concentration was determined by measuring absorbance of the solution at 280nm. L/P ratio of Tf conjugates was spectroscopically determined using Cu(II)-AAIII (0.15 M NH4OAc, pH 7.0). For ligand concentration, an aliquot of a Tf conjugate was added to the cuvette containing the Cu(II)-AAIII reagent, and the absorbance at 610nm was monitored over time. The reaction was complete after 15 min, and the concentration of N-NE3TA, C-NE3TA or C-NE3TA-Bn in the conjugate solution was calculated. The L/P ratio of N-NE3TA-Tf, C-NE3TA-Tf, C-NE3TA-Bn-Tf, and C-DOTA-Tf conjugate was found to be 4.5, 3.1, 6.6, and 2.9, respectively.</p><!><p>All HCl solutions were prepared from ultra-pure HCl. Ultra pure NH4OAc was purchased from Aldrich and used to prepare all NH4OAc buffer solutions (0.25 M, pH 5.5). The buffer solutions were chelexed, and filtered through 0.22 µm filter (Corning) prior to use. 64Cu was purchased from Washington University (St. Louis, MO). To a buffer solution (12.8~18.4 µL, pH 5.5) in a capped microcentrifuge tube was sequentially added a solution of each conjugate (20 µg) in PBS and 64Cu (0.05 M HCl, 60 µCi). The final volume of the resulting solution was around 20 µL, and the pH of the resulting reaction mixture was 5.5. The reaction mixture was agitated on the thermomixer set at 1,000 rpm at room temperature for 30 min. The labeling efficiency (%) was determined by TLC (solvent: 20 mM EDTA/ 0.15 M NH4OAc). The specific activity was calculated based on the activity of 64Cu and the amount of conjugate used in radiolabeling reaction. The activity was corrected by radiolabeling efficiency (%) and divided by amount of conjugate (mg) to determine the specific activity (mCi/mg). A solution of radiolabeled mixture (2 µL) was withdrawn at the designated time points (1 min, 10 min and 30 min), and DTPA (diethylenetriamine pentaacetic acid) solution (1 mM, 0.6 µL) was added to the mixture, and the resulting mixture was left for at least 20 min at RT. In TLC analysis, peaks for bound and unbound radioisotope appeared around 30 mm and 50 mm from the origin, respectively.</p><p>For radiolabeling of each conjugate at higher activity, 64Cu (30 µCi) was added to a solution of each conjugate (1 µg), and the final volume of the solution (pH 5.5) was adjusted to 20 µL. The reaction mixture was agitated on the thermomixer set at 1,000 rpm at room temperature for 1 h. The radiolabeling efficiency (%) was determined by TLC as described above.</p><p>A control experiment was conducted to check if 64Cu was bound to holo-transferrin (Tf). Radiolabeling of naïve holo-Tf (without NE3TA chelate attached) with 64Cu was repeated under the same reaction condition used for testing NE3TA-Tf conjugates. 64Cu (60 µCi) was added to a solution of holo-Tf (20 µg), and the final volume of the solution (pH 5.5) was adjusted to 20 µL. The reaction mixture was agitated on the thermomixer set at 1,000 rpm at room temperature for 30 min. A solution of radiolabeled mixture (2 µL) was withdrawn at the designated time points (1 min, 10 min and 30 min), and DTPA solution (1 mM, 0.6 µL) was added to the mixture, and the resulting mixture was left for at least 20 min at RT. The labeling efficiency (%) was determined by both TLC (solvent: 20 mM EDTA/ 0.15 M NH4OAc) and SE-HPLC (Phenomenex, BioSep-SEC S 3000, 7.8 × 30 cm, eluent: 0.05 M NaSO4 / 0.02 M NaH2PO4 / 0.05% NaN3, pH 6.8, flow rate: 1 mL/min).</p><!><p>Each 64Cu-radiolabeled NE3TA-Tf conjugate was prepared by reaction of a NE3TA-Tf conjugate with 64Cu at RT. 64Cu-C-DOTA-Tf conjugate was evaluated for comparison. The 64Cu-radiolabeled transferrin conjugates were purified using Biospin 6 column, and the purity of the conjugates was confirmed by HPLC. The reaction mixture containing 64Cu-radiolabeled Tf conjugate (7 µL, 15 µCi) was added into human serum (50 µL, Gemini Bioproducts, #100110) in a microcentrifuge tube. The stability of the conjugates was evaluated for 2 days. The serum stability of the radiolabeled complexes was assessed by measuring the transfer of the radionuclide from the complex to serum proteins using SE-HPLC (Phenomenex, BioSep-SEC S 3000, 7.8 × 30 cm, eluent: 0.05 M NaSO4 / 0.02 M NaH2PO4 / 0.05% NaN3, pH 6.8, flow rate: 1 mL/min). A solution of the radiolabeled complex in serum (2 µL) was withdrawn at the designated time point, treated with DTPA (1 mM, 0.6 µL), incubated at room temperature at least for 5 min and then diluted with the HPLC eluent (200 µL) prior to SE-HPLC analysis.</p><!><p>PC3 human prostate cancer cell line was obtained from American Type Culture Collection (ATCC). The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% CO2 at 37 °C. 64Cu was purchased from Essential Isotopes (Columbia, MO). Hela human cervix adenocarcinoma, HT 29 human colorectal and PC3 human prostate cancer cell lines were obtained from American Type Culture Collection (ATCC) and maintained and grown in exponential growth phase in RPMI-1640 medium (Life Technology), supplemented with 10% FBS, at 37°C and 5% CO2 incubator. Reaction buffer (0.25 M NH4OAc, pH 5.5), N-NE3TA [10], N-NE3TA-CA (cholic acid) [15], or NE3TA-transferrin conjugate (20 µg/ 0.5 –2 µl) and 64Cu (60 µCi/3 µl) were sequentially added into a reaction vial for final total volume of 20 µl. The reaction mixture was incubated at room temperature for 30 min with shaking at 1000 rpm, after which 1mM DTPA was added to quench the reaction. 2 µl of the reaction mixture was aliquoted and treated with DTPA (1mM, 0.6 µl) at room temperature for 20 min. Radio-TLC plates were developed with 20mM EDTA/0.15M NH4OAc, and the radiochemical purity was determined using Bioscan AR-2000 TLC Imaging Scanner. At this reaction condition, the radio-labeling efficiency was ≥ 95%. 64Cu-labeled Tf conjugate was diluted in RPMI-1640/0.2% bovine serum albumin (BSA) before addition to the cell suspension. An aliquot of 64Cu-labeled complex (10 µCi) in 0.1 mL medium was added to 3.0 × 106 PC3 cells in 5 mL of the medium. An aliquot of 0.2 mL of cell suspension was removed at various time points from 5 min to 90 min. Cells were centrifuged and rinsed twice with 2 × 1 ml ice-cold medium. The cell pellets were counted by Wallac Wizard 3" gamma counter to determine the percentage cellular uptake of 64Cu-labeled Tf conjugate.</p><!><p>All animal experiments were performed in compliance with the Guidelines for Care and Use of Research Animals established by the Division of Comparative Medicine and the Animal Studies Committee of Washington University School of Medicine. Female 3–4 weeks old SCID mice were purchased from Charles River Laboratories and housed under pathogen-free conditions in WUSM animal facility. Mice weighing 20–25 g were implanted subcutaneously on the rear right flank with 2.0 × 106 PC-3 cells suspended in 100 µl of saline. The tumors were allowed to grow for 3 weeks after implantation at which time they were about 150–250 mg. In vivo biodistribution studies were conducted in the Washington University Small Animal Imaging facility. In tumor bearing mice (n=4 per group), ~8 µCi of 64Cu-N-NE3TA-Tf conjugate was administrated via tail vein. After each time point (1, 4, and 4 h), mice were anesthetized with 1–2% isofluorane/oxygen mixture and sacrificed by cervical dislocation. Tumors and select normal tissues were collected, weighted, and radioactivity counted on a Beckman Gamma 8000 counter containing a NaI crystal (Beckman Instruments, Inc., Irvine, CA). Tumor and normal-tissue uptake were analyzed and measured as percentage injected dose per gram (%ID/g).</p><!><p>Quantitative data were expressed as mean ± SD. The Student t-test was used to determine significance at the 95% confidence level, P values of <0.05 were considered significantly different.</p><!><p>Introduction of a functional unit to the chelate backbone is known to affect stability and formation rate of metal complexes. [10,17–19] The three different bifunctional ligands N-NE3TA, C-NE3TA, and C-NE3TA-Bn (7-[2-[(carboxymethyl)amino]-2-(phenylmethyl)]-3-(4-nitrophenyl)propyl]-1,4,7-triazacyclononane-1,4-diacetic acid), containing the isothiocyanate (NCS) group for conjugation to transferrin, were prepared and evaluated in an effort to identify the best bifunctional version. N-NE3TA-NCS (3) and C-NE3TA-Bn-NCS (6) were synthesized by the same method used for preparation of C-NE3TA-NCS [20] (Scheme 1). Removal of t-butyl groups in 1 [15] by the treatment of 1 with 4M HCl/1,4-dioxane provided compound 2 which was further reacted with thiophosgene to provide N-NE3TA-NCS (3) in the isothiocyanate form. C-NE3TA-Bn containing the nitro group 4 [16] was subjected to hydrogenation to afford compound 5 containing the amino group which was converted to the NCS group in compound 6 using thiophosgene.</p><p>The NE3TA-based bifunctional chelates in the NCS form were conjugated to transferrin (Tf), and concentration of the corresponding transferrin conjugates was quantified by spectrometric method [20,21]. The known macrocyclic chelating agent C-DOTA was conjugated to Tf to produce C-DOTA-Tf for comparative serum stability studies. The UV-VIS spectrophotometric assay using Cu(II)-arsenazoIII (AAIII) was performed for the determination of the number of NE3TA ligands linked to Tf (L/P ratio). [21] The ligand to protein (L/P) ratio for N-NE3TA-Tf (7), C-NE3TA-Tf (8), C-NE3TA-Bn-Tf (9), and C-DOTA-Tf (10) was found to be 4.5, 3.1, 6.6, and 2.9, respectively.</p><!><p>The NE3TA-Tf conjugates were evaluated for radiolabeling reaction kinetics with 64Cu at room temperature (Table 1 and Supporting Information). Each conjugate (in 0.25 M NH4OAc, pH 5.5) was radiolabeled with 64Cu for 30 min at room temperature, and the radiolabeling efficiency (%) was determined using TLC. At the designated time points (1, 10, and 30 min), a sample of the reaction mixture was withdrawn and quenched with DTPA solution (10 mM). Copper-64-radiolabeled conjugates (Rf = 0.50) migrated less than unbound 64Cu (Rf = 0.75). The N-NE3TA-Tf conjugate (7) instantly bound to 64Cu, and almost complete radiolabeling of the conjugate was achieved in 1 min after the reaction. C-NE3TA-Tf (8) and C-NE3TA-Bn-Tf (9) were relatively slower in binding 64Cu as compared to N-NE3TA-Tf (7), and the respective radiolabeling efficiency of 99%, 95% and 80% at the 10 min time point was observed with N-NE3TA-Tf (7), C-NE3TA-Tf (8) and C-NE3TA-Bn-Tf (9). N-NE3TA-Tf (7), C-NE3TA-Tf (8), and C-NE3TA-Bn-Tf (9) were labeled with 64Cu for 30 min at specific activities of 2.98, 2.60, and 2.93 (mCi/mg), respectively.</p><p>A control experiment was conducted to check if 64Cu was bound to holotransferrin (Tf). A solution of naïve holo-Tf (20 µg) in 0.25 M NH4OAc buffer (pH 5.5) was mixed with 64Cu (60 µCi), and the resulting mixture was subjected for radiolabeling under the same reaction condition used for testing NE3TA-Tf conjugates for 30 min at room temperature. At the designated time points (1, 10, and 30 min), a sample of the mixture was withdrawn and quenched with DTPA solution (10 mM) and analysed using both radio-TLC and SE-HPLC. The data (Supporting information) clearly indicate that 64Cu was completely complexed with DTPA, and there was almost no bound 64Cu-transferrin present in the reaction mixture as quenched with DTPA.</p><p>The more efficient conjugates N-NE3TA-Tf (7) and C-NE3TA-Tf (8) were further evaluated for radiolabeling with 64Cu in higher specific activity. A dilute solution of C-NE3TA-Tf or N-NE3TA-Tf (1 µg) was tested for radiolabeling with 64Cu (30 µCi) at room temperature for 1 h (Supporting Information). N-NE3TA-Tf (7) and C-NE3TA-Tf (8) rapidly bound to 64Cu with respective specific activities of 29.2 mCi/mg (2.22 Ci/µmol) and 28.3 mCi/mg (2.15 Ci/µmol). As expected from the radiolabeling efficiency data (98.9% and 88.2% at 1 min for N-NE3TA-Tf and C-NE3TA-Tf, respectively), N-NE3TA-Tf (7) is slightly more efficient than C-NE3TA-Tf (8). This result suggests that the position of a functional group in the NE3TA backbone is an important factor affecting radiolabeling efficiency.</p><!><p>N-NE3TA-Tf (7) and C-NE3TA-Tf (8) conjugates that were efficient in binding 64Cu were further screened for complex stability in human serum and compared with C-DOTA-Tf (Figure 2). In vitro serum stability was performed to determine if the conjugates radiolabeled with 64Cu remained stable in human serum. Stability was assessed by measuring the transfer of 64Cu from the complex to human serum using HPLC. Copper-64-radioabeled conjugates were readily prepared from the reactions of the conjugates with 64Cu at room temperature and purified for serum stability studies (pH 7 and 37 °C). The 64Cu-radiolabeled complexes of N-NE3TA-Tf (7) were stable in human serum for 2 days, while a considerable amount of 64Cu (~5%) was released from 64Cu-C-NE3TA-Tf (8) (Figure 2 and Supporting Information). N-NE3TA-Tf (7) was shown to have complex stability comparable to C-DOTA-Tf. The radiolabeling kinetics and serum stability data indicate that among three NE3TA analogues tested, N-NE3TA is the most effective chelating agent of 64Cu.</p><!><p>N-NE3TA-Tf (7) and C-NE3TA-Tf (8) were evaluated for binding affinity to TfR using PC-3 cancer cells with elevated expression of TfR. The bifunctional chelate N-NE3TA in the nitro form [10] and N-NE3TA-CA (cholic acid) [15] containing a cholic acid moiety targeting a bile acid receptor were used as negative controls. Aliquots of 3 × 106 cells of PC-3 cells were incubated with 64Cu-radiolabeled Tf conjugate (10 µCi) at 37 °C, and the radioactivity bound to the cells was measured at designated time points. As shown in Figure 3, binding of 64Cu-N-NE3TA-Tf and 64Cu-C-NE3TA-Tf to the tumor cells was rapid and increased up to 30 min of incubation. 64Cu-N-NE3TA-Tf had higher uptake in PC3 cells than 64Cu-C-NE3TA-Tf. Both N-NE3TA-CA and N-NE3TA used as the negative controls displayed very low binding to PC3 cells.</p><!><p>Biodistribution studies were conducted to investigate the tumor and normal tissue uptake of 64Cu-N-NE3TA-Tf in SCID mice bearing PC-3 xenografts. As presented in Figure 4, accumulation of 64Cu-N-NE3TA-Tf in peripheral tissues like lung, liver, spleen and kidney was relatively high. The radioactivity in lung, liver, and kidney was shown to gradually decrease over the time points (11.22% ID/g, 11.54% ID/g, and 6.72% ID/g at 24 h position injection, respectively). Uptake of the tracer in muscle, bone, and pancreas remained low at all time points (24 h post injection, 3.03%, 2.90%, 4.00% ID/g, respectively). The tumor uptake increased in a sequential manner (Figure 4a), where the accumulation was 1.4 ± 0.4 %ID/g, 3.8 ± 0.2 %ID/g and 10.0 ± 1.5 %ID/g for 1, 4 and 24 h post-injection, respectively. As shown in Figure 4(b), the blood concentration decreased in a corresponding manner. All tumor and blood concentrations measured at 1, 4 and 24 h were observed to be statistically different with p < 0.05. With the continuing trend of decreasing blood and increasing tumor uptake values (Figure 4c), perhaps a longer evaluation time point, such as 48 h, might beneficial to determine the maximum tumor:blood ratio.</p><!><p>Bifunctional NE3TA-based chelating agents were synthesized and conjugated to transferrin (Tf) as a tumor targeting vector. The corresponding NE3TA-Tf conjugates were evaluated for the effect of the functional unit in the chelates on complexation with 64Cu. Among the three NE3TA-Tf conjugates tested, N-NE3TA-Tf conjugate was found to be the most effective in binding 64Cu. Radiolabeling of N-NE3TA-Tf with 64Cu was achieved with a high specific activity and extremely fast kinetics at room temperature. The lead 64Cu-N-NE3TA-Tf conjugate displayed rapid blood clearance and increased tumor uptake in prostate tumor-bearing mice over 24 h. The in vitro and in vivo data suggest that the bifunctional chelate N-NE3TA can be further evaluated for efficient 64Cu radiolabeling of tumor targeting antibodies or peptides for potential use in PET imaging.</p><!><p>This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.</p>