Amino Acids (2016) 48:1667–1675 DOI 10.1007/s00726-016-2227-y
ORIGINAL ARTICLE
Catabolism of 64Cu and Cy5.5‑labeled human serum albumin in a tumor xenograft model Choong Mo Kang1,2 · Hyunjung Kim1,2 · Hyun‑Jung Koo1 · Jin Won Park1,2 · Gwang Il An3 · Joon Young Choi1 · Kyung‑Han Lee1,2 · Byung‑Tae Kim1 · Yearn Seong Choe1,2
Received: 25 November 2015 / Accepted: 1 April 2016 / Published online: 20 April 2016 © Springer-Verlag Wien 2016
Abstract Human serum albumin (HSA), the most abundant protein in blood plasma, has been used as a drug carrier for the last few decades. Residualizingly radiolabeled serum albumin has been reported to be avidly taken up by tumors of sarcoma-bearing mice and to most likely undergo lysosomal degradation. In this study, we prepared 64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″tetraacetic acid (DOTA) and Cy5.5-conjugated HSA (dual probe), and evaluated its tumor uptake and catabolism. Two dual probes were prepared using different DOTA conjugation sites of HSA (one via Lys residues and the other via the Cys residue). 64Cu-DOTA-Lys-HSA-Cy5.5 (dual probeLys) exhibited higher uptake by RR1022 sarcoma cells in vitro than 64Cu-DOTA-Cys-HSA-Cy5.5 (dual probeCys). In RR1022 tumor-bearing mice, the two dual probes showed a similar level of tumor uptake, but uptake of dual probe-Lys was reduced in the liver and spleen compared to dual probe-Cys, probably because of the presence of a higher number of DOTA molecules in the former. At 24 and 48 h after injection, dual probe-Lys was intact or partially degraded in blood, liver, kidney, and tumor samples, but 64Cu-DOTA-Lys was observed in the urine using radioactivity detection. Similarly, Cy5.5-Lys was observed in the Handling Editor: G. J. Peters. * Yearn Seong Choe
[email protected] 1
Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Korea
2
Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul 06351, Korea
3
Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul 01812, Korea
urine using fluorescence detection. These results indicate that dual probe-Lys may be useful for predicting the catabolic fate of drug–HSA conjugates. Keywords Human serum albumin · PET/optical imaging · 64Cu-DOTA-Lys · Cy5.5-Lys · Tumor · Catabolism
Introduction Human serum albumin (HSA) is the most abundant protein in blood plasma and has long been used as a drug carrier (Peters 1996; Elsadek and Kratz 2012). HSA has 58 Lys residues and 1 Cys residue, which can serve as sites for conjugation of diverse functional groups. It is also non-immunogenic, biocompatible, biodegradable, and stable under a wide range of thermal and pH conditions (Yhee et al. 2015). HSA has been used to facilitate drug delivery by improving the solubility of drugs or by prolonging their half-lives. There are several ways to use HSA as a drug carrier. First, the drug can be covalently conjugated to HSA and then released in a certain environment. Second, the drug can be non-covalently bound to HSA. The first category includes doxorubicin substituted with a 6-maleimidocaproyl hydrazone group (aldoxorubicin) (Willner et al. 1993), which binds to HSA through Cys-34 shortly after intravenous administration. In the acidic environment of tumor tissues, doxorubicin is released from the HSA-bound form because of the presence of an acid-sensitive hydrazone group. This doxorubicin derivative demonstrated higher antitumor efficacy than doxorubicin itself in an AsPC-1 pancreatic tumor model and had a good safety profile in a Phase I study; further clinical trials are currently underway (Kratz 2011; Elsadek and Kratz 2012). The second category includes
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Abraxane®, which is FDA-approved albumin-stabilized paclitaxel in the form of 130-nm particles for treating metastatic pancreatic cancer, non-small cell lung cancer, and breast cancer. By using albumin as a carrier, toxicities derived from Cremophor®, the solvent for paclitaxel, are absent, and drug delivery to the tumor is increased (Green et al. 2006). Another example includes paclitaxel conjugated to 3-pentadecylglutaric acid, which mimics fatty acids. This conjugate is thought to bind non-covalently to HSA in vivo via the fatty acid mimic and be delivered to the tumor, where paclitaxel is released (Hackett et al. 2012). HSA can be also used in molecular imaging studies to increase tumor uptake of tumor targeting molecules. For example, near-infrared fluorescence (NIRF) imaging of U87MG tumor-bearing mice injected with cyclic Arg-GlyAsp peptide (RGD) conjugated to HSA-IRDye800 revealed higher tumor uptake and tumor-to-non-target uptake ratios with a prolonged circulation time than RGD-IRDye800 (Chen et al. 2009). 131I-tyramine-deoxisorbitol-labeled and 111In-diethylenetriaminepentaacetic acid (DTPA)labeled rat serum albumins were avidly taken up by and then trapped in tumors (Stehle et al. 1997; Wunder et al. 1997). It was also shown that 3H-raffinose-human albumin was taken up by lysosomes in major tissues at 48 h after injection into non-metastasizing sarcoma-bearing mice (C57BL/6J), because radioactivity was detected in partially purified lysosomes of tumor and liver tissue (Andersson et al. 1991). Furthermore, an 125I-labeled peak with the same retention time as that of 14C-tyrosine eluted on ion exchange chromatography when 125I-albumin was incubated with tumor and liver homogenates containing proteolytic activity (Andersson et al. 1991). However, catabolic degradation products of HSA in tumors have not been directly identified. Lysosomal degradation of glycoproteins has been successfully investigated using various residualizing labels. Galactosyl neoglycoalbumin (NGA) labeled with 99mTc via HYNIC(tricine)2 accumulated in the liver via receptormediated endocytosis, and the metabolite produced as a result of lysosomal degradation was evaluated (Ono et al. 1999). HPLC analysis of the liver homogenates showed the formation of 99mTc-HYNIC-Lys(tricine)2 in lysosomes, and this finding was further confirmed by analysis of liver homogenates treated with TPPMS, another co-ligand of 99m Tc-HYNIC, which has a similar retention time to that of 99mTc-HYNIC-Lys(tricine)(TPPMS). Another study showed that 111In-ethylenediaminetetraacetic acid (EDTA)benzyl-NGA and mannosyl-neoglycoalbumin (NMA) accumulated in the liver of mice and was metabolized to 111 In-EDTA-benzyl-Lys (Arano et al. 1994). Similarly, the use of 18F-labeled NGA for imaging of asialoglycoprotein receptors, which are located on the surface of hepatocyte membrane, was investigated in mice (Yang et al. 2009).
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F-Benzo-Lys was found in liver homogenates obtained 30 min after injection, indicating that the radiolabeled albumin was degraded to radiolabeled Lys in lysosomes. Lysosomal degradation of monoclonal antibodies (MAb) has also been reported; both 111In-DTPA-MAb 1A3 and MAb 1A3-F(ab’)2 were degraded to 111In-DTPA-Lys in the lysosomes of liver and kidney tissues (Rogers et al. 1995). A fluorescent residualizing label NN-dilactitolN′-floresceinylethyleneamine was also successfully used to identify the catabolites of rat serum albumin (Maxwell et al. 1990). In the present study, HSA was labeled with Cy5.5 and 64 Cu via DOTA, and its uptake and catabolic fate in a tumor xenograft model were investigated using radioactivity and fluorescence (FL).
Experimental methods Reagents and methods HSA and Chelex® 100 resin (50–100 mesh) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DOTANHS ester and maleimido-mono-amide-DOTA (DOTAmaleimide) were purchased from Macrocyclics (Dallas, TX, USA), and Cy5.5-NHS ester was obtained from Amersham Biosciences (Piscataway, NJ, USA). 64CuCl2 was kindly provided by KIRAMS (Seoul, Korea). PD-10 columns were purchased from GE Healthcare Life Sciences (Buckinghamshire, UK), BCA protein assay kits were from Pierce (Rockford, IL, USA) and Amicon filters were from Millipore (Billerica, MA, USA). All buffers used for synthesis and radiolabeling were pretreated with Chelex® 100 resin to make metal-free conditions. Fast atom bombardment (FAB) mass spectra were obtained using a JMS-700 Mstation (JEOL Ltd, Tokyo, Japan), and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry was performed on an AB SCIEX TOF/ TOF™ 5800 System (Framingham, MA, USA). For analysis of catabolites, thin layer chromatography (TLC) was performed on Merck F254 silica plates and plates were analyzed using a Bioscan radio-TLC scanner (Washington, DC, USA) or a Xenogen IVIS Spectrum (PerkinElmer, Waltham, MA, USA). Radioactivity was measured using a dose calibrator (Biodex Medical Systems, Shirley, NY, USA), and tissue radioactivity was measured using an automatic gamma counter (PerkinElmer). Micro-PET and optical images of mice were acquired at the Center for Molecular and Cellular Imaging, Samsung Biomedical Research Institute (SBRI, Seoul, Korea), using an Inveon micro-PET/CT scanner (Siemens Medical Solutions, Malvern, PA, USA) and a Xenogen IVIS Spectrum, respectively.
Catabolism of 64Cu and Cy5.5-labeled human serum albumin in a tumor xenograft model
Preparation of Cy5.5 and DOTA‑conjugated HSA HSA (2.2 mg, 33.1 nmol) and Cy5.5-NHS ester (373 μg, 330.8 nmol) were dissolved in 500 μL sodium carbonate buffer (0.1 M, pH 8.5), and this mixture was stirred overnight at room temperature. Cy5.5-conjugated HSA was purified using a spin column (MW cutoff: 7 K) to remove unreacted Cy5.5 NHS ester. Purified fraction was concentrated using an Amicon centrifugal filter (10 K), and simultaneously, the buffer was exchanged with Chelex®-treated distilled water. The purified product was divided and lyophilized. Aliquots were subjected to analysis by BCA protein assay and by MALDI-TOF mass spectrometry. HSA-Cy5.5 (903 μg, 13.6 nmol based on HSA) and DOTA-NHS ester (225.4 μg, 271.6 nmol) were dissolved in 400 μL sodium carbonate buffer (0.1 M, pH 8.5), and this mixture was stirred overnight at room temperature. The reaction mixture was then purified using a PD-10 column and concentrated using an Amicon centrifugal filter (10 K). Purified DOTA-Lys-HSA-Cy5.5 was divided and lyophilized. Aliquots were subjected to analysis by BCA protein assay and by MALDI-TOF mass spectrometry. DOTA-Cys-HSA-Cy5.5 was prepared as described above and characterized using a previously reported method (Kang et al. 2015). Briefly, HSA-Cy5.5 (717.5 μg based on HSA, 10.8 nmol) was stirred overnight with 10 equivalents of DOTA-maleimide (85 μg, 108 nmol) in 400 μL sodium phosphate buffer (0.01 M, pH 7.4), and the product was obtained after purification using a PD-10 column. The average number of DOTA per HSA was determined using a previously reported procedure (Meares et al. 1984; Cai et al. 2006a, b; Kang et al. 2015).
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calibrator. Serum stability was measured relative to the radioactivity loaded onto the column. Cell uptake RR1022 rat sarcoma cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10 % FBS, streptomycin (100 μg/mL), and penicillin (100 units/mL). Cells were incubated at 37 °C in a humidified 5 % CO2 incubator. RR1022 cells were seeded in 24-well plates at 4 × 104 cells/well, cultured for 45 h in RPMI 1640 medium, and serum-starved for 24 h in the same medium prior to study. Dual probes (111 kBq) dissolved in PBS were added to each well, and the cells in a total volume of 0.5 mL were incubated at 37 °C for 1, 16, 24, and 45 h. At the indicated time points, cells were washed three times with PBS and then lysed using 0.1 N NaOH, and the radioactivity in the resulting lysate was counted using a gamma counter. All experiments were performed in triplicate. Micro‑PET/optical imaging
DOTA-Lys-HSA-Cy5.5 (100 μg, 1.5 nmol) was dissolved in 200 μL sodium acetate buffer (0.1 M, pH 6.0) to which 64 CuCl2 (162.6 ± 1.3 MBq/50 μL of 0.01 N HCl) had been added. The reaction mixture was stirred at 40 °C for 30 min and then purified using a PD-10 column to give 64 Cu-DOTA-Lys-HSA (dual probe-Lys). 64Cu-DOTA-CysHSA (dual probe-Cys) was prepared as described above.
RR1022 cells (2 × 106) were subcutaneously inoculated into the right hind legs of 5-week-old BALB/c nude mice (male). When tumor size reached 612.5 ± 54.5 mm3, dual probes (10.2 ± 0.7 MBq/200 μL) in 0.01 M PBS were injected intravenously into mice via a tail vein (n = 3 per dual probe). Static PET images were acquired for 10 min at 16, 22, and 45 h after injection. Immediately after PET imaging, mice were subjected to optical imaging (excitation 675 nm, emission 720 nm) for 1 s. PET images obtained were reconstructed using 3-D ordered subset expectation maximization and then processed using Siemens Inveon Research Workplace 4.1 (IRW 4.1). Regions of interest (ROIs) were drawn over tumors in the right legs and muscles in the contralateral legs, and the average signal level in the ROIs was measured. ROIs from optical images were drawn over tumors, and signal levels were calculated using Living Image 3.2 software. Data are presented as photons/s/cm2/sr (sr: steradian).
In vitro serum stability
Ex vivo PET/optical imaging and biodistribution
Dual probe (238 MBq) in 1 mL of 0.01 M PBS (pH 7.4) was added to 50 % fetal bovine serum (FBS; Gibco, Brooklyn, NY, USA) and incubated at 37 °C for 48 h. Aliquots were taken from the incubation solution after 0, 2, 16, 24, and 48 h, diluted with PBS for a total volume of 1 mL, and then loaded onto PD-10 columns. Product fractions were eluted with 0.01 M PBS (pH 7.4) and counted using a dose
At the completion of PET/optical imaging, mice were killed and major tissues were immediately separated and subjected to micro-PET (10-min static scan) and optical imaging (1-s exposure). In the biodistribution study, the tissues of interest were weighed and the radioactivity of the tissues was counted. Data are expressed as the percent injected dose per gram of tissue (% ID/g).
Radiolabeling
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Catabolism studies Dual probe-Lys (19.6 ± 0.2 MBq/16.1 ± 0.1 μg) in 200 μL of PBS was injected into RR1022 tumor-bearing mice (tumor size: 742.9 ± 107.1 mm3) via a tail vein. At 24 and 48 h after injection, mice were killed and blood, tumor, liver, kidney, and urine samples were collected. Tissues (tumor, liver, and kidney) were homogenized in 1 mL of PBS and centrifuged. The supernatants of the three tissue homogenates as well as blood and urine samples were analyzed by radio-TLC using a 1:1 mixture of 10 % ammonium acetate and methanol as the mobile phase. The presence of free 64Cu in the supernatants was determined using a 1:1 mixture of 10 % ammonium acetate and MeOH containing 15 mM EDTA as the mobile phase (de Rosales et al. 2011). To help identify radiometabolites, 64Cu-DOTA-Lys and 64Cu-DOTA were synthesized as the reference standards (Thorpe et al. 1993). DOTA-Lys was synthesized by the two-step deprotection reactions of Fmoc-L-Lys-monoamide-DOTA-tris(t-butyl ester) (Macrocyclics). Bisprotected DOTA-Lys was stirred with 90 % trifluoroacetic acid in dichloromethane at room temperature overnight, and the reagents were removed using a rotary evaporator and the residue was dried in vacuo. The resulting Fmoc-protected DOTA-Lys was stirred with 20 % piperidine in dimethylformamide at room temperature for 1 h. After the reagents were removed using a rotary evaporator, the reaction mixture was washed three times with dichloromethane, and the aqueous layer was lyophilized. The product was purified by HPLC. Purified DOTA-Lys was identified using FAB mass spectrometry: (m/z) [M + H]+ calcd. 533.2935, C22H41N6O9; found 533.2941. DOTA-Lys-HSA-Cy5.5 (300 μg) in 200 μL of PBS was injected into RR1022 tumor-bearing mice via a tail vein. At 24 and 48 h after injection, mice were killed and the urine samples were obtained as described above. Urine samples and authentic standards (Cy5.5-Lys and Cy5.5) were analyzed by TLC using a 3:1 mixture of ethanol and ammonium hydroxide as the mobile phase. The TLC plates were then subjected to optical imaging (excitation 675 nm, emission 720 nm) for 1 s. Cy5.5-Lys was synthesized by reaction of Nα-(t-butoxycarbonyl)-l-lysine (TCI, Tokyo, Japan) with Cy5.5 NHS ester in DMSO in the presence of N,N-diisopropylethylamine, followed by treatment with trifluoroacetic acid. The product was purified by HPLC. Purified Cy5.5-Lys was identified using MALDI-TOF mass spectrometry: (m/z) [M + H]+ calcd. 1045.2698, C47H57N4O15S4; found 1045.2183. Statistical analysis Data are expressed as mean ± S.D. Data were analyzed using the unpaired, two-tailed Student’s t test, and
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differences at the 95 % confidence level (P
