Development of 99mTc-Labeled Human Serum Albumin withProlonged Circulation by Chelate-then-Click Approach: A PotentialBlood Pool Imaging AgentNadeem Ahmed Lodhi,†,§,# Ji Yong Park,†,‡,# Kyuwan Kim,‡ Young Joo Kim,† Jae Hwan Shin,⊥

Yun-Sang Lee,*,† Hyung-Jun Im,*,∥ Jae Min Jeong,† Muhammad Khalid,§ Gi Jeong Cheon,†

Dong Soo Lee,† and Keon Wook Kang†

†Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, 03080, Republic of Korea‡Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, 03080, Republic of Korea§Isotope Production Division, Pakistan Institute of Nuclear Science & Technology (PINSTECH), P. O. Nilore, 45650, Islamabad,Pakistan∥Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University,Seoul, 08826, Republic of Korea⊥Department of Chemistry, Graduate School, Kyung Hee University, Seoul, 02453, Republic of Korea

*S Supporting Information

ABSTRACT: Technetium-99m-labeled human serum albu-min (99mTc-HSA) has been utilized as a blood pool imagingagent in the clinic for several decades. However, 99mTc-HSAhas a short circulation time, which is a critical shortcoming fora blood pool imaging agent. Herein, we developed a novel99mTc-labeled HSA with a long circulation time using clickchemistry and a chelator, 2,2′-dipicolylamine (DPA), (99mTc-DPA-HSA). Specifically, we examined the feasibility ofcopper-free strain-promoted alkyne-azide cycloaddition(SPAAC) for the incorporation of HSA to the [99mTc(CO)3(H2O)3]

+ system by adopting a chelate-then-clickapproach. In this strategy, a potent chelate system, azide-functionalized DPA, was first complexed with [99mTc(CO)3(H2O)3]

+, followed by the SPAAC click reaction with azadibenzocyclooctyne-functionalized HSA (ADIBO−HSA)under biocompatible conditions. Radiolabeling efficiency of azide-functionalized DPA (99mTc-DPA) was >98%. Clickconjugation efficiency of 99mTc-DPA with ADIBO−HSA was between 76 and 99% depending on the number of ADIBOmoieties attached to HSA. In whole-body in vivo single photon emission computed tomography images, the blood pool uptakesof 99mTc-DPA-HSA were significantly enhanced compared to those of 99mTc-HSA at 10 min, 2, and 6 h after the injection (P <0.001, 0.025, and 0.003, respectively). Furthermore, the blood activities of 99mTc-DPA-HSA were 8 times higher at 30 min and10 times higher at 3 h after the injection compared to those of conventional 99mTc-HSA in ex vivo biodistribution experiment.The results exhibit the potential of 99mTc-DPA-HSA as a blood pool imaging agent and further illustrate the promise of the pre-labeling SPAAC approach for conjugation of heat-sensitive biological targeting vectors with [99mTc (CO)3(H2O)3]

+.

KEYWORDS: chelate-then-click, human serum albumin, SPECT, blood pool imaging, ADIBO−HSA

■ INTRODUCTIONBlood pool imaging by positron emission tomography andsingle photon emission computed tomography (SPECT) playsan important role in the detection of infection,1 to evaluatecardiac function,2,3 gastrointestinal bleeding,4 and assessingvascular permeability in cancerous tissues.5 At present,technetium-99m-labeled red blood cells (99mTc-RBCs) andtechnetium-99m-labeled human serum albumin (99mTc-HSA)are the two main tracers in clinical nuclear medicine for theblood pool imaging. However, 99mTc-RBCs has complicatedlabeling procedures and a potential risk of cross-contamination.The labeling method of 99mTc-HSA is convenient, however,

99mTc-HSA has a short circulation half-life (32 min), which is a

critical limitation for the purpose.6 More recently, a chelator-

based tracer, 99mTc-human serum albumin-diethylenetriamine-

pentaacetic acid (99mTc-HSA−DTPA), has been developed.7

However, generally, HSA should be exposed to a low pH of 4−

Received: December 3, 2018Revised: March 13, 2019Accepted: March 14, 2019Published: March 14, 2019

Article

pubs.acs.org/molecularpharmaceuticsCite This: Mol. Pharmaceutics 2019, 16, 1586−1595

© 2019 American Chemical Society 1586 DOI: 10.1021/acs.molpharmaceut.8b01258Mol. Pharmaceutics 2019, 16, 1586−1595

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5 for the direct chelator conjugation and the circulation half-life of 99mTc-HSA−DTPA has not been well evaluated.The long circulation time of HSA (serum half-life of 19−22

days) is the basis for using HSA as a blood pool agent.However, the circulation half-life of HSA can be easilycompromised according to the radiolabeling methods. Theconventional radiolabeling method of 99mTc-HSA is performedat low pH values of 2−4 and includes the addition of areducing agent (stannous solution) to cleave disulfide bonds ofHSA.6 We hypothesized that these harsh conditions ofradiolabeling are the reasons for the short circulation half-lifebased on previous studies that (1) higher amount of stannoussolution resulted in a shorter circulation time6 and (2)chelator-based methods without stannous solution showedlonger circulation times,8 whereas high pH conditions duringconjugation resulted in a shorter circulation time.9 Also, evenin the chelator-based method, HSA should be exposed to lowpH of 4−5.8,10 Therefore, we adopted a chelate-then-clickapproach which separates the chelation and conjugationprocess so that HSA is not exposed to any harsh conditionsduring the radiolabeling procedure.Click chemistry refers to a group of reactions that have a fast

reaction rate and good orthogonality.11 Click chemistry can beperformed in biocompatible aqueous conditions, which is ahuge advantage for the preservation of soft biologicalnanomaterials including HSA during the radiolabeling.12

Furthermore, click chemistry has good orthogonality whichenables the separation of the chelation process from theconjugation process and also precise control of the degree offunctionalization (DOF).13 Most recently, biocompatiblecopper-free stain-promoted alkyne-azide cycloaddition(SPAAC) has been widely used for the conjugation of softbiological materials.14

Herein, we utilized the chelate-then-click copper-freeSPAAC approach to radiolabel HSA using the [99mTc-(CO)3(H2O)3]

+ core under biocompatible conditions todevelop a novel 99mTc-labeled blood pool imaging agent. Forchelation, 2,2′-dipicolylamine (DPA) was selected as abifunctional chelator for the [99mTc(CO)3(H2O)3]

+ core dueto its favorable complexation and kinetic inertness.15−17

■ EXPERIMENTAL SECTIONGeneral. All commercially available chemicals were of

analytical grade and were used without purification. Bis(2-pyridylmethyl)amine, 4-(Boc-amino)butyl bromide, anhydrouspotassium carbonate, N,N-dimethylformamide (DMF), anhy-drous dichloromethane (DCM), trifluoroacetic acid (TFA),and N,N-diisopropylethylamine (DIPEA) were purchased fromSigma-Aldrich, Korea. Succinimidyl-4-azidobutyrate and aza-dibenzocyclooctyne-N-hydroxysuccinimidyl ester (ADIBO-NHS) were purchased from Futurechem. Co., Ltd (Korea).HSA was purchased from MP Biomedicals (China). Thecommercially available HSA kit containing reduced HSA (1mg), gluconate (300 μg), and SnCl2 (60 μg) was provided byCellBion. Co., Ltd (Korea). Na[99mTcO4] was eluted from a99Mo/99mTc generator using saline obtained from UnitechKorea. [99mTc(H2O)3(CO)3]

+ was prepared using an IsoLinkkit (Center for Radiopharmaceutical Science, Paul ScherrerInstitute, Villigen, Switzerland). Thin layer chromatography(TLC-SG) was performed with silica plates (Silica gel 60 F254,Merck Ltd., Korea), developed by system 1 (methanol:HCl(99:1, v/v)) and instant thin layer chromatography silica gel(ITLC-SG) was purchased from Agilent Technologies, Inc.

(CA), developed by system 2 (methanol:0.1 M citric acid (1:4,v/v)). Radio-TLC was scanned using a BioScan AR-2000system imaging scanner (Bioscan, WI). High-performanceliquid chromatography (HPLC) was performed with a Gilson,equipped with a 506C system interface, a 155 UV−visdetector, and 321 pumps. HPLC conditions: method A;column: XTerra RP18 10 μm (10 mm × 250 mm) (WatersCo. Milford, MA), mobile phase: A: water (0.1% TFA); B:acetonitrile. Gradient: 0−3 min, 100% A; 3−30 min, 0% A;30−35 min, 0% A; 35−36 min, 100% A; flow rate of 5 mL/min.Electrospray ionization mass spectrometry (ESI-MS) was

performed with a Thermo Fisher LTQ Velos Instrument usingthe positive ionization mode. 1H NMR spectra were recordedon an AL 300 FT NMR spectrometer (300 MHz for 1H; Jeol,Tokyo, Japan). Chemical shifts are expressed as δ values (partsper million). The coupling constant (J) are reported in Hertz(Hz); the following abbreviations are used for the descriptionof 1H NMR spectra in the Experimental Section: singlet (s),doublet (d), triplet (t), quartet (q), multiplet (m), doublet ofdoublets (dd), triplet of doublets (td), doublet of doublet ofdoublets (ddd), doublet of triplets (dt). Matrix-assisted laserdesorption ionization time-of-flight (MALDI-TOF) wasperformed using Voyager DE-STR (Applied Biosystems) in alinear mode. Micro-SPECT/CT imaging was performed usinga NanoSPECT/CT device. All animal studies were performedin accordance with the guidelines of the Association for theAssessment and Accreditation of Laboratory Animal CareInternational (AAALAC International, 2007) at the SeoulNational University Hospital, Seoul, Korea.

Statistical Analysis. Quantitative data were shown as themean ± standard deviation (SD). Means were compared usingStudent t-test. Data were considered statistically significantwhen P values were <0.05.

Chemistry. Synthesis of tert-Butyl (4-(Bis(pyridin-2-ylmethyl)amino)butyl)carbamate (1). Bis(2-pyridylmethyl)-amine (157.8 mg, 0.79 mmol) and anhydrous potassiumcarbonate (141.94 mg, 1.02 mmol) were suspended in DMF(2 mL). 4-(Boc-amino)butyl bromide (319 mg, 0.87 mmol)dissolved in DMF (1 mL) was added dropwise to the reactionmixture. After mixing the solution for 18 h at roomtemperature (rt) in an inert atmosphere, the reaction mixturewas filtered to remove K2CO3 and the filtrate was evaporatedin vacuo. The residue was purified by column chromatographyon silica gel eluted with a mixture of DCM:methanol (97:3 (v/v)) to obtain compound 1 (Figures S1 and S2) (175 mg, 60%)as a yellowish oil. 1H NMR (300 MHz, CDCl3): δ ppm 8.81(dd, 2H, J = 5.7, 1.6 Hz), 8.21 (td, 2H, J = 7.8, 1.7 Hz), 7.89(d, 2H, J = 8.0 Hz), 7.68 (ddd, 2H, J = 7.1, 5.5, 1.2 Hz), 4.38(s, 4H), 3.03 (t, 2H, J = 6.7 Hz), 2.88−2.77 (m, 2H), 1.62 (m,2H, J = 6.4, 5.2 Hz), 1.44 (t, J = 7.0 Hz, 2H), 1.41 (s, 9H).ESI-MS m/z [M + H]+ 371.

Synthesis of N1,N1-Bis(pyridin-2-ylmethyl)butane-1,4-di-amine (2). Anhydrous trifluoroacetic acid (1 mL) was addedto the solution of 1 (150 mg, 0.40 mmol) in dichloromethane(1 mL). The reaction mixture was stirred overnight (18 h) atrt. After removing the solvent under vacuum, the residue waswashed several times with methanol to remove traces of TFAto afford compound 2 (Figures S3 and S4) as a yellowish oil(quant). Proceed to the next step without further purification.1H NMR (300 MHz, CD3OD): δ ppm 8.71−8.65 (m, 2H),8.01 (td, 2H, J = 7.7, 1.8 Hz), 7.64−7.49 (m, 4H), 4.53 (s,4H), 3.22 (t, 2H, J = 8.0 Hz), 2.99−2.88 (m, 2H), 1.87 (d, 2H,

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J = 6.5 Hz), 1.69 (q, 2H, J = 8.1 Hz). ESI-MS m/z [M + H]+

271.Synthesis of 5-Azido-N-(3-(bis(pyridin-2-ylmethyl)amino)-

propyl)pentanamide (3). To a solution of 2 (27 mg, 0.10mmol) in anhydrous DCM (2 mL) was added DIPEA (39 μL,0.23 mmol) and stirred for 10 min in an inert atmosphere.Succinimidyl-4-azidobutyrate (34 mg, 0.10 mmol) dissolved inDCM (1 mL) was added slowly to the mixture and stirred at rtfor 1 h. After completion of the reaction, the solvent wasremoved under vacuum, the residue was purified by HPLC(method A). After removing the solvent by lyophilization,compound 3 was afforded (Figures S5 and S6) as a light yellowoil compound (34 mg, yield = 90%). 1H NMR (300 MHz,CDCl3) δ ppm 8.66 (ddd, 2H, J = 5.0, 1.8, 0.9 Hz), 7.84 (td,2H, J = 7.7, 1.8 Hz), 7.62 (dt, 2H, J = 7.8, 1.1 Hz), 7.40 (ddd,2H, J = 7.6, 5.0, 1.1 Hz), 5.79 (s, 2H), 4.48 (s, 4H), 3.31 (t,2H, J = 6.8 Hz), 3.24−3.17 (m, 4H), 3.13 (qd, 2H, J = 7.3, 4.2Hz), 2.26 (t, 2H, J = 7.3 Hz), 1.93−1.83 (m, 4H), 1.54−1.46(m, 4H), 1.32 (t, 2H, J = 7.3 Hz). ESI-MS m/z [M + H]+ 382.Synthesis of ADIBO-Functionalized HSA (ADIBO−HSA).

For the preparation of ADIBO−HSA, three samples of HSA (5mg/mL, 73.5 nmol each) were treated with differentequivalents of NHS-ADIBO (5, 10, 20 equiv) dissolved in2% dimethyl sulfoxide in phosphate buffer saline (PBS, pH 7.4,500 μL). The mixtures were vortexed and allowed to conjugatefor 2 h at rt. The unreacted ligand was removed by sizeexclusion chromatography (PD-10 column) using PBS (pH7.4) as the eluent. The molecular weight of products was

determined by MALDI-TOF. The ADBIO incorporation anddegree of conjugation of HSA were also determined usingMALDI-TOF and UV−vis spectrophotometer (NanoDrop,Thermo Fisher).

Radiochemistry. Preparation of 99mTc-DPA. The pre-cursor [99mTc(H2O)3(CO)3]

+ was prepared according to areported method with slight modification.18 Briefly, to anIsoLink kit containing sodium tetraborate decahydrated (2.9mg), sodium carbonate (7.8 mg), potassium sodium tartratetetrahydrate (9.0 mg), and disodium boranocarbonate (4.5mg) was added 99mTcO4

− (500−700 μL, 370−740 MBq) in asealed vial. The vial was heated for 30 min at 100 °C. Thereaction mixture was cooled to ambient temperature andsubsequently neutralized to pH ∼ 7.0 using 1 N HCl (200 μL).The radiochemical purity was determined by radio-TLC usingsystem 1. A freshly prepared solution of [99mTc-(H2O)3(CO)3]

+ (500−700 μL, 370−740 MBq) was addedto a vial containing precursor 3 in methanol (100 μL, 200nmol). The vial was heated for 30 min at 100 °C. Theradiochemical purity was determined by radio-TLC usingsystem 2.

Preparation of 99mTc-DPA-HSA (Click Method). 99mTc-DPA (500 μL, 185 MBq) was added to vials containing asolution of ADIBO−HSA (DOF = 4.8, 8.4, 16.4, 1 mL, 30nmol, each) in PBS (pH 7.4). The vails were kept at 37 °C for30 min in an incubator with gentle shaking. The radiotracerwas purified by PD-10 column once using PBS as the eluent.Radio-TLC using system 2 was used to determine radio-

Scheme 1. Syntheses of Precursors DPA and ADIBO−HSAa

aReagents and conditions for (A) (a) 4-(Boc-amino)butyl bromide, anhydrous K2CO3, DMF (b) TFA/DCM (1:1), and (c) succinimidyl-4-azidobutyrate, DIPEA, DCM; (B) (a) PBS (pH: 7.4).

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labeling efficiency. An aliquot (2 μL) of each sample wasspotted on ITLC/SG and developed in the mobile phasementioned in the Experimental Section. The conjugated andunconjugated albumin has different Rf values on a radio-ITLC/SG chromatogram. The developed radio-ITLC/SG was thencounted on a radio-TLC scanner. The area under the peak wasestimated by Winscan software attached to the radio-TLCscanner to achieve conjugation efficiency.Serum Stability. The in vitro stability was tested in human

serum. In brief, PD-10 column-purified 99mTc-DPA-HSA (50μL, 11.4 MBq) was added to human serum (250 μL) and keptup to 6 h at 37 °C. An aliquot of the solution (2 μL) was takenafter 1, 3, and 6 h and radiochemical purity were determinedby radio-TLC using system 2.In Vivo SPECT Imaging. The SPECT/CT images were

taken after the administration of 99mTc-DPA-HSA (DOF = 4.8,8.4, 16.4, 100 μL, 9−14 MBq) into the lateral tail vein ofnormal mice. Mice were anesthetized with isoflurane andscanned with nanoscan-SPECT/CT (Mediso Medical ImagingSystem, Hungary). The scanning parameters are as follows: aγ-ray energy window of 140 keV ± 10%, a matrix size of 256 ×256, an acquisition time of 5−15 s per angular step of 18° anda reconstruction algorithm of ordered subset expectationmaximization with nine iterations. CT scans are performedwith a tube voltage of 45 kVp, an exposure time of 1.5 s perprojection, and a reconstruction algorithm of cone-beamfiltered back-projection.Ex Vivo Biodistribution. The biodistribution of 99mTc-DPA-

HSA (DOF = 4.8, 8.4, 16.4) and commercially availableconventional 99mTc-HSA was evaluated in normal mice (22−25 g, n = 4). A volume of 100 μL purified 99mTc-DPA-HSA (37kBq) and conventional 99mTc-HSA was administered via alateral tail vein in each mice. The mice (n = 4) were sacrificedafter 30 min and 3 h post-injection. The blood and interestedtissues and organs were excised, weighed, and the percentageof the injected dose per gram (% ID/g) was calculated bycounting all tissues in γ-counter (Packard Cobra II automatedγ-counter). The standard solution was also prepared to

estimate the total dose injected per mice. Values wereexpressed as mean ± standard deviation (SD) for four animals.

■ RESULTSStudy Scheme. The clickable, 2,2′-dipicolylamine (DPA)

bifunctional chelator, was synthesized with a linker containingthe terminal azide moiety (Scheme 1A). The ADIBO-functionalized HSA (ADIBO−HSA) was synthesized afterthe introduction of a varying number of ADIBO side-chains byreacting HSA with azadibenzocyclooctyne-N-hydroxysuccini-midyl ester (ADIBO-NHS) at various molar excess (Scheme1B). The radiolabeling procedure involved the chelation of the[99mTc(H2O)3(CO)3]

+ core with DPA-linked azide, followedby the click reaction with ADIBO−HSA (Scheme 2).Purification by size exclusion chromatography using PD-10column results in the final 99mT-labeled albumin, which wasthen evaluated in vivo in healthy mice. The results thusobtained were compared with commercially available 99mTc-HSA.

Synthesis of DPA and ADIBO−HSA. The synthesis ofDPA, which served as a chelator was accomplished in threesteps (Scheme 1A). The first step involves the reaction ofbis(2-pyridylmethyl)amine 1 and 4-(Boc-amino)butyl bromidein the presence of potassium carbonate to afford intermediate2 in 60% yield after purification by silica gel columnchromatography. The Boc group was deprotected with TFAand DCM (2 mL, 1:1 (v/v)) at rt to yield dipicolylamine 2(quant.). Conjugation of compound 2 with succinimidyl-4-azidobutyrate in the presence of DIPEA base produced thefinal product DPA in 90% yield, which was purified by HPLCin chemical purity (>99%). ADIBO−HSA with variable DOFwas synthesized by mixing HSA with various equivalents ofADIBO-NHS (5, 10, and 20 equiv). Purification was achievedby PD-10 to afford ADIBO−HSA in >98 ± 2% chemicalpurity. The number of ADIBO moieties per molecule, thedegree of functionalization (DOF), of HSA was calculatedfrom MALDI-TOF data using a simple formula as follows;DOF = (molecular weight of modified HSA − molecularweight of unmodified HSA)/molecular weight of the ADIBO

Scheme 2. Chelate-then-Click Labeling Strategya

aReagents and conditions (a) [99mTc(H2O)3(CO)3]+, 100 °C, 30 min; (b) ADIBO−HSA, pH: 7, 37 °C, 30 min.

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group. The average DOF calculated was 4.8 ± 0.01, 8.4 ± 0.02,and 16.4 ± 0.03, respectively (Figure 1A). A UV−visspectrophotometer was used to further confirm the successfulconjugation of ADBIO to HSA (Figure 1B). Also, we obtainedvery good reproducibility in preparing ADIBO−HSA con-firmed by MALDI-TOF. For example, the coefficient ofvariation (CV) of measured molecular weight of modified HSA

(n = 3) from one batch (1st batch, Table S1) was extremelylow (0.004−0.025%). Moreover, the CV of the scaled-up batch(200−400 mg in one time, 2nd batch, Table S1) was also verylow (0.012−0.043%). Furthermore, the agreement of themeasurements between 1st and 2nd batch was excellent withan intraclass correlation coefficient of 0.998 (confidenceinterval = 0.691−0.999).

Figure 1. Characterization of albumin and modified albumin (A) MALDI-TOF mass spectrum of HSA and ADIBO-functionalized HSA by reacting5, 10, 20 equiv of NHS-ADIBO with HSA resulting in average 4.8 ± 0.01, 8.4 ± 0.02, and 16.4 ± 0.05 molecule of DBCO incorporated with HSA(n = 3). (B) UV−vis spectrometer absorption spectra of HSA−ADIBO, unmodified HSA, and ADIBO-NHS. The absorbance peaks were found at280 nm in albumin (blue) and at 280 and 309 (red) in albumin−ADIBO. The peak intensity of albumin−ADIBO was increased according to theDOF confirming successful conjugation of ADIBO with HSA.

Figure 2. (A) Radio-TLC chromatogram. Na[99mTcO4] (red line), TLC-SG, methanol:HCl (99:1 (v/v)); [99mTc(H2O)3(CO)3]+ (orange line),

TLC-SG, methanol:HCl (99:1 (v/v)); 99mTc-DPA (green line), ITLC-SG, methanol:0.1 M citric acid (1:4 (v/v)); PD-10 column-purified 99mTc-DPA-HSA (black line), ITLC-SG, methanol:0.1 M citric acid (1:4 (v/v)). (B) Click reaction conjugation efficiency of 99mTc-DPA with ADIBO−HSA (DOF = 4.8, 8.4, 16.4).

Figure 3. Maximal projection intensity SPECT/CT images at 5 min and 3 h after the injection of (A) 99mTc-DPA-HSA, (DOF = 4.8), (B) 99mTc-DPA-HSA, (DOF = 8.4), (C) 99mTc-DPA-HSA, (DOF = 16.4), and (D) conventional 99mTc-HSA.

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Radiolabeling and in Vitro Stability. The radiolabelingand SPAAC ligand conjugation reaction are presented inScheme 2. First, 99mTc-DPA was prepared by heating the DPAchelator (100 μL, 200 nmol) with [99mTc(H2O)3(CO)3]

+ atpH 7. These conditions consistently provide high radio-chemical yield (>98 ± 2%). Second, the pairing of 99mTc-DPAwith ADIBO−HSA (DOF = 4.8, 8.4, 16.4) was conducted atphysiological pH and temperature for 30 min. Labeled albumin(99mTc-DPA-HSA) was purified by size exclusion chromatog-raphy using PD-10 column, eluted with PBS (pH 7.4). Thesize exclusion chromatography completely removed theunreacted 99mTc-DPA from the final product (99mTc-DPA-HSA). Radio-TLC showed successful radiolabeling at each step(Figure 2A). Also, it has been observed that higher DOF,incubation time, and temperature enhances the click reactionconjugation efficiency (at 30 min, 76, 98, 99% for DOF = 4.8,8.4, 16.4, respectively) (Figure 2B and Table S2). Moreover,99mTc-DPA-HSA showed high stability in serum and there is

no sign of decomposition of the radiotracer over a time periodof 6 h (Figure S7).

In Vivo SPECT Image. To investigate the in vivopharmacokinetics of 99mTc-DPA-HSA (DOF = 4.8, 8.4,16.4), whole-body SPECT/CT imaging was performed forall conjugates in healthy BALB/C mice at 5 min and 2 h post-injection. For comparison purpose, we also performed SPECTimaging of conventional 99mTc-HSA at the same time points.99mTc-DPA-HSA SPECT images demonstrated that themajority of the radioactivity was observed in the blood pooland liver. The heart and both carotid arteries were clearlyvisualized not only at 5 min but also at 2 h after the injection.The blood pool uptake was decreased according to the increaseof DOF (Figure 3A−C). In contrast, conventional 99mTc-HSAshowed very limited uptake in the blood pool and intenseuptake in the liver (Figure 3D). Another set of SPECT imagingexperiment was performed for the quantitative comparisonbetween 99mTc-DPA-HSA (DOF = 4.8) (n = 3) and 99mTc-HSA (n = 3) at 5 min, 2, and 6 h after the injection. 99mTc-

Figure 4. In vivo SPECT images and quantification. (A) Higher blood pool uptake and lower liver uptake were seen in the 99mTc-DPA-HSA (n =3) compared to 99mTc-HSA (n = 3). Quantified uptakes in the blood pool (B) and liver (C). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. Ex vivo biodistribution studies of 99mTc-DPA-HSA (DOF = 4.8, 8.4, 16.4) and conventional 99mTc-HSA (A) 30 min post-injection, (B) 3h post-injection.

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DPA-HSA (DOF = 4.8) showed significantly higher uptake inthe blood pool than 99mTc-HSA (P = 0.0001, 0.025, and 0.003,respectively). The liver uptake was significantly lower in 99mTc-DPA-HSA (DOF = 4.8) than in 99mTc-HSA (P = 0.001,0.0009, and 0.002) (Figure 4).Ex Vivo Biodistribution. The biodistribution results of

99mTc-DPA-HSA (DOF = 4.8, 8.4, 16.4) and conventional99mTc-HSA are given in Figure 5 and Table S3. The blood poolhad the highest activity for all conjugates having DOF = 4.8,8.4, 16.4 (57.52 ± 0.69, 33.04 ± 3.69, and 22.73 ± 2.53% ID/g, respectively) at 30 min post-injection, which wassubsequently decreased (42.92 ± 1.52, 29.64 ± 2.75, and15.35 ± 1.16% ID/g, respectively) at 3 h post-injection. Theblood pool uptakes of 99mTc-HSA were 7.22 ± 0.79 and 4.13 ±0.25% ID/g at 30 min and 3 h, respectively, after the injection.Thus, the blood pool uptakes of 99mTc-DPA-HSA (DOF =4.8) were 8 times higher at 30 min post-injection and even 10times higher at 3 h post-injection than those of 99mTc-HSA.99mTc-DPA-HSA had the second highest activity in the liver(20.86 ± 0.45, 19.01 ± 1.66, and 31.93 ± 1.56, respectively).At 3 h post-injection, a slight increase in the activity wasobserved in the liver as well as in the intestine showing that theradiotracers are predominantly cleared from the systemthrough the hepatobiliary pathway. The conventional 99mTc-HSA exhibited the highest activity in the liver (57.77 ± 1.47and 58.11 ± 2.83 at 30 min and 3 h post-injection,respectively).

■ DISCUSSION

We developed a novel 99mTc-labeled HSA, 99mTc-DPA-HSA,which has an enhanced blood pool imaging ability using achelate-then-click strategy. The chelate-then-click strategyconsisted of two steps. First, 99mTc was chelated to azide-functionalized DPA (99mTc-DPA), and HSA was functionalizedusing ADIBO for click reaction. Then 99mTc-DPA and HSAwere conjugated using the SPAAC click reaction inbiocompatible conditions. Our 99mTc-DPA-HSA showedenhanced blood pool uptake with an order of magnitude at3 h after the injection compared to conventional 99mTc-HSA.Albumin, which is the most abundant, constitutes roughly

50% of the total protein content in blood plasma (3.5−5 g/dl).It is a 66.5 kDa globular protein with a primary sequence madeup of 585 amino acid residues.19−21 Traditionally, the direct99mTc-labeling of albumin (conventional 99mTc-HSA) as bloodpool imaging is the simplest procedure, the resulting complexis relatively weak and unstable in vivo when injected into aliving system, consequently leading to a nontarget tissueuptake.2 Therefore, lots of efforts have been put forward todevelop an appropriate radiotracer by conjugation of abifunctional chelator agent that remains within the intra-vascular space for a long duration. However, the slow reactionkinetics and unfavorable pharmacokinetics of albumin-basedradiotracers suggest that the albumin structure might bealternated during the conjugation and labeling procedure.2,10

For imaging purpose, HSA has also been labeled with severalradioisotopes including 68Ga,22,23 18F,24−27 125I,28 and 99mTc7

through diverse bifunctional chelators. Compared with otherradioisotopes, 99mTc is widely used in nuclear medicinebecause of its optimum photon energy (t1/2 = 6 h, Eγ = 140keV) for SPECT imaging, low cost, and its widespreadavailability from commercial generator system.29 Also, 99mTccan be used safely because of its low in vivo toxicity.30

Moreover, the development of 99mTc-based radiopharmaceut-ical is facilitated by the use of rhenium as a nonradioactiveanalogue having chemistry similar to that of technetium andproduced complexes having similar properties for standardchemical and structural characterization. Recently, the [99mTc-(CO)3(H2O)3]

+ core received much attention as a precursorfor 99mTc radiopharmaceuticals.31−33 The [99mTc-(H2O)3(CO)3]

+ core possesses a low oxidation state Tc(I)center and produced complexes that are kinetically inert andthermodynamically stable due to its small size.34 In addition, inthe [Tc(CO)3(H2O)3]

+ core, water molecules can be easilysubstituted by suitable ligands (bi or tridentate) in aqueoussolution.35 However, complexation between radiometal (99mTc< 1 μM) and ligand required high temperatures (>90 °C) anda large amount of ligand to attain high radiochemical yields inshort reaction times even with promising tridentate ligandsystems.36,37 Radiolabeling under these harsh conditions wouldnormally denature the temperature-sensitive biomolecules(e.g., protein, peptide, and antibodies).To circumvent the problem, several new labeling strategies

have been developed, the chelate-then-click approach, wherethe bifunctional chelator is first complexed with [99mTc-(H2O)3(CO)3]

+ followed by the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction with thetargeting moiety. As the CuAAC click reaction occurred atphysiological temperature and pH, this radiolabeling strategypermits the conjugation of sensitive biomolecules that requireharsh radiolabeling conditions.38,39 The main disadvantage ofthe CuAAC reaction is the contamination of the product withthe copper ion and its coordination to targeting biomole-cules.40 Additionally, the use of Cu(I) ions as catalystshindered their utility in clinical nuclear medicine due to strictcurrent good manufacturing practices.41 Therefore, otherbiorthogonal reactions that do not require copper catalysts,have been proposed.42−46 However, the lack of ideal selectivity,slow reaction kinetics, moderate to mild stability underaqueous conditions and reversible substitution in vivo arepotential drawbacks of these reactions.47−50 Most recently,biocompatible copper-free stain-promoted alkyne-azide cyclo-addition (SPAAC) has attracted lot of interest due to the rapidrate of the reaction and excellent biorthogonal properties,which makes it an excellent candidate for radiolabeling of heat-sensitive targeting molecules.14,51 Several 18F-labeled dibenzo-cyclooctyne (ADIBO) prosthetic groups have been reported inthe literature for in vitro labeling as well as in vivo pre-targetingof peptides for tumor imaging.41,52−54 Therefore, we adoptedthe chelate-then-click strategy with the SPAAC click reaction.For clinical translation of 99mTc-labeled HSA, standardiza-

tion and optimization of the radiolabeling procedure areessential. Therefore, we optimized the radiolabeling procedureto achieve high radiolabeling efficiency and stability. Theconjugation efficiency of 99mTc-DPA and ADIBO−HSA underoptimized conditions was high (between 76 and 99%)depending on the number of ADIBO moieties attached toHSA. The high conjugation efficiency is attributed to thestability of ADIBO and the azide group in aqueous solution.For the development of kit formulation, radiolabelingefficiency of over 95% is favorable. However, our 99mTc-DPA-HSA with DOF 4.8 showed 76% of radiolabelingefficiency. This needs to be further enhanced for thedevelopment of kit formulation that could be used in theclinic. We are currently testing another cyclooctyne clickablemoiety (ODIBO) to increase the radiolabeling efficiency.41

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Meanwhile, the blood pool uptakes were decreased in higherDOFs. The reason for diminished circulation properties inhigher DOF seems to be higher interaction with serumproteins caused by a higher degree of surface chemistryalteration including the charge.55

We also determine the nonspecific binding of 99mTc withHSA, for this purpose, 99mTc-DPA and 99mTc were incubatedwith unmodified HSA under the same conditions separately.The results showed that HSA does not bind with 99mTc and99mTc-DPA and thus there is no nonspecific interaction withHSA (Figure S8). Therefore, the present labeling methodenables the conjugation of HSA with the ligand underrelatively mild radiolabeling conditions to afford the desiredproduct with outstanding efficiency. Moreover, the pre-labelingstrategy also avoids nonspecific, low-affinity binding of 99mTcwith HSA.After successful conjugation, 99mTc-DPA-HSA was tested in

vivo. High retention and slow clearance of the radiotracer isone of the most important characteristics to be an ideal bloodpool imaging tracer.23 The biodistribution of 99mTc-DPA-HSAwas evaluated in healthy mice and compared withcommercially available conventional 99mTc-HSA. As expected,most of the radioactivity was retained in the vascular system,showing the promising radiotracer as a blood pool imagingagent. Even after 3 h, more than 40% ID/g was retained in theblood for HSA with DOF of 4.8. It has been reportedpreviously that higher DOF of HSA with a bifunctionalchelator may alter the intrinsic properties of the HSA moleculeresulting in higher accumulation in the liver.22 Our results alsoshowed higher accumulation of activity in the liver comparedto blood with an increase in DOF. Additionally, conventional99mTc-HSA showed low whole blood activity and high liveruptake. The SPECT images are in accordance with thebiodistribution results, which further validated the effectivenessof the developed HSA conjugate as a blood pool imagingagent. Finally, we demonstrate the application of clickchemistry for labeling of HSA, which showed an obviousadvantage over conventional 99mTc-HSA. This implies that thecurrent, chelate-then-click SPAAC method for labeling of HSAcan be applied to label other heat-sensitive molecules to the[99mTc(H2O)3(CO)3]

+ system.There are several limitations to this study. We have not

evaluated the performance as a blood pool agent in any diseasemodel. The mechanism of hepatobiliary excretion of the tracerhas not been elucidated yet. It is warranted to evaluate theadvantages of the 99mTc-DPA-HSA over conventional 99mTc-HSA in the clinical setting.

■ CONCLUSIONS

We developed and optimized a simplified chelate-then-clickSPAAC strategy to radiolabel HSA with 99mTc to make a bloodpool imaging agent. This approach enables the pairing of 99mTcand HSA under mild reaction conditions in excellentradiochemical yield. The number of ADIBO moieties onHSA has a dramatic effect on click reaction conjugationefficiency. 99mTc-DPA-HSA showed high stability in vivo,resulting in higher blood retention and visualization ofvasculatures in healthy mice up to 3 h compared withcommercially available conventional 99mTc-HSA. We con-cluded that the present radiolabeling method could beapplicable to other sensitive biomolecules.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.molpharma-ceut.8b01258.

1H NMR spectra, ESI-MS spectra, stability study, andnonspecific binding results (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: wonza43@snu.ac.kr. Tel: +82-2-2072-8906. Fax: 82-2-766-9083 (Y-S.L.).*E-mail: iiihjjj@gmail.com. Tel: +82-031-888-9187. Fax: 82-2-766-9083 (H-J.I.).ORCIDHyung-Jun Im: 0000-0002-4368-6685Keon Wook Kang: 0000-0003-2622-9017Author Contributions#N.A.L. and J.Y.P. contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank the Korea Institute of Radiological & MedicalSciences for providing radioisotopes. H.-J.I. was supported bythe National Research Foundation of Korea (NRF) funded bythe Ministry of Education (NRF-2017R1D1A1B03035556)and Ministry of Health and Welfare Korea (HI18C0886). Y.-S.L. was supported by the National Research Foundation ofKorea (NRF) funded by the Ministry of Science and ICT(2017M2A2A7A01021401 and 2017M3A9G5082640).

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