Radioiodinated Portable Albumin Binder as a Versatile Agent for in Vivo Imaging with Single-Photon Emission Computed Tomography

Article abstract of DOI:10.1021/acs.molpharmaceut.8b01116

In this study, radioiodinated 4-(p-iodophenyl)butyric acid ([¹³¹I]IBA) was synthesized and evaluated as a portable albumin-binder for potential applications in single photon emission computed tomography imaging of blood pool, tumor, and lymph n

Full text of DOI:10.1021/acs.molpharmaceut.8b01116

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Article
Radioiodinated Portable Albumin Binder as a Versatile Agent for in
vivo Imaging with Single-Photon Emission Computed Tomography
Xuejun Wen, Changrong Shi, Duo Xu, Pu Zhang, Zizhen Li, Jindian Li,
Xinhui Su, Rongqiang Zhuang, Ting Liu, Zhide Guo, and Xianzhong Zhang
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/
acs.molpharmaceut.8b01116 • Publication Date (Web): 03 Jan 2019
Downloaded from http://pubs.acs.org on January 3, 2019
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Radioiodinated Portable Albumin Binder as a Versatile Agent for in
vivo Imaging with Single-Photon Emission Computed Tomography
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Xuejun Wen,† Changrong Shi,† Duo Xu,† Pu Zhang,† Zizhen Li,‡ Jindian Li,† Xinhui
Su,# Rongqiang Zhuang,† Ting Liu,† Zhide Guo,*,† Xianzhong Zhang*,†
†
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics &
Center for Molecular Imaging and Translational Medicine, School of Public Health,
Xiamen University, 4221-116 Xiang’An South Rd, Xiamen 361102, China;
‡ National Institute of Diagnostics and Vaccine Development in Infectious Diseases,
Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell
Engineering, School of Life Sciences, Xiamen University, Xiamen361102, China;
#
ZhongshanHospital Affiliated to Xiamen University, Hubin South Road, Xiamen
361004, China.
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ABSTRACT
In this study, radioiodinated 4-(p-iodophenyl)butyric acid ([131I]IBA) was synthesized
and evaluated as a portable albumin-binder for potential applications in SPECT
imaging of blood pool, tumor and lymph node with significantly improved
pharmacokinetic properties. The [131I]IBA was prepared under the catalyst of
Cu2O/1,10-phenanthroline. After that, the albumin-binding capability of [131I]IBA was
tested in vitro, ex vivo and in vivo, respectively. [131I]IBA was obtained with very high
radiolabeling yield (> 99%) and good radiochemical purity (> 98%) within 10 min. It
binds to albumin effectively with high affinity (IC50= 46.5 μM) and has good stability.
The results of biodistribution indicated that the [131I]IBA was mainly accumulated in
blood with good retention (10.51±2.58%ID/g at 30 min p.i. and 4.63±0.17%ID/g at 4
h p.i.). In the SPECT imaging of mice models with [131I]IBA, blood pool, lymph node
and tumors could be imaged clearly with high target-to-background ratio. Overall, the
radioiodinated albumin binder of [131I]IBA with long blood half-life and excellent
stability could be used to decorate diversified albumin-binding radioligands and
developed as a versatile theranostic agent.
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Keywords: albumin binder, radioiodination, SPECT, animal models, in vivo imaging.
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INTRODUCTION
The development of molecular imaging has provided renewed hope for disease
diagnosis and therapy, which can offer the visualization, characterization, and
measurement of biological processes at the molecular level.1 During the design and
synthetic phase, by improving in vivo pharmacokinetics of drugs, researchers move
ever closer to the desired outcome. In general, getting the satisfactory target to
non-target ratios within a relatively short time is an effective way of achieving a rapid
diagnosis. While, in terms of traditional drugs, such as theranostic agents for
cardiovascular diseases and tumor, short blood circulatory half-life fails to live up to
their full potentials. Consequently, it is vital to research and develop effective drugs
with optimal in vivo pharmacokinetics.2 In such cases, just low dosages could achieve
and maintain therapeutic effects, which reduce toxicity and the possibility of
undesirable side effects.3
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Albumin was regarded as a prominent carrier to protect drugs from enzymatic
degradation and increase the half-lives.4,5 We also anticipate that a wide range of
biomedical applications for the albumin-binding moieties, particularly for the
amelioration of pharmacokinetic properties. Previous literatures reported that
truncated Evans Blue (EB) dye molecule could bind quantitatively to serum albumin
in vivo and had been used for lymph node (LNs), tumor and blood pool imaging.6,7
For example, 68Ga (t1/2=68 min) or 18F labeled NEB has been used for PET imaging.8,9
Dumelin et al.10 reported a class of albumin binders that displayed stable
noncovalent binding interaction with human serum albumin (HSA). Among these
binders, 4-(p-iodophenyl)butyricacid (4-IBA) was one of the promising structures,
and has aroused great interest among the researchers in the molecular imaging field.
Cristina Müller’ team developed a series of DOTA/NOTA-folate conjugates
containing the albumin-binding IBA entity to improve the tumor to kidney ratio as a
consequence of a prolonged blood circulation time.11-13 Likewise, some other agents,
such as 177Lu labeled prostate-specific membrane antigen (PSMA)-targeting ligands,
also benefited from the albumin-binding motif.14,15
The goal of present work is to radioiodinate 4-IBA without destroying the
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molecular structure and evaluate its potential application for SPECT imaging with
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long blood half-life and excellent in vivo stability. Compared with 99mTc or F, both
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125I (t1/2=60 d) and 131I (t1/2=8.03 d) have longer physical half-life, which have been
widely used as therapeutic radionuclides to label albumin and other molecular
structures.16,17 Furthermore, the versatile characteristics of radioiodine isotopes (such
as 123I, 124I, 125I and 131I) ensure the maximum satisfaction of theragnostic purposes.
Therefore, radioiodinated 4-IBA was prepared using a convenient and quantitative
radioiodination method via copper-mediated cross-coupling of aryl boronic acids.18,19
In addition to the amelioration of pharmacokinetic properties of different
molecules, the radioiodinated IBA agent can be used for the examination and
prediction of multiple diseases. In the present study, preliminary applications of
radioiodinated IBA in the blood pool, tumor and lymphatic imaging with SPECT
were performed.
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EXPERIMENTAL SECTION
Reagents and Instruments
All chemicals were obtained commercially and used without further purification.
The Na131I were obtained from Zhongshan Hospital Affiliated to Xiamen University.
The polyacetamide film was purchased from Pall Life Sciences and silica gel 60 F254
aluminum plates were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd.
The labeling efficiency and radiochemical purity were tested by Mini-Scan radio-TLC
Scanner (BioScan, USA) and Dionex Ulti-Mate 3000 high-performance liquid
chromatography (HPLC, Thermo Scientific, USA) with flow-counter radioactivity
detector (BioScan, USA). The radioactivity was measured with γ-counter (WIZARD
2480, Perkin-Elmer, USA) and CRC-25R Dose Calibrator (CAPIN-TEC. Inc, USA).
SPECT imaging was performed by using a nanoScan SPECT/CT scanner (Mediso,
HUNGARY).
Animal Models
All the mice were obtained from the Laboratory Animal Center of Xiamen
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University. Animal studies were approved by the national laws and carried out in
compliance with the conduct of animal experimentation.
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Inflamed LNs models: BALB/c mice were used to develop the hind limb LNs
inflammation models. Mouse received a bilateral injection of 30 μL complete Freund
adjuvant (Sigma Chemical) into the dorsal footpad, causing bilateral lymphadenitis. A
few days later, SPECT imaging of inflamed LNs was performed. After imaging, EB
dye was injected to identify lymphadenitis models.
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4T1 murine breast tumor models: The right rear flanks of BALB/c mice were given
a suspension of 4T1 murine breast cancer cells (5 × 106 cells of 4T1 in 50 μL PBS)
subcutaneously. The mice were subjected to SPECT imaging when the tumor volume
reached about 250 mm3 (about 10 days after inoculation).
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Orthotopic U87MG glioblastoma models: U87MG glioblastoma cells (1 × 105)
stably transfected with a luciferase expression plasmid (U87MG-Luc) were suspended
in sterile PBS, and stereotactically implanted in the right cerebral striatum of
immunocompromised nude mice. Tumor growth was quantified weekly by
bioluminescence imaging using a Xenogen In Vivo Imaging System after
intraperitoneal injection of 110 mg/kg D-luciferin. The mice were subjected to
SPECT imaging when the tumor volume reached about 100 mm3.
Chemistry and Radiochemistry
The preparations of the desired compound [131I]IBA and control compound
4-iodobenzoic acid ([131I]IA) are shown in Figure 1a and Figure S13a.
Octanol/Water Partition Coefficient
To determine the hydrophilicity of [131I]IBA, octanol/water partition coefficient
(expressed as logP) was measured by using the reported method previously.20,21
Briefly, 100 µL radiotracer solution was diluted with 2.9 mL PBS and 3 mL
1-octanol. After vortex blending for 3 min, the mixtures were centrifuged at 6000 rpm
for 5 min. Then 100 µL organic layer was removed, and 2.9 mL 1-octanol and 3 mL
PBS were added for another vortex blending and centrifugation. Repeat the process
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and then the radioactive counts in the organic layer and inorganic layer (100 µL) were
determined by γ-counter, respectively. The following equation was used to calculate P
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=
(activity in octanol phase-background activity)/(activity in aqueous
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phase-background activity). All the experiments were performed with three
independent measurements and reported as mean ±SD.
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Interactions between [131I]IBA and Proteins
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The interaction of [131I]IBA with albumin was tested using dialysis experiment.
Dialysis membranes (molecular weight cut-off (MWCO) value of 7 kDa) containing
radiolabeled compounds (about 18 MBq) and HSA (or BSA) were submerged in
phosphate buffer (PB, pH 7.4) to remove the unbounded tracer. Radioactivity in PB
and remained in membranes were measured by γ-counter and CRC-25R dose
calibrator at different times, respectively. [131I]IBA dialyzed against PB without HSA
was performed for comparison. To explore the binding specificity, [131I]IBA was
added to the various type of proteins (HSA, hemoglobin and immunoglobulin) and
incubated for 1 h at 37 °C. Then, the mixture was dialyzed in PB to remove the
unbounded [131I]IBA as described above. Furthermore, as a supplement, ultrafiltration
was carried out to further validate the binding specificity. All experiments were
performed with triplicate samples and reported as mean ± SD.
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To learn more about the interaction between [131I]IBA and protein, molecular
docking technique was further employed to confirm binding site, binding force and
energy of [131I]IBA with HSA.
To determine whether the IBA and EB were in the same binding site, a competitive
blocking experiment was performed. Briefly, [131I]IBA and increasing concentrations
of EB were incubated with albumin for 1 h at 37 °C. After incubation, an
ultrafiltration membrane with an MWCO value of 10 kDa was used to separate
unbounded [131I]IBA. Thereafter, the radioactivity bound to HSA in ultrafiltration
centrifuge was measured by γ-counter. Binding values were calculated by fitting the
data with nonlinear regression using GraphPad Prism. Experiments were performed in
triplicate.
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In Vitro Binding Affinity Assays
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In vitro albumin binding affinity of [131I]IBA was measured via competition with
nonradioactive IBA, by following the same steps as the above dialysis experiment.
For comparison, EB was radioiodinated with 131I using the iodogen method and the in
vitro albumin binding affinity of [131I]EB was measured as well (see Supplementary
Information). Furthermore, the influences of precursor 4-(4-boronophenyl)butyric
acid (4-BBA) on the albumin binding of [131I]IBA were studied also. Briefly,
[131I]IBA (185 kBq) was incubated in albumin with the presence of increasing
concentrations of 4-BBA for 1 h at 37 °C. After incubation, an ultrafiltration
membrane with an MWCO value of 10 kDa was used to separate HSA bounded and
unbounded [131I]IBA. Thereafter, the radioactivity bound to HSA in ultrafiltration
centrifuge was measured by γ-counter. The results were normalized in terms of the
total added radioactivity (% of total activity). All the experiments were performed
with triplicate samples and reported as mean ± SD.
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Biodistribution Study
The biodistribution of [131I]IBA was evaluated in normal and 4T1 tumor-bearing
BALB/c mice (18-20 g, female), which were randomly divided into 7 and 4 groups
(n=3), respectively. Approximately 370 kBq of the radiotracer (100 µL) was
administered via a lateral tail vein of each mouse. Then the normal mice were
sacrificed at 1 min, 30 min, 1 h, 2 h, 4 h, 12 h, and 24 h post-injection (p.i.) and the
tumor-bearing mice were sacrificed at 2 h, 4 h, 6 h, and 12 h p.i. The interested tissues
and organs were excised, weighed and counted by γ-counter. The results were
calculated as a percentage of the injected dose per gram of tissues (%ID/g).
The biodistribution of [131I]IA(370 kBq) in normal BALB/c mice was also
performed at 1 min, 1 h, 4 h, 24 h p.i. (n=3/group) for comparison.
In Vivo SPECT/CT Imaging Study
About 7.4 MBq radiotracer (in 100 μL saline) was injected intravenously into each
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4T1 tumor-bearing mouse and orthotopic U87MG model mouse, respectively, for
SPECT/CT imaging studies using a nanoScanSPECT/CT preclinical scanner (Mediso,
HUNGARY) at 15 min, 1 h, 2 h, 6 h, 24 h (4T1 tumor-bearing mice) or 2 h, 6 h, 12 h
(U87MG glioblastoma models) p.i. The acquiring parameters were as follows: 364
keV energy peak for 131I, window width of 20%, matrix of 256×256, medium zoom,
and 48 frames (non-rotational acquisition of multipinhole SPECT for 30 s/frame).
During acquisition, anesthesia was induced with 1.5% isoflurane to maintain
spontaneous breathing of mice.
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For SPECT imaging of lymphatic node, 0.74 MBq [131I]IBA in 10 μL saline was
injected into the footpad of each side of BALB/c mice. Then the mice were imaged at
30 min and 1 h p.i. with imaging parameters described above.
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SPECT imaging of 4T1 tumor-bearing mice with [131I]IA were conducted for
comparison. About 7.4 MBq [131I]IA (in 100 μL saline) was injected intravenously
into each mouse and SPECT/CT images were acquired at 15 min, 2 h, 6 h and 24 h
p.i.
Biological Safety
The toxicity of precursor 4-BBA and nonradioactive IBA were tested by MTT
(methyl thiazolyl tetrazolium) experiments in 293T (human embryonic kidney cells)
and LO2 cells (normal human hepatocyte). For the radiation safety, the medical
internal radiation dose (MIRD) of [131I]IBA was estimated based on biodistribution
data by using OLINDA/EXM 1.1 code.
RESULTS
Chemistry and Radiochemistry
As shown in Figure 1a, [131I]IBA-NHS was synthesized in the presence of Cu2O
and 1,10-phenanthroline according to a procedure reported by Zhang et al.19 The
4-BBA and its active ester were characterized by 1H-NMR and 13C-NMR (Figure S1
and Figure S2). The labeling progress of [131I]IBA-NHS monitored by TLC was
shown in Figure S3. The radioactive peak area of [131I]IBA-NHS was exceeded 99%
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within 10 min indicating high radiolabeling yield. As shown in Figure S4-S7, the
radiochemical purity (>98%), specific activity (300 MBq/μmol) and stability were
also determined by TLC and HPLC. The HPLC retention times of [131I]IBA-NHS and
[131I]IBA were 5.23 and 4.16 min, respectively. [131I]IBA was stable after incubated
24 h in saline or PB at different pH (from 4.5 to 8.5). The logP of [131I]IBA was
calculated as 1.01±0.03, indicating its moderate lipophilicity.
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The protein-ligand interactions (including [131I]IA and 4-BBA) were discussed in
Figure 1b. Radioiodine labeled [131I]EB was also obtained for comparison study, and
its radiochemical purities (>99%) were confirmed by HPLC (Figure S10).
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Interactions between [131I]IBA and Proteins
As shown in Figure S8a-c, [131I]IBA in dialysis bag without HSA was readily
dialyzed into PB solution (88.46±1.28% of radioactivity at 24 h) and only
1.62±0.41% radioactivity remained in the bag after 24 h. In the presence of HSA,
long-time radioactivity retention of [131I]IBA in dialysis bag could be detected,
indicating the effectively albumin-binding of [131I]IBA. To further understand the
binding specificity of [131I]IBA, it was incubated with hemoglobin and
immunoglobulin, respectively. The dialysis (Figure S8d-f) and filtered (Figure
S9a-b) fraction of [131I]IBA incubated with hemoglobin samples contained significant
portions of radioactivity, indicating that most of the [131I]IBA was bound to
hemoglobin. While the [131I]IBA-binding capacity of immunoglobulin was much
weaker than that of HSA and hemoglobin. The binding specificity of [131I]IBA was
similar to [131I]EB (Figure S9c).
According to the modeling results of HSA with IBA and IA (Figure 2a), the
principal regions of intermolecular binding on HSA are located in hydrophobic
cavities, which are corresponding to sites 1 and 2. The more detailed information of
protein-ligand interaction was also evaluated by estimating energies of binding
(shown in Figure 2b). The binding energies between IBA and HSA were found to be
-14.62 kcal/mol (site 1) and -16.75 kcal/mol (site 2), which indicated that the
interaction between IBA and HSA was highly spontaneous and energetically
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favorable. As a comparison, binding of IA-HSA was also studied by molecular
docking, and the binding energy was found to be -7.38 kcal/mol (site 1) and
-6.16 kcal/mol (site 2), which were higher than those of IBA-HSA. Moreover, as
shown in Figure 2c, IBA and EB dye could bind HSA competitively, indicating the
IBA and EB had similar binding sites.
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In vitro albumin binding affinity of the [131I]IBA was assessed via competition with
different concentration of nonradioactive 4-IBA. As shown in Figure S11a, the
[131I]IBA showed eminent albumin-binding affinity with an IC50 value of 46.5 μM,
which comparable to the radioiodinated EB ([131I]EB, IC50 = 25.1 μM, Figure S11b).
Furthermore, the result of ultrafiltration experiment (Figure S12) showed that the
albumin-binding affinity of [131I]IBA was basically impregnable in different
concentration of 4-BBA.
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Biodistribution Study
The biodistribution result of [131I]IBA in normal mice was shown in Figure 3a. At
1 min p.i., much higher blood radioactivity (20.70±0.53%ID/g) was observed than the
other tissues, indicating an initial accumulation in the circulation system. After 30 min
injection, about half of the radioactivity uptake was cleared from blood and other
major organs. The blood uptake was decreased to 10.51±2.58%ID/g, and the uptakes
of the liver, kidney, lung were decreased from 5.47±0.38%ID/g, 8.83±1.40%ID/g,
11.18±4.76%ID/g to 2.93±0.11%ID/g, 4.47±0.13%ID/g, 5.68±0.40%ID/g,
respectively. At 24 h p.i., the blood remained the highest accumulation
(1.15±0.12%ID/g) when compared with the other tissues. As shown in Table S1, the
ratios of blood to tissues were relatively high during the observation period indicating
it might be a useful agent in blood pool imaging. Furthermore, the radioactivity in the
thyroid was steadily low, confirming the good in vivo stability of the
radiopharmaceuticals.
The biodistribution of [131I]IBA in 4T1 tumor-bearing mice showed high tumor
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uptake with good retention (Table S2). The sustained high tumor uptake (about
3%ID/g from 2 to 10 h p.i.) might due to the slow blood clearance of tracer. For the
other organs, the biodistribution showed a similar trend with the results of normal
mice.
[131I]IA was selected as a control compound to validate the influence of molecular
structure on the albumin binding ability. As illustrated in Figure S13b, the blood
radioactivity was decreased to an extremely low level (0.57±0.08%ID/g) at 30 min
p.i., the very fast blood clearance reflect very low albumin binding affinity of [131I]IA
when compared to [131I]IBA (see the Figure 3b and 3c). This result confirmed that
the efficient binding of [131I]IBA to the proteins was due to the particular structure of
4-IBA.
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In Vivo SPECT/CT Imaging Study
In vivo SPECT/CT scans of 4T1 tumor mice were shown in Figure 4a. After i.v.
injection of [131I]IBA, long lasting high radioactivity accumulation in the heart and
blood vessels allowed the visualization of radioactive distribution with SPECT. At the
same time, increased tumor uptake of [131I]IBA was observed at later time points. The
low uptakes in the background and non-target organs leading to high SPECT image
contrast. Furthermore, the negligible thyroid radioactivity reaffirmed the good in vivo
stability of [131I]IBA. After SPECT imaging, the tumor region was further identified
by in vivo MRI imaging and in vitro haematoxylin and eosin (H&E) staining to
discriminate the necrosis part from the active tumor area (Figure 4b and 4c). As
shown in Figure 4d, there was no obvious difference between the tumor and
surrounding muscle at 15 min p.i., while the T/NT ratios (tumor/liver and
tumor/muscle ratios were increased obviously at later time points (from 1.65 and 3.35
to 2.76 and 3.87 from 1 h to 6 h p.i.). The enhanced tumor uptake might due to the
slow blood clearance of [131I]IBA. These imaging results are in accordance well with
that of biodistribution. The fast blood clearance and negligible tumor radioactivity of
control agent [131I]IA (Figure S13c) further demonstrated the low albumin binding
affinity of 4-iodobenzoic acid.
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The successful imaging of subcutaneous tumor motivated us to further explore
more complex applications, especially for an orthotropic brain tumor and inflamed
LNs models. For SPECT imaging of brain tumor (Figure 5a), due to the low level of
radioactivity in the normal brain and high uptake in tumor regions, these
circumstances resulted in a high tumor-to-brain ratio. The ratio of tumor-to-brain
reached to 5.75±0.32 at 12 h p.i. (Figure 5b). As shown in Figure 5c, the
orthotopically implanted Luc-expressing U87MG glioma tumor models were further
verified by bioluminescence 750 imaging.
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To verify the potential of [131I]IBA for LNs imaging, inflamed LNs models were
established. The hotspot was displayed through maximum intensity projection (MIP).
Fusion of SPECT/CT images confirmed the anatomic location of the popliteal LNs.
As shown in Figure 6, inflamed popliteal LNs on both sides were clearly seen on
SPECT images, with a high signal to background ratio at all time points examined.
Based on the analysis of these images, we found that LNs in different states had
obviously different tracer uptakes. To understand the sources of this difference, after
imaging, EB dye was injected into soles to identify lymphadenitis models (Figure
6a(ii), b(ii) and c(ii)). The LNs with the significant signal intensity of dye matched
exactly with the hotspots in SPECT images. The mouse was sacrificed after SPECT
imaging, and the bilateral LNs were dissected and observed. Through the anatomical
analysis in Figure 6b(ii) and 6c(ii), it was found that the left popliteal LNs were
enlarged due to the inflammatory stimulation, which was consistent with the messages
in Figure 6b(i) and 6c(i). Quantification of the SPECT images showed that uptake of
[131I]IBA in the inflamed LN was 0.21%ID, which was significantly higher than that
in the contralateral LN (0.02%ID) at 1 h p.i. (Figure 6b). Along with the
inflammation development, the activity-rich region was expanded (Figure 6c).
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Biological Safety
MTT assay of precursor 4-BBA and nonradioactive IBA at various concentrations
in 293T and LO2 cells was tested. The results showed that 4-BBA and IBA had little
effect on cell viability at concentrations below 0.1 mg/mL (Figure S14). To estimate
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the safe dosage for clinical use, total absorbed radiation dosage based on
biodistribution data was estimated by OLINDA/EXM software (as shown in Table
S3). The administration of [131I]IBA was well tolerated, with no marked effects on
vital signs. This dosage provided a more accurate estimate of the dosimetry clinically
for human scans in the future.
DISCUSSION
As we all know, there are several agents for the circulation system or tumor
imaging have been explored and developed. The 99mTc (t1/2=6.02 h) labeled red blood
cells (99mTc-RBCs) as the major imaging agent for radionuclide angiography, had
been used clinically as early as 1958 for determining total blood and plasma
volume.22,23 99mTc-Sulfurcolloid (99mTc-SC) has long been used in the imaging of
LNs. However, it is plagued by slow transport from the injection site.24 It is also a pity
that, in the cases of radionuclide therapy, these radiotracers are powerless. Relatively,
radioiodine isotopes are clearly the more versatile and favorable radionuclides.
Radioiodinated albumin can be used as a blood pool imaging agent to determine the
circulating plasma volume, amount of blood circulation and blood circulation time,
etc.25 Unfortunately, the common methods of radiolabeling albumin in vitro (such as
chloramine-T method for radioiodine labeling) come with many disadvantages like
the instability of product, protein degeneration, product contamination and
immunological rejection reaction. The unfavorable pharmacokinetics of these tracers
could be crucial factors degrading the quality of imaging. Among the in vivo and in
vitro albumin-labeled molecules, EB dye is a representative one. However, the
preparation of EB derivatives requires harsh conditions and tedious procedures.6
Moreover, EB molecule can’t be labeled with radionuclides (18F, 68Ga or 177Lu)
directly, chemical modification with bifunctional chelators such as NOTA or DOTA
is needed to chelate diagnostic or therapeutic radionuclides, which add new
complexities.9,26,27
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Based on the results of previous literature,10 we develop the in vivo
albumin-labeling method to meet the needs for both preclinical evaluation and clinical
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application. The preparation procedure of radioiodinated 4-IBA is rapid, efficient and
simple, which has great potential for clinical translation. As mentioned above,
nonradioactive IBA has been utilized as an albumin binding molecule to modulate
pharmacokinetics for therapeutic purposes. [131I]IBA, compared with EB dyes, has
several eminent advantages: mild labeling conditions, simple operational processes
and stable chemical structure, no obvious deiodination is observed in vivo and in vitro
experiments, which is agreed well with the previous studies.18,19,28 In contrast, as
shown in Figure S15, [131I]EB has shortage instability, which degenerates the images
seriously. Tedious purification processes were always bottlenecks restricting the next
application of drug candidates. And at the same time, radioactivity loss during
purification processes was not to be ignored. The facile radioiodination of aromatic
and heteroaromaticboronic acid systems provided ultra-high labeling yield and
excellent radiochemical yield. Furthermore, after radiolabeling, superfluous precursor
boronic acid substrate had no impact on the albumin-binding affinity of the aimed
product, which meant further purification was unnecessary (see Figure 1b and Figure
S12). And more importantly, the radioiodinated portable IBA-NHS can serve as a
“Bolton-Hunter” reagent or a versatile intermediate, which could be conveniently
converted into the desired targeting or therapeutic molecules through the simplest
chemical reaction at RT, without requiring extra functional chelating agents. Stability
properties and long half-life provide a foundation for transport transfer.
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In the present work, the potential application of [131I]IBA were demonstrated in the
blood pool, tumor and LNs imaging. High radioactivity retained in the blood
circulatory system after i.v. injection verify the feasibility of using the tracer as a
blood-pool imaging agent. More importantly, the high accumulation of [131I]IBA in
the tumor is afforded by albumin binding and resulted in enhanced imaging contrast.
As shown in Figure 4, this agent could also be used to distinguish tumor and necrosis
sites, detect tumor activity and monitor the progress of tumor. And as expected, the in
vivo labeling strategy can also be applied to evaluate the lymphatic system under both
physiologic and pathologic circumstances (such as inflamed LNs). Better yet, these
encouraging results are not mediated and affected by receptors expressed in cancer
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cells. By contrast, the albumin binding affinity of [131I]EB has a narrow lead over that
of [131I]IBA (IC50: 25.1 µM vs. 46.5 µM). Whereas, relatively high thyroid and
stomach uptakes were achieved because [131I]EB is unstable in vivo. From these
results, we can see that radioiodinated IBA is comparable to radiolabeled EB tracers
for tumors and LNs imaging.10,24,29 Thus, radioiodinated IBA is a capable
albumin-binding radiotracer like EB agents, which has been successfully
demonstrated in the modification of several radiolabeled targeting molecules (RGD 3,
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PMSA , Octreotide , Exendin-4 , etc.). In some ways, IBA provides even more
extensive features.
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CONCLUSIONS
A novel radioiodinated portable albumin binding moiety was obtained with
high-efficiency synthesis processes and mild reaction conditions. Our study indicated
that this moiety has long blood half-life and good stability, which is well-tolerated
with regard to the interactions of the compound with serum proteins. Hence, the
radioiodinated IBA has great potential for clinical application in the blood pool,
tumors and LNs imaging. In addition, this radioiodinated albumin binder is
transferable to other molecules or targeting agents to facilitate the design of
albumin-binding radioligands and perfect their pharmacokinetics. This will be the
main contents in future research.
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ASSOCIATED CONTENT
Supporting Information
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The details of chemical synthesis routes, characterization of compounds and
biodistribution were described in the supplementary file. This material is available
free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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*Corresponding authors
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Xianzhong Zhang (Ph.D., Professor) and Zhide Guo (Ph.D.). Center for Molecular
Imaging and Translational Medicine, School of Public Health, Xiamen University,
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4221-116
Xiang’An
South
Rd,
Xiamen
361102,
China.
E-mail:
zhangxzh@xmu.edu.cn; gzd666888@xmu.edu.cn.
Author contributions
Xianzhong Zhang, Zhide Guo and Xuejun Wen were responsible for the conception
and design of the study, the acquisition and analysis of the data, the drafting of the
manuscript, and final approval of the version to be published. Ting Liu and
Rongqiang Zhuang contributed to critical revision for important intellectual content,
and final approval of the version to be published. Changrong Shi, Duo Xu, Pu Zhang,
Zizhen Li and Jindian Li contributed to acquisition, analysis and interpretation of
data. Xinhui Su contributed to critical revision of the manuscript for important
intellectual content and material support.
Notes
The authors declare no competing financial interest.
Acknowledgments
This study was financially supported by the National Natural Science Foundation of
China (81471707), Postdoctoral Science Foundation of China (2018M630732), China
Postdoc Innovation Talent Supporting Program (BX201700142) and Science
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Foundation of the Fujian Province, China (Grant No. 2016J05200).
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ABBREVIATIONS
The following abbreviations are used in this manuscript:
IBA, 4-(p-iodophenyl)butyric acid
4-BBA, 4-(4-boronophenyl)butyric acid
IA, 4-iodobenzoic acid
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EB, Evans Blue
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LNs, lymph nodes
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HSA, human serum albumin
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PSMA, prostate-specific membrane antigen
MWCO, molecular weight cut-off
MTT, methyl thiazolyl tetrazolium
MIRD, medical internal radiation dose
H&E, haematoxylin and eosin staining
SPECT, single photon emission computed tomography
PET, positron emission tomography
MRI, magnetic resonance imaging
PB, phosphate buffer
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%ID/g, percentage of injected dose per gram
ROIs, regions of interests
T/NT, tumor-to-non-target-tissue
MIP, maximum intensity projection
HPLC, High Performance Liquid Chromatography
RBCs, red blood cells
99mTc-SC, 99mTc-Sulfurcolloid
DOTA, 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
NOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid
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The legends of tables and figures:
Figure 1. (a) Chemical synthesis route and radiolabeling process of [131I]IBA. (b)
Illustration for the protein-ligand interactions.
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Figure 2. (a) Docking of IBA and IA to HSA. (b) The binding energy of probes and
HSA. (c) The albumin binding ratios of IBA were obtained in the presence of
different EB dye concentrations.
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Figure 3. (a) The biodistribution results of [131I]IBA in normal mice (%ID/g,
mean±SD, n=3). (b) Blood elimination curve of [131I]IBA and [131I]IA. (c) The
percentage curve of blood elimination rate of [131I]IBA and [131I]IA.
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Figure 4. (a) MIP SPECT/CT images of 4T1 tumor mice after intravenous injection
of 7.4 MBq [131I]IBA. (b) After SPECT imaging, the tumor region was further
identified by MRI. (c) H&E staining was used to discriminate necrosis and tumor. (d)
T/NT ratios at different time-points. Data are shown as mean ± SD (n=3).
Figure 5. (a) SPECT/CT images of brain tumor mice after intravenous injection of
7.4 MBq [131I]IBA (2 h, 6 h and 12 h p.i.). (b) Tumor/brain ratios at different
time-points. Data are shown as mean ± SD (n=3). (c) The tumor region was further
identified by bioluminescence imaging.
Figure 6. (a(i), b(i), c(i)) MIP SPECT/CT images of inflamed LNs models at different
stages. The inflamed popliteal LNs were indicated by red arrows. Quantitative
analysis of [131I]IBA uptake in LNs was calculated as a percentage of the injected
dose. (a(ii), b(ii), c(ii)) LNs mapping with EB dye in inflamed LNs models. Red
arrows indicate popliteal LNs with blue color, and black arrowheads indicate the
injection site.
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Figure 1. (a) Chemical synthesis route and radiolabeling process of [131I]IBA. (b)
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Figure 2. (a) Docking of IBA and IA to HSA. (b) The binding energy of probes and
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Figure 3. (a) The biodistribution results of [131I]IBA in normal mice (%ID/g,
mean±SD, n=3). (b) Blood elimination curve of [131I]IBA and [131I]IA. (c) The
percentage curve of blood elimination rate of [131I]IBA and [131I]IA.
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Figure 4. (a) MIP SPECT/CT images of 4T1 tumor mice after intravenous injection
of 7.4 MBq [131I]IBA. (b) After SPECT imaging, the tumor region was further
identified by MRI. (c) H&E staining was used to discriminate necrosis and tumor. (d)
T/NT ratios at different time-points. Data are shown as mean ± SD (n=3).
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Figure 5. (a) SPECT/CT images of brain tumor mice after intravenous injection of
7.4 MBq [131I]IBA (2 h, 6 h and 12 h p.i.). (b) Tumor/brain ratios at different
time-points. Data are shown as mean ± SD (n=3). (c) The tumor region was further
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Figure 6. (a(i), b(i), c(i)) MIP SPECT/CT images of inflamed LNs models at different
stages. The inflamed popliteal LNs were indicated by red arrows. Quantitative
analysis of [131I]IBA uptake in LNs was calculated as a percentage of the injected
dose. (a(ii), b(ii), c(ii)) LNs mapping with EB dye in inflamed LNs models. Red
arrows indicate popliteal LNs with blue color, and black arrowheads indicate the
injection site.
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