Synthesis and preliminary evaluation of a 99mTc-labeled folate-PAMAM dendrimer for FR imaging

Article abstract of DOI:10.1111/cbdd.12899

Folate receptor is an ideal target for tumor-specific diagnostic and therapeutic. The aim of this study was to synthesize ^99mTc-labeled folate-polyamidoamine dendrimer modified with 2-hydrazinonicotinic acid (^99mTc-HP₃FA)

Full text of DOI:10.1111/cbdd.12899

Received Date : 20-Aug-2016
Revised Date : 29-Sep-2016
Accepted Date : 30-Oct-2016
Article type
: Research Article
Synthesis and Preliminary Evaluation of a 99mTc-labeled Folate-PAMAM
Dendrimer for FR Ifmaging
Manli Song1, Zhide Guo1,2, Mengna Gao1, Changrong Shi1, Duo Xu1, Linyi You1, Xiaowei Wu1,
Xinhui Su3*, Rongqiang Zhuang1, Weimin Pan4*, Ting Liu1, Xianzhong Zhang1*
1 Center for Molecular Imaging and Translational Medicine, State Key Laboratory of Molecular Vaccinology and Molecular
Diagnostics, School of Public Health, Xiamen University, Xiang’an South Rd, Xiamen 361102, China
2 Department of Isotope, China Institute of Atomic Energy, P. O. Box 2108, Beijing 102413, China
3 Department of Nuclear Medicine, Zhongshan Hospital Affiliated of Xiamen University, Hubin South Road, Xiamen
361004, China
4 Department of Nuclear Medicine, the Affiliated Hospital of Hainan Medical College, 31 Longhua Rd, Haikou 570102,
China
*For correspondence contact:
Xianzhong Zhang, PhD, Professor
Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen
University
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12899
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Xiang’an South Rd., Xiang’an district, Xiamen 361102, China.
E-mail: zhangxzh@xmu.edu.cn; Phone: +86(592)2880645, Fax: +86(592)2880645
Dr. Xinhui Su
E-mail: suxinhuii@163.com
Dr. Weimin Pan
E-mail: 18907576957@163.com; Tel/Fax: +86(898)66761893
 These authors contributed equally to this work.
Manli Song, graduate student
E-mail: songmanli199142@126.com; Phone: +86(592)2880646, Fax: +86(592)2880646
Zhide Guo, PhD candidate
E-mail: guozhide0518@gmail.com; Phone: +86(592)2880646, Fax: +86(592)2880646
Running Title: PAMAM dendrimer based 99mTc-radiofolate
Keywords: Technetium-99m, folate receptor, PAMAM dendrimer, SPECT imaging, KB tumor
bearing mice
Abstract
Folate receptor is an ideal target for tumor-specific diagnostic and therapeutic. The aim of this
study is to synthesize 99mTc-labeled folate-PAMAM dendrimer modified with 2-hydrazinonicotinic
acid (99mTc-HP3FA) for FR imaging. The 99mTc-HP3FA conjugate was prepared using
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N-tris-(hydroxymethyl)-methylglycine and trisodium triphenylphosphine-3,3',3"-trisulfonate as
co-ligands. Physicochemical properties, in vitro cell uptake study and in vivo micro-SPECT/CT
imaging were performed. The radiolabeled 99mTc-HP3FA conjugate was prepared with high
radiolabeling yield, good stability and water-solubility (logP=-1.70±0.21). In cell uptake study, the
radiolabeled conjugate showed high uptakes in the FR-abundant KB cells, and could be blocked
significantly by excess folic acid. The 7721 cells which served as control group substantially had no
uptakes. The results of micro-SPECT/CT imaging exhibited that high accumulation of activity was
found in FR overexpressed KB tumor, and the tumor to muscle ratio was approximately 25.78. While,
using free FA as inhibitor, the uptakes of 99mTc-HP3FA in KB tumor and kidney were obviously
inhibited. In summary, a new radio-compound was synthesized successfully with specifically FR
targeting ability. The feasibility of 99mTc-HP3FA for early diagnosis of FR-positive tumors with
noninvasive single photon emission computed tomography imaging was demonstrated and the
possibility of imaging-guided drug delivery based on multifunctional PAMAM will be studied in the
future.
Introduction
Nuclear medicine relies on collaboration of radiopharmaceuticals and nuclear medicine
technologies. Substantial effort has been focused on the development of molecular probes to achieve
tumor-specific therapeutic and diagnostic agents (1). Antibody, sugar, folic acid (FA), transferrin,
epidermal growth factor and Arg-Gly-Asp peptide are tumor targeting ligands used to give an active
targeting-ability to drug carriers (2,3). Of these ligands, FA has attracted wide attention because of its
high stability, low-cost, nonimmunogenic character, low molecular weight, and simple chemical
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modification. Moreover, a number of tumor cell exhibit high levels of the folate receptors (FRs), and
its expression is restricted in FR-negative tissues (4,5). These preferable properties of FA recommend
it as a good candidate of targeting groups. Among various folate-probes, one typical case is
micromolecule with single target (6-8). The other part is macromolecule-based multimer (9-11).
An important aspect for successful development of the multimeric folate derivatives is to get a
suitable spacer and bifunctional chelator (BFC). Polyamidoamine (PAMAM) dendrimer offer such a
carrier system, which has a unique branching structure like a tree (12,13). Based on PAMAM
dendrimer, an approach to increase the targeting and labeling efficiency has been focused on the
construction with more targeting and chelating agents. PAMAM dendrimer has been used for a
variety of biomedical applications due to its favourable biocompatibility, controllable properties and
carrying capacity (14-17). There are a large number of surface functional groups on the dendrimer’s
outer shell, which can be modified with a variety of interesting guest molecules.
Positron emission tomography (PET) and single photon emission computed tomography (SPECT)
are sensitive imaging techniques for FR-targeted imaging. They have high sensitivity and the
possibility to perform functional imaging (18, 19). Several folate conjugates labeled with 67/68Ga,
111In, 64Cu, and 99mTc are under pre-clinical and clinical stages (20-22). To perform radiolabeling, the
labeling efficiency of dendrimer was precisely controlled by choosing different BFC agents on the
surface of PAMAM dendrimer. In most cases, a hydrazino nicotinic chelator (HYNIC) was attached
to the FA to bind 99mTc using coligands N-tris-(hydroxymethyl)-methylglycine (tricine) and trisodium
triphenylphosphine-3,3',3"-trisulfonate (TPPTS) (23). HYNIC is a very efficient BFC for 99mTc
labeling at a relatively low concentration. 99mTc (t1/2 =6 h) is a suitable radionuclide for SPECT
imaging due to its ideal physical decay characteristics, low cost and easy availability from a
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99Mo-99mTc generator system (24-26). Efficient labeling process and high specific activity come from
the perfect match of HYNIC and 99mTc.
Herein, we described the synthesis of 99mTc-labeled folate-PAMAM dendrimer for FR targeting
and evaluated the effects of the probe properties on diagnostic efficacy. Further, the possibility of
imaging-guided drug delivery based on multifunctional PAMAM dendrimer will be studied in the
future.
Materials and methods
Chemicals and equipments
Generation-3 (G3) PAMAM dendrimer was purchased from Weihai CY Dendrimer Technology
Co., Ltd. (China). G3 PAMAM dendrimer has molecular weight of 6909 Da and 32 primary surface
amine groups. N-Hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), tricine, TPPTS and
other reagents were purchased from J&K Chemical Ltd. All other reagents and solvents were
purchased from commercial suppliers and used without further purification. The eluent Na99mTcO4
were obtained from Zhongshan Hospital Affiliated of Xiamen University. Instant thin-layer
chromatography silica gel strips (ITLC-SG) were purchased from Pall Life Sciences. The
radioactivity counts were measured with CRC-25R Dose Calibrators (CAPINTEC. INC, USA). The
ITLC strips were detected with Mini-Scan radio-TLC Scanner (BioScan, USA). Imaging study was
performed by a nanoScan-SPECT/CT preclinical imager (Mediso, HUNGARY). The UV
spectroscopy was measured by using microplate spectrophotometer (Multiskan GO, Thermo Fisher,
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USA). 1HNMR spectra were measured on a Bruker (600 MHz) spectrometer. Chemical shifts are
reported in δ (ppm) values.
Preparation of HP3FA
Synthesis procedures to obtain HP3FA can be classified into three main steps. FA-NHS and
HYNIC-NHS were synthesized according to the procedure similar to that reported by Guo et al. (27,
28).
HYNIC-NHS was prepared as follows: 22 mL 85% hydrazine hydrate containing 1.5 g
6-chloronicotinic acid was heated at 100 °C for 6 h. Then the reaction mixture was concentrated to
afford a white solid. The solid was dissolved in 20 mL water and the pH was adjusted to 5.5 by
addition of HCl until a yellow precipitate was formed. The precipitate was collected by filtration,
washed with 95% ethanol and ether and dried under high vacuum to afford 1.17 g of
6-hydrazinopyridine-3-carboxylic acid. Then 332 mg 6-hydrazinopyridine-3-carboxylic acid was
dissolved in 10 mL dimethyl sulphoxide (DMSO) containing 350 mg 4-dimethylaminobenzaldehyde.
The reaction mixture was stirred for 8 h at room temperature (RT). Then 374 mg NHS and 670 mg
DCC was added into the flask and the stirring was continued for 18 h. After reaction, the mixture was
filtered to give a reddish-brown filtrate containing HYNIC-NHS.
FA-NHS was prepared as follows: In brief, 455 mg DCC and 254 mg NHS were added into 10
mL DMSO containing 650 mg FA. The mixture was stirred at RT for 24 h in the dark. The byproduct
was removed by filtration to give a reddish-brown filtrate containing FA-NHS.
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HP3FA was prepared as follows: Filtrate containing HYNIC-NHS was added slowly into 10 mL
DMSO containing 1 g PAMAM (G3). The mixture was stirred at RT for 3 d. After that, filtrate
containing FA-NHS was added slowly into the mixture and the reaction was continued for another 3 d
at RT in the dark. To remove unreacted FA-NHS and HYNIC-NHS, the reaction solution was
dialyzed (cellulose membrane with 3.5 k MWCO) against DMSO for 3 d. Then the canary yellow
solution in dialysis bag was dropped into ether. The precipitate was collected by filtration, then dried
under vacuum to afford the crude product.
Radiolabeling of the compound with 99mTc
The formulation and route for radiolabeling was shown in Figure 2A. In brief, 500 μL tricine
solution (80 mg/mL in phosphate-buffered saline (PBS), pH 8), 200 μL HP3FA solution (1 mg/mL in
saline, 10 μL 1 M NaOH ), 200 μL TPPTS (5 mg/mL in PBS, pH 8), 20 μL SnCl2 solution (2 mg/mL
−
in 1 M HCl) and 370 MBq 99mTcO4 were added to a 10 mL vial. The vial was oscillated at 100 °C for
15 min in a lead-shielded water bath and then cooled to RT. ITLC on silica gel was carried out by
running a sample on a 10 cm strip using ACD (acid-citrate-dextrose buffer, 0.068 mol/L citrate, 0.074
mol/L glucose, pH 5.0) as mobile phase. The radiochemical purity and the labeling efficiency were
analysed using MiniScan radio-TLC. For in vitro stability test, 99mTc-HP3FA was incubated in saline
and serum at 37 °C for 8 h. The stability was monitored using ITLC/ACD system.
Octanol/water partition coefficient
The partition coefficient of 99mTc-HP3FA was measured by using the method reported by Guo et
al. (27, 28). In brief, 100 μL radiotracer solution was mixed with 2.9 mL of PBS (0.05 mol/L,
pH=7.4) and 3 mL of 1-octanol in a micro centrifuge tube. The tube was shocked for 5 min and
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centrifuged at 8000 rpm for 5 min. The counts in 100 μL aliquots of both organic and inorganic layers
were counted using -counter. Experiments were performed with triplicate samples. The partition
coefficient value (expressed as log P) was calculated using the following equation: P = (activity in
octanol phase-background activity)/(activity in aqueous phase-background activity).
Cell Uptake Study
The cell uptake study described below is based on a published procedure (27, 28). 24 h before
the study, the KB cells (a human epithelial carcinoma cell line) were seeded in 24-well plates (5×105
cells/well, using FA-free RPMI medium) and incubated at 37 °C. In the study, 99mTc-HP3FA (11.1
kBq per 100 μL FFRPMI medium) was added to each well and incubated at 37 °C for increasing
periods of time. After incubation, the medium was removed and the cells were gently washed three
times with cold PBS (pH 7.4, including 0.2% bovine serum albumin) to determine total radiofolate
uptake. Cellular internalization of the 99mTc-HP3FA was assessed by additionally washing with 500 μL
stripping buffer (0.82 g NaAc, 2.25 g NaCl, 0.5 g bovine serum albumin, 250 mL H2O, adjust the pH
to 4 with acetic acid) two times. The stripping buffer was then transferred to tubes. Finally, the cells
were lysed by treatment with 1 M NaOH for 5-10 min and transferred to tubes. The blocking studies
were performed by addition of free FA solution (10 μL, 1 mg/mL). Then, 100 μL radiotracer was
added, and the well plates were incubated at 37 °C for 1 h, 2 h and 4 h, respectively. After incubation,
the medium were removed, and the cells were rinsed with cold PBS. Finally, the cells were lysed by
treatment with 1 M NaOH for 5-10 min and transferred to tubes. Each sample was counted for
radioactivity using a -counter. The cell uptake was normalized in terms of the total added
radioactivity (% of total activity).
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SPECT Imaging
The experimental procedures and the animal use and care protocols were approved by the
Institutional Animal Care and Use Committee of Xiamen University. All experimental protocols were
carried out in accordance with the relevant guidelines.
The possibility of FR-targeted SPECT imaging was investigated after radiotracer (18.5 MBq/100
μL) was injected intravenously through tail vein of each mouse. The SPECT imaging was performed
with a nanoScan-SPECT/CT preclinical scanner equipped with low energy high-resolution
collimators. Anesthesia was induced with isoflurane and spontaneous breathing was maintained
during the scan procedure. Computed tomography (CT) data were acquired using an X-ray voltage
biased to 50 kVp with a 670 μA anode current, and the projections were 720°. The acquiring
parameters of SPECT imaging were as follows: energy peak of 140.5 keV for 99mTc, window width of
20%, matrix of 256 × 256, medium zoom, and frame: 45 s. The uptakes in different tissues were
calculated by drawing regions of interests (ROIs) on the SPECT/CT images.
Results
Chemistry
The synthesis routes and chemical structure of HP3FA are shown in Scheme S1 and Figure S1A
(in supporting information), respectively. To attach HYNIC and FA to PAMAM dendrimer (G3),
carboxyl groups of the HYNIC and FA were activated by DCC and NHS. The reaction occurs mainly
at the γ-carboxylic group due to it has higher reactivity than that of α-carboxylic group of folate (4,
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27, 28). The structure and size of end-product HP3FA were characterized by UV-spectroscopy (Figure
1A), 1H NMR (Figure 1B) and dynamic laser scattering (DLS, Figure 1C). The FA has an absorption
peak at the wavelength 296 nm, and the absorption at 368 nm is relatively weak. The absorption peak
of HYNIC is at 380 nm. We measured the UV absorbance of the tracer HP3FA. The HP3FA has two
characteristic absorptions at 286 nm and 365 nm. The result suggested that we had successfully
synthesized the HP3FA. Furthermore, the absorbance values of FA and HYNIC in UV
spectrophotometer were used to quantify the number of FA and HYNIC per PAMAM. Average of 6.5
FA and 7.3 HYNIC moieties per PAMAM were calculated by plotting the standard curve of FA and
HYNIC. The structure of HP3FA was confirmed by 1H NMR in DMSO to verify functional groups.
The characterization of FA, HYNIC and dendrimer by 1H NMR spectrum also indicated the
successful synthesis of HP3FA. In DMSO, protons from FA found at 4.0-4.5 ppm and protons on the
HYNIC at 7.0-7.5 ppm were found in the 1H NMR spectrum of final product. By comparing the
integration of chemical shifts of the compounds, we speculated that the HYNIC and FA molecules
were conjugated to the dendrimer platform. The conjugation of FA and HYNIC groups on PAMAM
could also be confirmed by 1H-NMR of HP3FA (Figure S1B). FA/HYNIC ratio (about 0.91) could be
obtained according to the integral ratios of FA and HYNIC protons (4.0-4.5 ppm for FA; 2.8-3.0 and
7.8-8.0 ppm for HYNIC) to the multiple methylene protons of PAMAM (-CH2CO-, at 2.5-2.8 ppm).
Approximate 7.3 FA and 8.0 HYNIC molecules were calculated on each PAMAM dendrimer. This
NMR based results are slightly larger than that of UV spectroscopy. Furthermore, the average
molecular weight of HP3FA could be calculated as about 12125 Da. According to the DLS (dynamic
laser scattering) results, the size of HP3FA was about 1.27 nm.
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Radiolabeling
99mTc-HP3FA could be prepared via HYNIC with high labeling efficiency and good stability.
Both of labeling efficiency and radiochemical purity of 99mTc-HP3FA are greater than 95% by ITLC
method (Figure 2B). 99mTc-HP3FA remained stable over 8 h when incubated both in serum and saline
(Figure S2 in supporting information). The logP of 99mTc-HP3FA is -1.70±0.21 indicating the
hydrophilicity of the labeled complex. The specific activity of 99mTc-HP3FA was calculated as ~ 22
GBq/mol at the end of synthesis.
Cell uptake study
In cell uptake study, as further proof of specific binding of the 99mTc-HP3FA to FRs, FR-negative
7721 cell line and FR-positive KB cell line were used and incubated from 30 min to 4 h. The
99mTc-HP3FA conjugate shows high uptakes in the FR-abundant KB cells (Figure 3A). While the 7721
cells which served as control group substantially had no uptakes (Figure 3B). At 1 h after incubation,
maximum total binding ratio (27%) of 99mTc-HP3FA radioactivity was observed on KB cell line. As
the surface binding curve showed, 99mTc-HP3FA can be specifically binding to KB cells via the FRs
on the surface of cells. Moreover, the competitive blocked effect of free FA was examined on
FR-positive KB cells, which clearly demonstrated that 99mTc-HP3FA can target cancer cells through
FR-mediated effect.
SPECT imaging
Firstly, SPECT imaging of healthy BALB/c mice with 99mTc-HP3FA were performed to visualize
the distribution of the radiotracer in normal mice (shown in Figure S3). The radiotracer was observed
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mostly in FR-abundant kidney. In the other organs, such as liver and lung, the uptake of the conjugate
kept at low level. This is a common feature of most folate conjugates.
The distribution of the radiotracer 99mTc-HP3FA was further confirmed by using KB-tumor
bearing nude mice in SPECT imaging study. The sagittal, coronal, and transaxial sections of
SPECT/CT images were shown in Figure 4, the accumulation of 99mTc-HP3FA conjugates is visible in
kidneys and the KB tumor xenografts localized on both shoulders. The tumor focus was also
confirmed through CT scan. Mutually matches relations existed between the SPECT and CT images.
More clearly three-dimensional SPECT/CT images were also shown in Figure 4. The results of
micro-SPECT/CT imaging showed that 99mTc-HP3FA conjugate exhibited high uptakes in KB tumor
and kidneys. And the tumor-to-muscle and tumor-to-kidney ratios were approximately 25.78 and 0.78
(Table S1), respectively. For the blocking group, we used the FA as inhibitor. The sagittal, coronal,
and transaxial sections of SPECT/CT imaging showed that the KB tumor and kidney uptakes of the
99mTc-HP3FA conjugates were obviously inhibited. The ratios of tumor-to-muscle and
tumor-to-kidney were decreased obviously to 1.31 and 0.25, respectively. FR-mediated uptake and
renal excretion pathway lead to high radioactivity accumulation in the kidney. Injection of excess FA
resulted in an almost complete blockade of the FR-specific tumor and kidney uptake, while
non-FR-mediated renal excretion can’t be reduced by injecting excess FA. For this reason, in the
presence of FA, the tumor-to-kidney ratio was decreased from 0.78 to 0.25 (Table S1).
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Discussion
PAMAM dendrimer has drawn great attention and often used as platform to deliver therapeutics
and imaging agents (3, 29, 30). As large number of primary amino groups of on the surface of
dendrimer, the end groups of PAMAM have high chemical activity and can be modified with many
functional molecules, such as therapeutic drugs, targeting groups and imaging agents (31-33). With
the development of PAMAM, the conception of the "extensible and modular platform" has become
more clearly and come into our research field. To investigate the concept and achieve ideal targeting
and imaging results, we chose FA and HYNIC as the candidates in our study.
FR targeting plays a very important role in tumor detection with the assistance of nuclear
medicine imaging. As reported in previous studies, to meet the imaging need, some radiolabeled FA
derivatives based on dendrimer were synthesized (17, 29, 34, 35). As we know, radiolabeled low
molecular weight folate conjugates offer advantages of high tumor to background ratio and faster
systemic blood clearance kinetics. Unfortunately, it is a difficult challenge for macromolecule-based
folate conjugates. Some studies have demonstrated that FA conjugated to PAMAM dendrimer could
target the FR. While, some radiolabeled PAMAM conjugates have the disadvantages of multi-step
synthesis, low radiochemical yields and time-consuming purification process, which are not suitable
for large-scale productions as needed for clinical application. Moreover, due to the slow clearance of
radioactivity from liver, spleen, blood or other non-target tissues, undesired tumor-to-background
contrast was achieved, which affected the tumor visualization (34). In our study, the HYNIC could
likely improve the pharmacokinetic profiles by increasing labeling efficiency and chelating capability
at low HYNIC-conjugate concentrations. Fortunately, the 99mTc-HP3FA has excellent tumor targeting
ability with minimum uptake by the liver, lung and other organs, implying that it has great potential
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not only for targeting the primary cancer site but also metastatic sites. As the in vitro experiment and
imaging study have already reflected the good properties of 99mTc-HP3FA, biodistribution study was
not performed in our study. A direct comparison between 99mTc-HP3FA and other radiolabeled folate
conjugates using percentage of injected dose per gram (%ID/g) was not offered.
Different generation dendrimers show different in vivo characteristics. Low generation
dendrimer can find tumor quickly and be cleared from the bloodstream rapidly. High generation
PAMAM are more likely to accumulate in liver while low generation PAMAM tend to localize in
kidney. There have more active-sites on the surface of higher generation dendrimer which could be
modified with functional group (3, 30). In this study, PAMAM (G3) can provide sufficient primary
amino groups to attach HYNIC and FA, but also has excellent pharmacokinetic profiles. It is
particularly gratifying that based on the unique structural characteristics of PAMAM dendrimer, by
replacing HYNIC or FA with other corresponding functional groups, this versatile strategy can be
readily extended to other sensitive multimodal imaging and/or enhanced therapy of different cancers.
In other words, it is feasible to synthesize mono-, di-, and even multi-functionalized conjugates using
PAMAM dendrimer as platform with facile modification methods. By different components'
assembly, targeting groups, therapeutic elements, and imaging reagents were mixed with PAMAM
dendrimer platform simultaneously or sequentially to give mono-, di-, and multi-functional conjugates
for different uses (33, 36, 37). These achievements have shown brilliant future to the final
implementation of "extensible and modular platform". Further studies are under way to implement
these ideas by using more sophisticated functional groups basing on more efficient synthetic scheme.
We anticipate these attempts will facilitate further advancement of multifunctional PAMAM.
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Conclusion
A new radio-compound of 99mTc-HP3FA was synthesized successfully with specifically FR
targeting ability. It shows promising characteristics in comparable with many FA-modified PAMAM
dendrimers. This new delivery system overcomes the obstacles of the non-specific uptake in liver and
lung. The feasibility of 99mTc-HP3FA for early diagnosis of FR-positive tumors with noninvasive
SPECT imaging was demonstrated in this study. The possibility of imaging-guided drug delivery or
multimodal imaging system based on this versatile strategy will be studied in the follow-up
experiment.
Acknowledgments
This study was financially supported by the National Key Basic Research Program of China
(2014CB744503), National Natural Science Foundation of China (21271030, 81471707) and partially
by the Fundamental Research Funds for the Central Universities of China (20720150063,
2013SH009).
Conflicts of Interest: The authors declare no conflict of interest.
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The legends of tables and figures:
Figure 1. (A) The UV spectroscopy of HYNIC, FA and HP3FA. (B) 1H-NMR spectrums of FA,
HYNIC, PAMAM and HP3FA. (C) The average size for HP3FA measured by DLS was 1.27 nm.
Figure 2. (A) Radiolabeling procedure of 99mTc-HP3FA. (B) The radiolabeling efficiency and
radiochemical purity of 99mTc-HP3FA were tested by ITLC-SG/ACD. 99mTc-HP3FA stayed at the point
-
of origin (Rf: 0-0.1), whereas hydrolyzed 99mTc and 99mTcO4 moved to the front (Rf: 0.8-1.0).
Figure 3. The cell binding properties of 99mTc-HP3FA in the KB cells (A) and the 7721 cells (B). The
cell binding (cell total binding, internalization and blocked by excess FA, P < 0.05) was expressed as
percentage of total added radioactivity.
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Figure 4. (A and B) The SPECT/CT imaging of the KB-tumor bearing nude mice at 2 h. (C and D)
Blocking imaging study performed by received 100 μg FA 10 min prior to the injection of
99mTc-HP3FA.
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