Preparation and Evaluation of Novel Folate Isonitrile 99mTc Complexes as Potential Tumor Imaging Agents to Target Folate Receptors

In order to seek novel technetium-99m folate receptor-targeting agents, two folate derivatives (CN5FA and CNPFA) were synthesized and radiolabeled to obtain [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA complexes, which exhibited high radiochemical purity (>95%) without purification, hydrophilicity, and good stability in vitro. The KB cell competitive binding experiments indicated that [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA had specificity to folate receptor. Biodistribution studies in KB tumor-bearing mice illustrated that [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA had specific tumor uptake. Compared with [99mTc]Tc-CN5FA, the tumor/muscle ratios of [99mTc]Tc-CNPFA were higher, resulting in a better SPECT/CT imaging background. According to the results, the two 99mTc complexes have potential as tumor imaging agents to target folate receptors.


Introduction
Tumors are pathological changes caused by the uncontrolled and continuous proliferation of cells under the influence of various factors [1]. Folate receptor (FR) is a 38 kDa glycoprotein linked to the cell membrane by glycan phosphatidylinositol. It is overexpressed on the surface of the cell membrane in the majority of human tumors. FR mainly consists of α, β, and γ subtypes [2]; in particular, folate α-receptors are highly conserved in healthy cells but highly expressed in many epithelial-derived malignancies, such as ovarian, breast, endometrial, lung, and nasopharyngeal cancer cells. Thus, the α-subtype of the folate receptor has become a hotspot of radiation-targeted drug research for the diagnosis and therapy of high-expression tumors [3,4].
In recent years, nuclear medicine imaging has shown the advantages of being sensitive, specific, noninvasive, and able to detect changes in molecular biological behavior and pathophysiology in vivo. It has become one of the most important diagnostic methods for cancer. With the widespread use of single-photon emission computed tomography (SPECT) and positron emission tomography (PET), radionuclide tumor imaging has become one of the advantages of nuclear medicine [5][6][7]. 99m Tc is the most widely used SPECT radionuclide with good nuclide properties, including a half-life of 6.02 h, emission of 140 keV γ-rays, and the advantages of coordinated chemical diversity. Therefore, 99m Tc complexes are often used in the clinical diagnosis of some diseases, accounting for more than 85% of nuclear medicine SPECT images [8,9]. Recently, a number of 99m Tc-labeled folate-based conjugates have been developed as potential folate receptor imaging agents [10][11][12][13][14]. Although these complexes have shown high tumor uptake, most of the reported radiopharmaceuticals targeting folate receptor require purification by high-performance liquid chromatography (HPLC), thus limiting their application in clinical practice. Therefore, developing novel 99m Tc-labeled folate-based conjugates for folate receptor imaging with no further purification is worthwhile.
For the design of bifunctional radiopharmaceuticals, there are four important components: targeting group, linker, bifunctional chelator (BFC), and radionuclide [15]. Isocyanate (-NC) is a strong coordination group that can form a stable 99m Tc complex with six coordination sites. Because the 99m Tc-labeled isocyanate-based complex had six isocyanate ligands which contain targeting group, its targeting ability to the target could be enhanced [16][17][18]. Recently, preclinical studies of 99m Tc-CN5DG and 99m Tc-CN7DG demonstrated that they could be promising candidates for tumor imaging [19,20]. The introduction of an ethylene glycol (PEG) chain can increase the hydrophilicity of the drug, thus improving its metabolic pathway in vivo [21][22][23].
Considering the evidence above, we aimed to develop novel 99m Tc-labeled folatebased conjugates as tumor imaging agents. We synthesized two folate-based conjugates with an isonitrile group (CN5FA and CNPFA) and labeled them with 99m Tc to produce [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA. Their potential for tumor imaging was evaluated for the first time.

Synthesis
The reaction route is shown in Scheme 1. require purification by high-performance liquid chromatography (HPLC), thus limiting their application in clinical practice. Therefore, developing novel 99m Tc-labeled folatebased conjugates for folate receptor imaging with no further purification is worthwhile. For the design of bifunctional radiopharmaceuticals, there are four important components: targeting group, linker, bifunctional chelator (BFC), and radionuclide [15]. Isocyanate (-NC) is a strong coordination group that can form a sTable 9 9m Tc complex with six coordination sites. Because the 99m Tc-labeled isocyanate-based complex had six isocyanate ligands which contain targeting group, its targeting ability to the target could be enhanced [16][17][18]. Recently, preclinical studies of 99m Tc-CN5DG and 99m Tc-CN7DG demonstrated that they could be promising candidates for tumor imaging [19,20]. The introduction of an ethylene glycol (PEG) chain can increase the hydrophilicity of the drug, thus improving its metabolic pathway in vivo [21][22][23].
Considering the evidence above, we aimed to develop novel 99m Tc-labeled folatebased conjugates as tumor imaging agents. We synthesized two folate-based conjugates with an isonitrile group (CN5FA and CNPFA) and labeled them with 99m Tc to produce [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA. Their potential for tumor imaging was evaluated for the first time.

Synthesis
The reaction route is shown in Scheme 1.

Radiolabeling
[ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA (the proposed structures of the radiolabeled tracers are shown in Scheme 2) were easily prepared in high radiochemical purity (RCP > 95%) without purification using a developed kit formulation in 20 min at 100 °C. The resulting solutions were analyzed by instant thin-layer chromatography silica gel strips (ITLC/SG) and high-performance liquid chromatography (HPLC

Stability Study
As shown in Figure 1, after incubation in the radiolabeling solution at room temperature for 4 h or in serum at 37 °C for 4 h, the RCPs of the complexes remained at more than 95%. Nearly no decomposition of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA was found, indicating their good stability in vitro.

Partition Coefficient
The partition coefficient values, log P (P = the radioactivity of the organic phase/the radioactivity of the water phase) of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA were −1.92 ± 0.15 and −2.28 ± 0.02, respectively. This result suggested that both complexes were

Stability Study
As shown in Figure 1, after incubation in the radiolabeling solution at room temperature for 4 h or in serum at 37 °C for 4 h, the RCPs of the complexes remained at more than 95%. Nearly no decomposition of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA was found, indicating their good stability in vitro.

Partition Coefficient
The partition coefficient values, log P (P = the radioactivity of the organic phase/the radioactivity of the water phase) of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA were −1.92 ± 0.15 and −2.28 ± 0.02, respectively. This result suggested that both complexes were

Stability Study
As shown in Figure 1, after incubation in the radiolabeling solution at room temperature for 4 h or in serum at 37 • C for 4 h, the RCPs of the complexes remained at more than 95%. Nearly no decomposition of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA was found, indicating their good stability in vitro.

Partition Coefficient
The partition coefficient values, log P (P = the radioactivity of the organic phase/the radioactivity of the water phase) of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA were −1.92 ± 0.15 and −2.28 ± 0.02, respectively. This result suggested that both complexes were hydrophilic  Figure 2).

In Vitro Binding with KB Cells
To confirm the FR binding property of acquired 99m Tc-labeled complexes, in vitro competitive binding experiments were designed with KB cells. In vitro competitive binding curves of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA are shown in Figure 3. These findings imply that the IC50 values of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA were 37.92 ± 0.41 nM and 3.80 ± 0.07 nM with KB cells, respectively. This result suggests that both complexes specifically bound to the folate receptor.

Biodistribution
The biological distribution results in KB tumor-bearing mice are shown in Table 1 The results showed that the tumor uptakes of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA were 2.42 ± 0.44 %ID/g and 2.53 ± 0.12 %ID/g at 2 h post injection (p.i.), respectively, which were significantly reduced to 1.53 ± 0.10 %ID/g and 1.62 ± 0.08 %ID/g at 2 h p.i. by preadministration of free folate acid (FA) (about 4 × 10 4 µg/kg). This indicates that they were specific to the folate receptor. As the results demonstrate, the tumor/muscle ratios of [ 99m Tc]Tc-CNPFA were 2.82 at 0.5 h p.i. and 3.01 at 2 h p.i., which were better than 1.76 at

In Vitro Binding with KB Cells
To confirm the FR binding property of acquired 99m Tc-labeled complexes, in vitro competitive binding experiments were designed with KB cells. In vitro competitive binding curves of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA are shown in Figure 3. These findings imply that the IC 50 values of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA were 37.92 ± 0.41 nM and 3.80 ± 0.07 nM with KB cells, respectively. This result suggests that both complexes specifically bound to the folate receptor.

In Vitro Binding with KB Cells
To confirm the FR binding property of acquired 99m Tc-labeled complexes, in vitro competitive binding experiments were designed with KB cells. In vitro competitive binding curves of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA are shown in Figure 3. These findings imply that the IC50 values of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA were 37.92 ± 0.41 nM and 3.80 ± 0.07 nM with KB cells, respectively. This result suggests that both complexes specifically bound to the folate receptor.

Biodistribution
The biological distribution results in KB tumor-bearing mice are shown in Table 1. The results showed that the tumor uptakes of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA were 2.42 ± 0.44 %ID/g and 2.53 ± 0.12 %ID/g at 2 h post injection (p.i.), respectively, which were significantly reduced to 1.53 ± 0.10 %ID/g and 1.62 ± 0.08 %ID/g at 2 h p.i. by preadministration of free folate acid (FA) (about 4 × 10 4 µg/kg). This indicates that they were specific to the folate receptor. As the results demonstrate, the tumor/muscle ratios of [ 99m Tc]Tc-CNPFA were 2.82 at 0.5 h p.i. and 3.01 at 2 h p.i., which were better than 1.76 at 0.5 h p.i. and 1.91 at 2 h p.i. for [ 99m Tc]Tc-CN5FA (Figure 4).

Biodistribution
The biological distribution results in KB tumor-bearing mice are shown in Table 1. The results showed that the tumor uptakes of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA were 2.42 ± 0.44 %ID/g and 2.53 ± 0.12 %ID/g at 2 h post injection (p.i.), respectively, which were significantly reduced to 1.53 ± 0.10 %ID/g and 1.62 ± 0.08 %ID/g at 2 h p.i. by pre-administration of free folate acid (FA) (about 4 × 10 4 µg/kg). This indicates that they were specific to the folate receptor. As the results demonstrate, the tumor/muscle ratios of  (Figure 4).  For other organs, the radioactive uptake in the kidney is relatively high due to the high FR expression in kidney proximal tubule cells. In addition, the radioactive concentrations in the liver and intestines are also appreciable.

SPECT/CT Imaging Study
SPECT/CT imaging studies of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA in KB tumorbearing mice are shown in Figure 5. The SPECT/CT imaging results were consistent with the biodistribution results. The tumor lesions were clearly visualized by both probes at 2 h post injection. Kidney and tumor uptake significantly decreased in the blocking experiments by pre-administration of excess FA (about 4 × 10 4 µg/kg). Interestingly, the imaging effect of [ 99m Tc]Tc-CNPFA was superior to that of [ 99m Tc]Tc-CN5FA because the former had a higher T/NT ratio than the latter.

SPECT/CT Imaging Study
SPECT/CT imaging studies of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA in KB tumorbearing mice are shown in Figure 5. The SPECT/CT imaging results were consistent with the biodistribution results. The tumor lesions were clearly visualized by both probes at 2 h post injection. Kidney and tumor uptake significantly decreased in the blocking experi ments by pre-administration of excess FA (about 4 × 10 4 µg/kg). Interestingly, the imaging effect of [ 99m Tc]Tc-CNPFA was superior to that of [ 99m Tc]Tc-CN5FA because the former had a higher T/NT ratio than the latter.

Discussion
In recent years, receptor-mediated targeted molecular probes have become the focus of research and development of radiopharmaceuticals. Compared with other targeted transport systems, the folate molecule has the advantages of strong affinity, small molec ular weight, low cost, easy structural modification, good chemical and biological stability and no immunogenicity. Folate receptor drugs have become a hot topic of targeted drug research. Folate and its analogues were coupled with a radionuclide to form tracers, which can specifically bind to FR and selectively concentrate in tissues rich in FR expression Because of the significant difference in the distribution of FR in tumor and nontumor tissues, FR-mediated tracers can obtain high-contrast images of tumor and normal tissues to diagnose, locate, and evaluate the effect of chemotherapy [24,25]. At present, 66/67/68 Ga, 111 In 99m Tc, and 18 F have been reported as radionuclides for preparing targeted folate receptor radiopharmaceuticals [26][27][28][29].

Discussion
In recent years, receptor-mediated targeted molecular probes have become the focus of research and development of radiopharmaceuticals. Compared with other targeted transport systems, the folate molecule has the advantages of strong affinity, small molecular weight, low cost, easy structural modification, good chemical and biological stability, and no immunogenicity. Folate receptor drugs have become a hot topic of targeted drug research. Folate and its analogues were coupled with a radionuclide to form tracers, which can specifically bind to FR and selectively concentrate in tissues rich in FR expression. Because of the significant difference in the distribution of FR in tumor and nontumor tissues, FR-mediated tracers can obtain high-contrast images of tumor and normal tissues to diagnose, locate, and evaluate the effect of chemotherapy [24,25]. At present, 66/67/68 Ga, 111 In, 99m Tc, and 18 F have been reported as radionuclides for preparing targeted folate receptor radiopharmaceuticals [26][27][28][29].
At present, most studies on folate-mediated radiopharmaceuticals are limited to the preclinical studies, and most folate imaging agents need to be separated and purified by HPLC. To promote clinical application, finding a better labeling method and better linker, simplifying the preparation process, and improving drug targeting in vivo are the problems to be solved in the future.
In this work, we used folate as a target molecule, isonitrile as a BFC, methylene chain and PEG chain as linkers, and 99m Tc as a radionuclide to discover novel tracers for folate receptor imaging. Two 99m Tc-labeled complexes ([ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA) were successfully prepared, and the RCP of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA was over 95% without purification.
In the biodistribution experiment stage, because of the poor solubility of ligand CN5FA, the biodistribution results showed obvious concentrations in the liver (52.35 ± 1.19 %ID/g at 2 h p.i.) and other abdominal organs when the labeled condition was under weak acid. Therefore, to reduce the concentration of radioactivity in the liver and other abdominal organs, the pH of the labeling solution was changed to weakly alkaline in the follow-up experiments so that the ligand CN5FA could be better dissolved.
As shown in Table 1, the tumor uptake of [ 99m Tc]Tc-CN5FA was 3.03 ± 0.10 %ID/g and that of [ 99m Tc]Tc-CNPFA was 4.18 ± 0.36 %ID/g at 0.5 h p.i., suggesting that [ 99m Tc]Tc-CNPFA had a higher tumor uptake. However, there was no significant difference in tumor uptake between the two complexes at 2 h p.i. The muscle uptake of [ 99m Tc]Tc-CNPFA (0.84 ± 0.04 %ID/g) was lower than that of [ 99m Tc]Tc-CN5FA (1.27 ± 0.03 %ID/g), thus leading to a higher tumor/muscle ratio of the former than that of the latter. In the inhibition experiments, with early injection of excessive FA, the tumor and kidney uptake of both complexes was shown to have an inhibitory effect of more than one-third at 2 h p.i.
In 2019, Lodhi et al. developed a folate receptor tracer [ 99m Tc]Tc-10 using isonitrile (-NC) as the BFC [14]. In contrast to our work, the results showed that [ 99m Tc]Tc-10 had higher tumor uptake (4.02 ± 0.80 %ID/g) and lower liver concentration (5.14 ± 1.79 %ID/g) at 2 h post injection. However, the preparation of [ 99m Tc]Tc-10 was a two-step process in which the [ 99m Tc(H 2 O) 3 (CO) 3 ] + intermediate needs to be prepared first before the product required to be purified by HPLC. Compared with the method of direct labeling of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA, the preparation process of [ 99m Tc]Tc-10 was tedious, time-consuming, and not suitable for clinical application.
As shown in Figure 5, SPECT/CT results showed that both complexes had significant radioactivity concentrations in tumor and kidney sites, and radioactivity concentrations in the liver and intestinal tract were also observed, suggesting that, in addition to urine metabolism, the liver and intestine may also be involved in metabolism to a certain extent. At the same time, inhibition assays demonstrated that these two probes could bind specifically to KB tumor with high FR expression.

Materials and Methods
All chemical reagents and solvents were purchased from commercial sources and were used without further purification. Blank kits were acquired from Beijing Shihong Pharmaceutical Center, Beijing Normal University, China. The 99 Mo/ 99m Tc generator was obtained from Atomic High Tech Co. Ltd., China. NMR spectra were recorded on a 400 MHz spectrophotometer (JEOL, Tokyo, Japan). HRMS spectra were recorded on a Bruker microTOF-QII mass spectrometer and a Bruker solariX ESI mass spectrometer. HPLC was acquired from a SHIMADZU system (CL-20AVP) equipped with an SPD-20A UV detector (λ = 254 nm) and a Bioscan flflow count 3200 NaI/PMT γ-radiation scintillation detector. The SPECT/CT imaging studies were obtained with a Triumph SPECT/CT scanner (TriFoil Imaging,   The precursor N-(2-aminoethyl)-folate acid was prepared according to the previously reported method [14]. Under nitrogen atmosphere, N-Boc-ethylenediamine (247 mg, 1.5 mmol) and DCC (536 mg, 2.6 mmol) were added to the DMSO solution containing FA (662 mg, 1.5 mmol, 1). The reaction took place overnight at room temperature and in the dark. The reaction liquid was filtered to remove the residue, and the filtrate was collected. The yellow solid product 2 was obtained by repeatedly washing the filtrate with cold ether. 1  2,3,5,6-Tetrafluorophenyl 6-isocyanohexanoate (4) and 2,3,5,6-tetrafluorophenyl 3-(2isocyanoethoxy)propanoate (5) were synthesized according to the literature [20].     The complexes were determined by ITLC/SG of the ACD system and radio-HPLC. The ITLC/SG trip was cut into 10 equal parts, and radioactive counting of ITLC strips was determined by a γ-counter. ITLC results of both complexes were as follows: in ACD solution, [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA stayed at the origin (R f = 0-0.1), while 99m TcO 4 − (R f = 0.9-1.0) and 99m TcO 2 ·nH 2 O (R f = 0.8-1.0) were moved to the front. Radio-HPLC was analyzed by a mobile phase system consisting of 0.1% TFA water (solvent A) and 0.1% TFA acetonitrile (solvent B) with a gradient of 0−2 min 10% B, 2−5 min 10%−90% B, 10−20 min 90% B, and 20−25 min 90%−10% B (system B) at a flow rate of 1 mL/min. All experiments were performed in triplicate.

Stability Study
The stability of [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA in vitro was evaluated by measuring the RCP of the complexes. Stability in saline was achieved by holding the solution at room temperature for 4 h. Stability in the serum was achieved by mixing 100 µL of [ 99m Tc]Tc-CN5FA or [ 99m Tc]Tc-CNPFA with 200 µL of mouse serum at 37 • C for 4 h. After precipitation using acetonitrile, centrifugation, and concentration, the solutions were analyzed by radio-HPLC.

Determination of Partition Coefficient
The partition coefficient was measured as follows: 1.9 mL of phosphate buffer solution (0.025 M, pH 7.4), 2.0 mL of 1-octanol, and 0.1 mL of 99m Tc-CN5FA or 99m Tc-CNPFA solution were added to the centrifuge tube, and the sample was swirled for 5 min and centrifuged for 5 min (3000 r/min). Then, the radioactivity counts of the two phases were measured by taking 0.1 mL from the organic phase and the water phase. The partition coefficient P was calculated. The final partition coefficient was expressed as log P ± SD.

In Vitro Binding with KB Cells
KB cells were cultured with FFRPMI 1640 medium 24 h in advance on a 48-well plate with 5 × 10 4 cells per well. The labeling solution was diluted to 5 µCi/mL with the same medium after growing, and the inhibitor was prepared at different concentrations. The 48-well plates were divided into eight groups, and each group was incubated with 100 µL of inhibitor solution, 100 µL of diluted labeled solution (5 µCi/mL), and 200 µL of medium at 37 • C for 2 h.
After removing the cell plate from the incubator, the medium was removed, and the cells were washed with PBS (0.1 M, pH 7.4). The washed cells were lysed with NaOH (1 M) for 5 min. Pyrolysates were collected in the hoses, and the radioactivity count of each hose was measured by a γ-counter. The data were analyzed with GraphPad Prism 5.0 and nonlinearly fit to the log IC 50 ± SD value.

Biodistribution Study in KB Tumor-Bearing Mice
The biodistribution experiments were divided into normal groups and inhibition groups (n = 3), in which the inhibition group was injected with inhibitor (about 4 × 10 4 µg/kg) 30 min in advance. The KB tumor-bearing mice were decapitated 30 and 120 min after the tail vein injection of 0.1 mL labeled solution (about 7.4 × 10 4 Bq). The heart, liver, lung, kidney, spleen, stomach, bone, muscle, intestine, tumor, blood, and other related tissues and organs were wiped clean and weighed, and the radioactivity count was measured. Each tissue per gram of injection dose (%ID/g) was calculated.

SPECT/CT Imaging Study in KB Tumor-Bearing Mice
The SPECT/CT imaging experiments were also divided into a normal group and an inhibition group (n = 3). The labeled solution (about 18.5 MBq) was injected into the tail vein of nude mice bearing KB tumor. The inhibition group was injected with 100 µg FA (about 4 × 10 4 µg/kg) 30 min in advance. After 2 h, the SPECT/CT scan was performed in anesthetized mice by using 1.5% isoflurane. The SPECT acquisition (peak value 140 keV, 20% width, 90 • rotation, MMP 919 Collimator) was performed after 4 min of CT scanning (512 views, 2 × 2 binding, 75 kV, exposure time 230 ms). SPECT/CT images were made using HiSPECT software and vivoquant 2.5 software.

Statistical Analysis
All quantitative data were expressed as the mean ± SD. Excel 2016 software was used for statistical analysis by the t-test. The statistical data were evaluated using a two-sided test (* p < 0.05 and ** p < 0.01 were considered statistically significant).

Conclusions
In this work, we successfully developed two novel molecular probes, [ 99m Tc]Tc-CN5FA and [ 99m Tc]Tc-CNPFA, to target FR without further purification. In vitro and in vivo studies demonstrated the high affinity and specificity of these probes for targeting FR. As they can be prepared as a kit formulation, they are promising candidates as tumor imaging agents to target folate receptors.
Funding: This work was financially supported, in part, by the National Natural Science Foundation of China (21771023, 22076013) and the project of the Beijing Municipal Science and Technology Commission (Z181100002218033).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.