[99mTc]Tc-Labeled Plectin-Targeting Peptide as a Novel SPECT Probe for Tumor Imaging

Certain receptors are often overexpressed during tumor occurrence and development and closely correlate with carcinogenesis. Owing to its overexpression on the cell membrane and cytoplasm of various tumors, plectin, which is involved in tumor proliferation, migration, and invasion, has been viewed as a promising target for cancer imaging. Hence, plectin-targeting agents have great potential as imaging probes for tumor diagnosis. In this study, we developed a [99mTc]Tc-labeled plectin-targeted peptide (PTP) as a novel single-photon emission computed tomography (SPECT) probe for tumor imaging and investigated its pharmacokinetics, biodistribution, and targeting ability in several types of tumor-bearing mouse models. The PTP had good biocompatibility and targeting ability to tumor cells in vitro and could be readily labeled with [99mTc]Tc after modification with the bifunctional chelator 6-hydrazino nicotinamide (HYNIC). Furthermore, the prepared [99mTc]Tc-labeled PTP ([99mTc]Tc-HYNIC-PTP) showed high radiochemical purity and excellent stability in vitro. In addition, favorable biodistribution, fast blood clearance, and clear accumulation of [99mTc]Tc-HYNIC-PTP in several types of tumors were observed, with a good correlation between tumor uptake and plectin expression levels. These results indicate the potential of [99mTc]Tc-HYNIC-PTP as a novel SPECT probe for tumor imaging.


Introduction
Molecular imaging can reflect biological events at the cellular and molecular levels during the occurrence and progression of diseases and has been widely applied for cancer diagnosis [1][2][3]. Because of the overexpression or activation of specific receptors in the tumorigenesis process, various tumor-targeting ligands, including antibodies, affibodies, nanobodies, peptides, and small-molecule compounds, have been labeled with appropriate radionuclides in the past several decades to develop molecular imaging probes for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging [4][5][6][7][8][9]. Among them, radiolabeled peptides are the best candidates for clinical translation. Some have been successfully applied in clinical practice, such as arginine-glycine-aspartate (RGD) for integrin receptors in solid tumors and octreotide for somatostatin receptors in neuroendocrine tumors [10][11][12]. This type of receptor-targeted nuclear medicine imaging shows an enormous value for tumor diagnosis and management, including but not limited to detection, staging, noninvasive quantification of receptor expression, therapy response monitoring, risk stratification, and patient selection [12][13][14]. Therefore, developing tumor-targeting imaging probes based on specific receptors is important for clinical applications.

Materials
The PTP was manufactured by ChinaPeptides Co., Ltd. (Shanghai, China). During the synthesis process, the N-terminus of PTP was modified with fluorescein isothiocyanate (FITC)

Cells and Animals
PANC-1, BxPC-3, C6, U87, 4T1, A549, mouse β-TC-6 insulinoma cells, and human lung epithelial BEAS-2B cells obtained from the Chinese Academy of Sciences (Shanghai, China) were incubated in media and treated under the conditions recommended by the supplier. The animal experiments conformed to the National Institutes of Health Guidelines and were approved by the ethical committee of Shanghai General Hospital. Four-week-old female BALB/c nude mice (18-20 g) and healthy ICR mice (20-22 g) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). Animal models were established according to previously published protocols [34,35]. The mice were subcutaneously injected in their right-side flanks with 2 × 10 6 C6, 1 × 10 7 U87, 5 × 10 6 4T1, 1 × 10 7 BxPC-3, or 1 × 10 7 A549 cells suspended in 100 µL PBS. When the tumor nodules reached a 0.8-1.2 cm diameter, tumor-bearing mice were used for animal experiments.

Cytotoxicity Assay In Vitro and Toxicity Study In Vivo
CCK-8 assays were used to assess the potential cytotoxic effects of PTP on normal and tumor cell lines. Briefly, taking BxPC-3 cells as an example, the cells were seeded into 96-well plates at a density of 1 × 10 4 per well with RPMI-1640 medium and 10% FBS. After 24 h, the medium was replaced with 100 µL of fresh medium containing different concentrations of PTP (0-200 µg/mL) for another 24 h. Next, 10 µL of CCK-8 solution was added, and the cells were subsequently cultured for 1.5 h. Absorbance at 450 nm was measured using a Varioskan Flash multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The cytocompatibility of PTP was also assessed in C6 and BEAS-2B cells using the same method.
To assess the preliminary safety in vivo, five BALB/c nude mice were treated with PTP at a single dose (1 mg, 100 µL), and another five mice treated with saline were used as the control group. The body weight, physical activity, and death of each mouse were recorded during the following seven days. The major organs of the mice, including the heart, liver, spleen, lung, and kidneys, were harvested for histological examination.

Western Blot Assay and Immunofluorescence Staining
Plectin expression in BxPC-3 cells was confirmed using western blotting. PANC-1 and β-TC-6 cells were set as the positive and negative controls, respectively. Total proteins from BxPC-3, PANC-1, and β-TC-6 cells were extracted using RIPA buffer supplemented with PMSF and quantified using a BCA protein assay kit. The protein samples were separated by a 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. After blocking with 5% non-fat milk in TBST for 1 h, the bands were incubated overnight at 4 • C with the primary antibody at a concentration of 1:1000 (anti-glucose transporter plectin antibody and tubulin used as endogenous controls). After washing three times in TBST, the membranes were incubated with goat anti-rabbit secondary antibodies at a concentration of 1:1000 for 1.5 h at room temperature. After washing in TBST, the protein bands were identified using ECL reagents. The blots were quantified using the ImageJ software.

Flow Cytometry Analysis and Confocal Microscopy
The ability of PTP to target tumor cells in vitro was evaluated by flow cytometry. Briefly, taking C6 cells as an example, cells were seeded into 6-well plates at a density of 2 × 10 5 cells/well. After 24 h, the medium was replaced with 1 mL of fresh serum-free medium containing FITC-PTP at different concentrations (0-40 µM). After another 4 h, the cells were washed, trypsinized, resuspended in 200 µL PBS containing 2% FBS, and measured using BD AccuriTM C6 Flow Cytometer (BD Biosciences, USA). A minimum of 10,000 events were recorded for each sample. The targeting abilities of PTP towards BEAS-2B (negative control), U87, 4T1, A549, BxPC-3, and PANC-1 (positive control) in vitro were evaluated according to a similar method.
The specificity of PTP towards tumor cells (U87, C6, 4T1, A549, and BxPC-3) in vitro was also tested by confocal microscopy. BEAS-2B cells were used as the negative control. Briefly, taking BxPC-3 cells as an example, the cells were seeded at a 5 × 10 4 cells/mL density in glass-bottom cell culture dishes. The cells were incubated in a medium containing 10 µM FITC-PTP for 4 h. The cells were then rinsed, fixed, counterstained, and observed under a fluorescence microscope (Leica SP8, Wetzlar, Germany).

Ex Vivo Fluorescent Imaging
Fluorescent imaging was performed in five types of tumor-bearing mice (U87, C6, 4T1, A549, and BxPC-3) to test the in vivo targeting ability of FITC-PTP. Tumor-bearing mice were intravenously injected with a PBS solution of FITC-PTP (150 µL, 1 mg/mL), and one mouse was injected with PBS as a control. Considering the limited penetration of FITC, tumor-bearing mice were sacrificed to harvest the main organs and tumors at 1 h post-injection for ex vivo fluorescent imaging. Fluorescent images were acquired using IVIS Spectrum optical imaging (IVIS Lumina Series III, PerkinElmer, Waltham, MA, USA) with excitation at 535 nm and emission at 580 nm.

Quality Control
The [ 99m Tc]Tc-labeled PTP was characterized using an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a UV-vis detector (λ = 220 nm) and a radioactive flow detector (BioScan, Poway, CA, USA). A SunFire C18 column (5 µm, 4.6 × 250 mm, Waters, Japan) was used at a 1 mL/min flow rate with the following gradient method: 0.1% trifluoroacetic acid in H 2 O and CH 3 CN (0-20 min, 15-45% CH 3 CN). The RCP of [ 99m Tc]Tc-HYNIC-PTP was determined by radio-HPLC and could also be rapidly analyzed by ITLC in a system consisting of silica gel 60 F254 TLC plates (Merck, Germany) and 50% acetonitrile as mobile phase. To assess the stability in vitro, the formed [ 99m Tc]Tc-HYNIC-PTP (500 µL, 1 mCi) was mixed with 500 µL of PBS (0.1 M, pH = 7.4) and 500 µL of cysteine solution (100-fold molar excess over the PTP) at room temperature, and 500 µL of FBS at 37 • C. The RCPs were tested by ITLC at different time intervals (0-6 h).

Pharmacokinetics
The pharmacokinetic profile of [ 99m Tc]Tc-HYNIC-PTP was investigated in healthy ICR mice. The mice were randomly divided into nine groups (n = 3), and each mouse was intravenously injected with [ 99m Tc]Tc-HYNIC-PTP at a dose of 20 µCi in a 200 µL solution. One hundred microliters of blood from each mouse was immediately collected and weighed at designated times (1,2,5,15,30,60,120,240, and 360 min), and radioactivity was measured using a γ-counter (CAPINTEC, USA) to calculate the percent uptake of the injected dose per gram (%ID/g). In addition, the pharmacokinetic data were analyzed by DAS 2.0 (Shanghai, China) using a two-compartment model to calculate the half-life of [ 99m Tc]Tc-HYNIC-PTP in the blood.

SPECT Imaging In Vivo and Immunohistochemistry Assays
The feasibility of [ 99m Tc]Tc-HYNIC-PTP as a SPECT probe for tumor imaging was examined using established subcutaneous tumor models. Briefly, [ 99m Tc]Tc-HYNIC-PTP solution (200 µL, [ 99m Tc]Tc = 10 mCi/mL, corresponding to 2 µg of HYNIC-PTP) was intravenously injected into tumor-bearing mice. SPECT images were acquired 0.5, 1, 2, and 4 h after injection using a SPECT imaging system equipped with a Xeleris 2.0 workstation and low-energy general-purpose collimators (Infinia, Denver, CO, USA). After SPECT imaging, the tumors and muscle tissues were excised to confirm plectin expression levels by immunohistochemistry. Briefly, excised specimens were fixed in formalin and embedded in paraffin. The sections (5 µm) were subjected to heat treatment in a citrate solution for antigen retrieval and blocking with 3% bovine serum albumin for 30 min. Then the sections were incubated with anti-plectin primary antibody (1:200) overnight at 4 • C. After incubation with secondary antibody (K5007, DAKO, Carpinteria, CA, USA) at room temperature for 50 min, the slides were treated with 1:100 DAB (K5007, DAKO, USA) and counterstained with hematoxylin. At least three sections of each specimen were chosen, and 5-10 high-power visual fields were randomly selected to calculate the positive area of staining. The average areas were then used to plot histograms.

Biodistribution
Mice bearing C6 or 4T1 tumor xenografts were intravenously injected with [ 99m Tc]Tc-HYNIC-PTP at a dose of 20 µCi in 200 µL solution (corresponding to 20 ng of HYNIC-PTP) to evaluate biodistribution in tumors and major organs (n = 3). The mice were sacrificed 0.5, 1, 2, and 4 h after injection to collect blood and tissue samples, including the heart, lung, liver, stomach, intestine, spleen, kidneys, muscle, and tumor. These samples were immediately weighed, and their radioactivity was measured using a γ-counter. The amount of radioactivity is expressed as %ID/g.

Statistical Analysis
Data are presented as the mean ± standard deviation, and one-way analysis of variance was performed to evaluate the significance of the data. A p-value of 0.05 was selected as the threshold of significance, and the data were denoted with (*) for p < 0.05, (**) for p < 0.01 and (***) for p < 0.001.

Preliminary Toxicity Assessment
Biocompatibility of the PTP peptide was investigated in different cell lines in vitro and in normal nude mice in vivo. The results showed that the cell viabilities after treatment with PTP at the studied concentrations remained more than 90% at 24 h ( Figure S1), suggesting excellent cytocompatibility in vitro. None of the mice died in the toxicity study, and no significant difference in body weight and physical activity of mice treated with PTP was found compared with the saline group within seven days. Moreover, biosafety after treatment with PTP was checked by observing the H&E staining of the main organs, including the heart, liver, lung, spleen, and kidneys ( Figure S2). Unsurprisingly, no significant difference in tissue damage, necrotic areas, or abnormalities was found between the PTP and saline groups, suggesting the excellent biocompatibility of PTP in vivo.

Plectin Expression in Tumor Cells
We examined the expression of plectin in BxPC-3 cells using western blotting. PANC-1 and β-TC-6 cells were used as the positive and negative controls, respectively. As presented in Figure 1A,B, PANC-1 and BxPC-3 cells exhibited high plectin expression levels with no statistical difference, while no obvious plectin was expressed in β-TC-6 cells, consistent with the literature [30]. Furthermore, the plectin expression levels in different tumor cells (U87, C6, A549, 4T1, and BxPC-3) were compared with those in BEAS-2B cells (negative control) using immunofluorescence staining assays. The results revealed strong fluorescence signals in the U87, C6, A549, 4T1, and BxPC-3 cells compared with BEAS-2B cells, suggesting plectin overexpression in these tumor cells. Quantitative analysis showed that plectin expression levels in the selected tumor cells followed the order from lowest to highest: BEAS-2B, U87, C6, A549, 4T1, and BxPC-3.

Plectin Expression in Tumor Cells
We examined the expression of plectin in BxPC-3 cells using western blotting. PANC-1 and β-TC-6 cells were used as the positive and negative controls, respectively. As presented in Figure 1A,B, PANC-1 and BxPC-3 cells exhibited high plectin expression levels with no statistical difference, while no obvious plectin was expressed in β-TC-6 cells, consistent with the literature [30]. Furthermore, the plectin expression levels in different tumor cells (U87, C6, A549, 4T1, and BxPC-3) were compared with those in BEAS-2B cells (negative control) using immunofluorescence staining assays. The results revealed strong fluorescence signals in the U87, C6, A549, 4T1, and BxPC-3 cells compared with BEAS-2B cells, suggesting plectin overexpression in these tumor cells. Quantitative analysis showed that plectin expression levels in the selected tumor cells followed the order from lowest to highest: BEAS-2B, U87, C6, A549, 4T1, and BxPC-3.

Specificity of PTP to Tumor Cells In vitro
The ability of the PTP peptide to target different tumor cells in vitro was examined by flow cytometry. We first performed a series of flow cytometry assays using C6 cells at different FITC-PTP concentrations (0.5-40 µM) to confirm the appropriate concentration. As shown in Figures 2A and S3, the fluorescence intensity of C6 cells after 4 h of incubation gradually increased with the FITC-PTP concentration, and no changes in the fluorescence intensities could be found between the PBS group and FITC-PTP at relatively low concentrations of 0.5 and 1 µM. When the concentration ranged from 2-40 µM, the fluorescence

Specificity of PTP to Tumor Cells In Vitro
The ability of the PTP peptide to target different tumor cells in vitro was examined by flow cytometry. We first performed a series of flow cytometry assays using C6 cells at different FITC-PTP concentrations (0.5-40 µM) to confirm the appropriate concentration. As shown in Figure 2A and Figure S3, the fluorescence intensity of C6 cells after 4 h of incubation gradually increased with the FITC-PTP concentration, and no changes in the fluorescence intensities could be found between the PBS group and FITC-PTP at relatively low concentrations of 0.5 and 1 µM. When the concentration ranged from 2-40 µM, the fluorescence intensity was almost proportional to the concentration of FITC-PTP. Based on these results, the concentration of FITC-PTP used in flow cytometry for other tumor cells was 10 µM. As shown in Figure 2B, the fluorescence intensities in tumor cells after 4 h of incubation with FITC-PTP were significantly higher than those of the negative control cell (BEAS-2B). Notably, after quantitative analysis, the fluorescence intensities followed the same order as their plectin expression levels in tumor cells, and BxPC-3 cells showed the highest FITC signal intensities. Similarly, confocal microscopy also showed stronger FITC fluorescence intensities in tumor cells than in BEAS-2B cells ( Figure 3). Together, these data from flow cytometry and confocal microscopy demonstrated the high specificity of the PTP peptide for tumor cells.
tion with FITC-PTP were significantly higher than those of the negative control cell (BEAS-2B). Notably, after quantitative analysis, the fluorescence intensities followed the same order as their plectin expression levels in tumor cells, and BxPC-3 cells showed the highest FITC signal intensities. Similarly, confocal microscopy also showed stronger FITC fluorescence intensities in tumor cells than in BEAS-2B cells ( Figure 3). Together, these data from flow cytometry and confocal microscopy demonstrated the high specificity of the PTP peptide for tumor cells.   µM. As shown in Figure 2B, the fluorescence intensities in tumor cells after 4 h of incubation with FITC-PTP were significantly higher than those of the negative control cell (BEAS-2B). Notably, after quantitative analysis, the fluorescence intensities followed the same order as their plectin expression levels in tumor cells, and BxPC-3 cells showed the highest FITC signal intensities. Similarly, confocal microscopy also showed stronger FITC fluorescence intensities in tumor cells than in BEAS-2B cells (Figure 3). Together, these data from flow cytometry and confocal microscopy demonstrated the high specificity of the PTP peptide for tumor cells.

Ex Vivo Fluorescent Imaging
The specificity of PTP to the studied tumor cells was investigated in vivo using fluorescence imaging. Five types of tumor-bearing mice (U87, C6, A549, 4T1, and BxPC-3) were intravenously injected with FITC-PTP, and the major organs and tissues, including the heart, liver, lung, spleen, kidneys, tumor, and muscle, were collected 1 h after injection. As shown in Figure 4, a high accumulation of FITC-PTP was found in tumors, but low fluorescence intensities were observed in major organs, such as the liver and kidneys. The preferential accumulation in the tumor made a clear distinction from other tissues, suggesting the good specificity of PTP for tumor imaging. These data further verified that PTP could specifically deliver imaging agents to tumors, making it a promising agent for tumor diagnosis.
fluorescence intensities were observed in major organs, such as the liver and kidneys preferential accumulation in the tumor made a clear distinction from other tissues, gesting the good specificity of PTP for tumor imaging. These data further verified PTP could specifically deliver imaging agents to tumors, making it a promising agen tumor diagnosis.  (7) tumor, re tively.

Radiochemistry
PTP could be effectively radiolabeled with [ 99m Tc]Tc via the bifunctional che HYNIC, using tricine and EDDA as co-ligands. The RCPs of [ 99m Tc]Tc-HYNIC-PTP analyzed by radio-HPLC and ITLC. As the radio-HPLC results show in Figure 5A Figure 5B). To investigate the op labeling conditions, a series of experiments were performed using different dos HYNIC-PTP for radiolabeling. Although the RCPs were all over 90% in the range o 200 µg, the RCP could be more than 95% when the dose of HYNIC-PTP for radiolab was above 50 µg ( Figure 5C), suggesting no need for further purification proced Therefore, [ 99m Tc]Tc-HYNIC-PTP used in subsequent experiments was prepared u the optimum reaction conditions of 50 µg HYNIC-PTP, 50 mCi Na[ 99m Tc]TcO4, and 5 SnCl2, with a specific radioactivity of more than 1000 Ci/g. Furthermore, no ev changes in RCPs were found in PBS in the presence of a 100-fold molar excess of cys

Radiochemistry
PTP could be effectively radiolabeled with [ 99m Tc]Tc via the bifunctional chelator HYNIC, using tricine and EDDA as co-ligands. The RCPs of [ 99m Tc]Tc-HYNIC-PTP were analyzed by radio-HPLC and ITLC. As the radio-HPLC results show in Figure 5A Figure 5B). To investigate the optimal labeling conditions, a series of experiments were performed using different doses of HYNIC-PTP for radiolabeling. Although the RCPs were all over 90% in the range of 10-200 µg, the RCP could be more than 95% when the dose of HYNIC-PTP for radiolabeling was above 50 µg ( Figure 5C), suggesting no need for further purification procedures. Therefore, [ 99m Tc]Tc-HYNIC-PTP used in subsequent experiments was prepared under the optimum reaction conditions of 50 µg HYNIC-PTP, 50 mCi Na[ 99m Tc]TcO 4 , and 50 µg SnCl 2 , with a specific radioactivity of more than 1000 Ci/g. Furthermore, no evident changes in RCPs were found in PBS in the presence of a 100-fold molar excess of cysteine at room temperature and FBS at 37 • C within 6 h, suggesting high stability of [ 99m Tc]Tc-HYNIC-PTP in vitro ( Figure 5D). ceutics 2022, 14, x FOR PEER REVIEW 9 at room temperature and FBS at 37 °C within 6 h, suggesting high stability of [ 99m T HYNIC-PTP in vitro ( Figure 5D).

Pharmacokinetics
The radioactivity-time curve is shown in Figure S4. At 1 min post-injection, the oactivity in blood was 14.52%ID/g, which rapidly decreased to 1.35%ID/g at 30 min injection. Less than 0.01%ID/g could be recovered from the blood pool at 360 min injection. The distribution-phase half-life (t1/2 alpha) and clear-phase half-life (t1/2 be [ 99m Tc]Tc-HYNIC-PTP were estimated to be 0.88 and 9.17 min, respectively.

Targeted SPECT Imaging of Tumors In Vivo and Immunohistochemistry Assays
To evaluate the feasibility of [ 99m Tc]Tc-HYNIC-PTP as a probe for tumor detecti vivo, SPECT imaging was conducted in five types of tumor-bearing mice (U87, C6, A 4T1, and BxPC-3). As shown in Figure 6A, the probe exhibited a similar distribution tern in these mice. The main radioactivity was found in the kidneys and bladder, with uptake in the heart, lung, liver, spleen, intestines, and muscle, suggesting that [ 99m T HYNIC-PTP was predominantly cleared through the urinary system. Notably, fast ance was observed in the blood and lungs, with background radioactivity levels rem ing at 2 h post-injection. Conversely, evident uptake in the tumors was observed d the study period. The tumor accumulation of [ 99m Tc]Tc-HYNIC-PTP was observed h post-injection and sustained with time. The tumor-to-muscle (T/M) SPECT signal r at different time points were measured to determine the optimal time point for SP imaging ( Figure 6B). Although satisfactory tumor conspicuity was detected in thes

Pharmacokinetics
The radioactivity-time curve is shown in Figure S4. At 1 min post-injection, the radioactivity in blood was 14.52%ID/g, which rapidly decreased to 1.35%ID/g at 30 min post-injection. Less than 0.01%ID/g could be recovered from the blood pool at 360 min post-injection. The distribution-phase half-life (t 1/2 alpha) and clear-phase half-life (t 1/2 beta) of [ 99m Tc]Tc-HYNIC-PTP were estimated to be 0.88 and 9.17 min, respectively.

Targeted SPECT Imaging of Tumors In Vivo and Immunohistochemistry Assays
To evaluate the feasibility of [ 99m Tc]Tc-HYNIC-PTP as a probe for tumor detection in vivo, SPECT imaging was conducted in five types of tumor-bearing mice (U87, C6, A549, 4T1, and BxPC-3). As shown in Figure 6A, the probe exhibited a similar distribution pattern in these mice. The main radioactivity was found in the kidneys and bladder, with low uptake in the heart, lung, liver, spleen, intestines, and muscle, suggesting that [ 99m Tc]Tc-HYNIC-PTP was predominantly cleared through the urinary system. Notably, fast clearance was observed in the blood and lungs, with background radioactivity levels remaining at 2 h post-injection. Conversely, evident uptake in the tumors was observed during the study period. The tumor accumulation of [ 99m Tc]Tc-HYNIC-PTP was observed at 0.5 h post-injection and sustained with time. The tumor-to-muscle (T/M) SPECT signal ratios at different time points were measured to determine the optimal time point for SPECT imaging ( Figure 6B). Although satisfactory tumor conspicuity was detected in these five tumor types at 1 h post-injection, the highest T/M ratios occurred at different time points. The highest T/M ratios in U87 and C6 tumors were observed at 2 h post-injection, while the T/M ratios in A549, 4T1, and BxPC-3 continued to increase during the study period. After SPECT imaging, the plectin expression levels in tumors were confirmed by immunohistochemistry to analyze the correlation with tumor uptake, and muscle tissues were set as the negative control. As shown in Figure 6C and Figure S5, the levels of plectin expression showed high consistency between immunohistochemistry and immunofluorescence staining. Tumors with higher plectin expression levels had better T/M ratios, indicating a higher tumor uptake. This correlation further validated the specificity of [ 99m Tc]Tc-HYNIC-PTP for the plectin receptor in vivo.

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After SPECT imaging, the plectin expression levels in tumors were confirmed by immunohistochemistry to analyze the correlation with tumor uptake, and muscle tissues were set as the negative control. As shown in Figures 6C and S5, the levels of plectin expression showed high consistency between immunohistochemistry and immunofluorescence staining. Tumors with higher plectin expression levels had better T/M ratios, indicating a higher tumor uptake. This correlation further validated the specificity of [ 99m Tc]Tc-HYNIC-PTP for the plectin receptor in vivo.

Biodistribution
A biodistribution experiment was performed in C6 and 4T1 tumors to validate the correlation between tumor uptake and plectin expression. Similar to the SPECT results, the major radioactivity accumulated in the kidneys, with relatively low accumulation in other organs and rapid clearance from the blood and lungs (Figure 7). Importantly, as expected, tumors with higher expression levels of plectin possessed better tumor uptake and T/M ratios. For example, the tumor uptake and T/M ratio in C6 tumors at 1 h postinjection were 0.22 ± 0.02%ID/g and 2.78 ± 0.25, lower than those of 4T1 tumors (0.45 ± 0.02%ID/g and 4.93 ± 0.48). Although both the tumor uptake in C6 and 4T1 tumors decreased at 4 h post-injection (0.13 ± 0.01%ID/g and 0.25 ± 0.03%ID/g), a higher T/M ratio was displayed in 4T1 tumors when compared with that in C6 tumors at the same time point (3.37 ± 0.57 vs. 5.05 ± 0.97).

Biodistribution
A biodistribution experiment was performed in C6 and 4T1 tumors to validate the correlation between tumor uptake and plectin expression. Similar to the SPECT results, the major radioactivity accumulated in the kidneys, with relatively low accumulation in other organs and rapid clearance from the blood and lungs (Figure 7). Importantly, as expected, tumors with higher expression levels of plectin possessed better tumor uptake and T/M ratios. For example, the tumor uptake and T/M ratio in C6 tumors at 1 h post-injection were 0.22 ± 0.02%ID/g and 2.78 ± 0.25, lower than those of 4T1 tumors (0.45 ± 0.02%ID/g and 4.93 ± 0.48). Although both the tumor uptake in C6 and 4T1 tumors decreased at 4 h post-injection (0.13 ± 0.01%ID/g and 0.25 ± 0.03%ID/g), a higher T/M ratio was displayed in 4T1 tumors when compared with that in C6 tumors at the same time point (3.37 ± 0.57 vs. 5.05 ± 0.97).

Discussion
The design and development of molecular imaging probes for nuclear medic attracted significant interest. As a result, an increasing number of receptors ha explored in this field, such as fibroblast activation proteins, prostate-specific m antigens, and somatostatin receptors [2]. In addition, several types of targeting m have been investigated for their receptors [4][5][6][7][8][9]. Because of their attractive advan constructing molecular imaging probes, including high affinity and specificity, fa clearance, low immunogenicity, and easy modification, many peptides have been with radionuclides for tumor receptor imaging, and some are being widely used [12]. Given these successes, peptide-based probes have become established stra molecular imaging, further prompting the discovery and development of novel based imaging agents for disease diagnosis [11].
In this study, the heptapeptide PTP was selected as a targeting molecule to a new peptide-based probe for tumor imaging because of its high binding affi specificity for the plectin receptor, which is widely overexpressed in various tum has great potential as a biomarker for tumor detection. We first tested the cytocom ity in vitro and conducted a preliminary toxicity assessment of PTP peptides in expected, no remarkable cytotoxicity to the tumor or normal cells and no abnorm normal mice were detected, indicating the high safety of the peptide. Although m ports have proven the specificity of the PTP peptide for plectin, most studies have on pancreatic cancer. Hence, the targeting abilities of PTP to tumor cells (U87, C 4T1, and BxPC-3) used in the present study were evaluated in vitro. In the flow cy study, FITC-PTP exhibited significantly higher fluorescence signals in these plec

Discussion
The design and development of molecular imaging probes for nuclear medicine have attracted significant interest. As a result, an increasing number of receptors have been explored in this field, such as fibroblast activation proteins, prostate-specific membrane antigens, and somatostatin receptors [2]. In addition, several types of targeting molecules have been investigated for their receptors [4][5][6][7][8][9]. Because of their attractive advantages in constructing molecular imaging probes, including high affinity and specificity, fast blood clearance, low immunogenicity, and easy modification, many peptides have been labeled with radionuclides for tumor receptor imaging, and some are being widely used in clinics [12]. Given these successes, peptide-based probes have become established strategies in molecular imaging, further prompting the discovery and development of novel peptide-based imaging agents for disease diagnosis [11].
In this study, the heptapeptide PTP was selected as a targeting molecule to develop a new peptide-based probe for tumor imaging because of its high binding affinity and specificity for the plectin receptor, which is widely overexpressed in various tumors and has great potential as a biomarker for tumor detection. We first tested the cytocompatibility in vitro and conducted a preliminary toxicity assessment of PTP peptides in vivo. As expected, no remarkable cytotoxicity to the tumor or normal cells and no abnormality in normal mice were detected, indicating the high safety of the peptide. Although many reports have proven the specificity of the PTP peptide for plectin, most studies have focused on pancreatic cancer. Hence, the targeting abilities of PTP to tumor cells (U87, C6, A549, 4T1, and BxPC-3) used in the present study were evaluated in vitro. In the flow cytometry study, FITC-PTP exhibited significantly higher fluorescence signals in these plectin-overexpressing tumor cells than in BEAS-2B cells (negative control), and the fluorescence intensities showed good correlations with their plectin expression results from immunofluorescent staining. To provide an easy method for the [ 99m Tc]Tc-labeled PTP preparation, HYNIC was attached to the N-terminus of the peptide, and HYNIC-PTP was labeled with [ 99m Tc]Tc using tricine and EDDA as co-ligands. The main reason for using HYNIC as a bifunctional chelator was the facile conjugation to the peptide with high RCP in [ 99m Tc]Tc radiolabeling.
Using EDDA and tricine as co-ligands could improve the hydrophilicity and pharmacokinetics of [ 99m Tc]Tc-labeled PTP for rapid elimination through the urinary system, which was demonstrated in a subsequent SPECT imaging study. Hence, [ 99m Tc]Tc-HYNIC-PTP could be acquired in 15 min with high RCP and specific radioactivity in this study. This method was easy to perform because the preparation was achieved in one pot, and the RCP was greater than 90% without further purification, even in 10 µg HYNIC-PTP. The convenience of radiolabeling makes the kit formulation available. Moreover, [ 99m Tc]Tc-HYNIC-PTP displayed favorable stability in vitro in PBS, FBS, and cysteine solution for 6 h, supporting further investigations in animal models for tumor-targeting SPECT imaging.
SPECT images of mice injected with [ 99m Tc]Tc-HYNIC-PTP displayed fast accumulation and relatively high uptake in the tumors. The accumulation of [ 99m Tc]Tc-HYNIC-PTP in the five types of tumor-bearing mice was observable 0.5 h post-injection and maintained during the study period. However, these mice's T/M ratios at different time points showed inconsistent trends, probably related to the tumor plectin status. The highest T/M ratios of tumors with relatively low plectin expression, such as U87 and C6, were observed at 1 and 2 h after injection, respectively. In contrast, the T/M ratios continuously increased in the tumors with higher plectin expression levels (4T1, A549, and BxPC-3). SPECT imaging was in accordance with the immunohistochemistry results, indicating good specificity of PTP to different tumors in vivo and enhanced retention of [ 99m Tc]Tc-HYNIC-PTP in tumors with higher plectin expression levels. The biodistribution experiment further supported this, which showed that 4T1 tumors exhibited better tumor uptake and T/M ratio at each time point than C6 tumors. Additionally, the biodistribution and pharmacokinetic data revealed that [ 99m Tc]Tc-HYNIC-PTP had a rapid elimination from the blood and muscle but a relatively slow clearance in tumors, which allowed sufficient conspicuity of tumor imaging within 2 h after injection. Furthermore, except for the kidneys and bladder, the accumulation of radioactivity in other organs was low. However, rapid clearance of [ 99m Tc]Tc-HYNIC-PTP was still found in some normal organs, such as the lungs, which was beneficial in avoiding unnecessary radiation dose burden. Notably, both [ 99m Tc]Tc-HYNIC-PTP and FITC-PTP showed distinct tumor uptake, while different biodistribution in liver and kidneys due to their structural differences. The hydrophilicity of HYNIC led to a high accumulation of [ 99m Tc]Tc-HYNIC-PTP in kidneys. Still, FITC-PTP had an obvious fluorescence signal in the liver, which was in line with the literature [30]. This suggested that the introduction of FITC or HYNIC probably changed the biodistribution and pharmacokinetics of PTP, with no significant influence on targeting ability.
The PTP peptide has been utilized as a targeting molecule to enhance the specificity of imaging agents and the efficacy of drug delivery systems in various studies, such as PTP-combined RGD peptides as a bispecific molecular probe for pancreatic cancer imaging and PTP-modified nanopolyplexes for pancreatic cancer therapy [30,31]. Although nanoplatform-based probes have many unique characteristics for tumor imaging, they often suffer from high accumulation in the reticuloendothelial system and undesired toxicological risks. Therefore, the development of radionuclide-labeled PTP shows great advantages because of the probe's high sensitivity and tracer amount, which is beneficial for clinical translation. However, exploration of the utility of PTP-based radiopharmaceuticals for tumor diagnosis is still lacking. Only one previous study reported [ 111 In]In-labeled tetrameric PTP peptide ([ 111 In]In-tPTP) as a SPECT agent for pancreatic cancer imaging [18]. In that study, [ 111 In]In-tPTP showed good specificity and selectivity in distinguishing pancreatic cancer from its metastases in an orthotopic and liver metastasis mouse model. Nevertheless, the RCP of [ 111 In]In-tPTP was unsatisfactory, and post-labeling purification was required. Furthermore, the low availability and high production cost of [ 111 In]In radionuclides limit further clinical application. In contrast to [ 111 In]In, [ 99m Tc]Tc is the most commonly used radionuclide for SPECT imaging in the clinic because of its excellent physicochemical properties and convenient acquisition from commercial generators at a low cost, making it suitable for peptide-based probes. This study designed and successfully synthesized [ 99m Tc]Tc-HYNIC-PTP to develop a plectin-targeting probe for tumor imaging. Plectin overexpression has been identified in a wide range of cancers because of its involvement in various cellular activities in tumors, such as cell proliferation, survival, migration, and invasion [15]. This motivated us to evaluate the feasibility of [ 99m Tc]Tc-HYNIC-PTP as a SPECT imaging agent for multiple cancers, not just pancreatic carcinoma. Although [ 99m Tc]Tc-HYNIC-PTP displayed satisfactory imaging performance in selected tumors, its radiation dosimetry was unclear, and its extended application for cancer imaging was also uncertain. Further studies are required to address these issues.

Conclusions
In summary, plectin has been proven to be overexpressed in various tumors, serving as a specific target for cancer diagnosis. In this study, we prepared a plectin-targeting imaging agent, [ 99m Tc]Tc-HYNIC-PTP using a simple method with high RCP and stability and evaluated its feasibility as a SPECT probe for tumor imaging. The data demonstrated a distinct accumulation of [ 99m Tc]Tc-HYNIC-PTP in five types of tumor-bearing mice with favorable biodistribution and pharmacokinetics, and tumor uptake correlated with their plectin expression levels. Although this probe holds great potential as a novel strategy for tumor imaging, further studies are needed to verify its validity in other plectin-overexpressing tumors and acquire sufficient preclinical data before clinical trials.