A Preclinical Study of an 125I-Labeled PSMA Ligand for Prostate-Cancer Puncture

Purpose: Prostate cancer (PCa) is characterized by high expression of prostate-specific 1membrane antigen (PSMA), a type II transmembrane protein. Prostate-specific membrane antigen positron emission tomography (PSMA PET) has high sensitivity and specificity and can therefore be potentially used to detect PCa. Exploiting the advantages of PSMA PET imaging, in this study, we aim to develop a novel radiopharmaceutical to facilitate biopsy punching of PCa. Methods: We synthesized a high-affinity radiopharmaceutical of PSMA (125I-PSMA-7). We evaluated the properties of 125I-PSMA-7, including the purity, stability, affinity, partition coefficient, and toxicity. (PSMA+) 22Rv1 and (PSMA−) PC3 cell lines were used to evaluate 125I-PSMA-7 in vitro. BALB/c nude mice bearing 22Rv1 and PC3 xenografts were used for biodistribution and imaging. The uptake of the main organs was evaluated in vivo using single photon emission computed tomography (SPECT). Results: 125I-PSMA-7 had a purity of 99.6% and remained stable for seven days and was therefore always safe to use. 125I-PSMA-7 had a Ki of 4.037 × 10−11 and a partition coefficient of −1.80. The results of in vitro cellular experiments showed a high uptake by 22Rv1 cells (ranging from 2.88 ± 0.14 IA%/106 at 5 min to 61.98 ± 3.43 IA%/106 at 24 h, where the internalization was 46.1% at 1 h and 88.06% at 24 h). However, the uptake of PC3 cells was very low (ranging from 0.34 ± 0.08 IA%/106 at 5 min to 1.60 ± 0.15 IA%/106 at 24 h). The tumors’ uptake of 125I-PSMA-7 ranged from 9.02 ± 0.30 ID%/g at 1 h to 4.11 ± 1.04 ID%/g at 7 d and the tumor/muscle ratios and tumor/blood ratios increased over time. In addition, we used γ-counter to measure cpm per milligram of tumor and muscle on days 4 and 7. The background on day 4 is 42 cpm and the tumor is 1739 cpm/mg and the muscle is 45 cpm/mg, and the background on day 7 is 74 cpm and the tumor is 1404cpm/mg and the muscle is 32 cpm/mg. At 1 h post-injection, the high uptake of 125I-PSMA-7 resulted in clear delineation of 22Rv1-derived tumors upon imaging. By comparison, 22Rv1-blocking mice took up less 125I-PSMA-7. Conclusions: These results show that 125I-PSMA-7 is a promising radiotracer that could be used to puncture the prostate. 125I-PSMA-7 could be applied to targeted biopsy, reducing the need for saturated biopsy.


Introduction
PCa is one of the most common malignant tumors and poses a severe threat to men's health [1]. It has been estimated that in the United States in 2022, nearly 0.27 million new cases of PCa will emerge and nearly 35 thousand men could die from PCa [2]. The development of imaging examinations has resulted in the increasing use of multiparametric MRI (mpMRI) and positron emission tomography (PET) for diagnosing PCa. Whether a repeat biopsy needs to be performed along with an mp-MRI-negative remains controversial [3]. However, the mpMRI currently misses 35% of clinically significant PCa (csPCa) [4]. If a

Synthesis and Radiochemistry
The synthetic and radiolabeling steps are shown in Figure 1; Figure 2, respectively. repeat biopsy needs to be performed along with an mp-MRI-negative remains controversial [3]. However, the mpMRI currently misses 35% of clinically significant PCa (csPCa) [4]. If a patient's previous biopsy is negative, but there is constant suspicion of PCa, the European Association of Urology (EUA) guidelines recommend mpMRI as well as a biopsy [5]. Over the past decade, the advent of PSMA-targeted probes has enabled PSMAtargeted PET/CT and PET/MRI to accurately display the location of primary PCa [6][7][8].
PET is superior to mpMRI in terms of diagnostic efficacy, even though PET is not recommended in current clinical guidelines [9]. For the diagnosis or exclusion of PCa, prostate biopsy, with all its limitations, complications and risks, remains a mandatory step. However, up to 30% of csPCa are missed through standard biopsy protocols with 10-12 systematic transrectal ultrasound-guided biopsies (TRUS-GB) [10,11]. The results of using MRI-guided biopsy techniques, such as MRI-guided transrectal-targeted prostate biopsy (MRGB), cognitive target biopsy, and fusion biopsy, were compared against those obtained using TRUS-GB. The results showed that MRI-guided techniques were superior to TRUS-GB, although the difference between the results of the two techniques was not significant [12]. Another study demonstrated that compared with mpMRI, the volume of primary PCa depicted by PSMA PET was highly consistent with postoperative pathologic results7. PSMA PET showed good sensitivity for detecting primary prostatic lesions compared with MRI and TRUS [13,14]. However, a limited number of studies have been performed on the real-time detection of prostate puncture. Therefore, we exploited the advantages of PET precision imaging to evaluate a novel probe synthesized 125 I-PSMA-7 for the real-time detection of prostate puncture. This 125 I-PSMA-7 probe was used for in vitro and in vivo studies of PCa puncture.

Synthesis and Radiochemistry
The synthetic and radiolabeling steps are shown in Figure 1; Figure 2, respectively.

HPLC Purity Identification and MS Analysis
125 I-PSMA-7 was purified by HPLC to a purity of 99.6% (Figure 3a). The chromatogram of the mixture of Compound 7 and 125 I-PSMA-7 is shown in Figure 3b. MS analysis was used to determine the molecular weights of three important compounds: Compound 4, Compound 6, and Compound 7. The peaks of these three compounds correspond to

Determination of PSMA Inhibitory Activity
The Ki of 125 I-PSMA-7 is 4.037 × 10 −11 ( Figure 5) and indicates a high binding affinity to PSMA recombinant protein.

In Vitro Stability and Partition Coefficient
The log D value of 125 I-PSMA-7 is −1.80. Performing HPLC on 125 I-PSMA-7 for 1 h, 6 h, 12 h, 1 d, 3 d, 5 d, and 7 d at room temperature or 37 • C produced chromatograms with a single peak ( Figure 6).

Acute Toxicity Test
Over a 14-day observation period, no significant differences were observed in the diet, activity, mental state, skin or body weight between the experimental and control groups (p > 0.05). The results of HE staining ( Figure 7) and blood tests were normal (p > 0.05).

Determination of PSMA Inhibitory Activity
The Ki of 125 I-PSMA-7 is 4.037 × 10 −11 ( Figure 5) and indicates a high binding to PSMA recombinant protein.

In Vitro Stability and Partition Coefficient
The log D value of 125 I-PSMA-7 is −1.80. Performing HPLC on 125 I-PSMA-7 fo h, 12 h, 1 d, 3 d, 5 d, and 7 d at room temperature or 37 °C produced chromatogram a single peak ( Figure 6).

Acute Toxicity Test
Over a 14-day observation period, no significant differences were observed diet, activity, mental state, skin or body weight between the experimental and groups (p > 0.05). The results of HE staining ( Figure 7) and blood tests were norm 0.05).

In Vitro Stability and Partition Coefficient
The log D value of 125 I-PSMA-7 is −1.80. Performing HPLC on 125 I-PSM h, 12 h, 1 d, 3 d, 5 d, and 7 d at room temperature or 37 °C produced chroma a single peak ( Figure 6).

Acute Toxicity Test
Over a 14-day observation period, no significant differences were ob diet, activity, mental state, skin or body weight between the experimenta groups (p > 0.05). The results of HE staining ( Figure 7) and blood tests we 0.05).   Results of acute toxicity tests on mice. The investigated tissues included the heart, liver, spleen, lung, kidney, bladder, brain, salivary glands, and small intestine. Histopathological analysis of mice by HE staining after a high-dose injection of 125 I-PSMA-7 (top) and normal saline for the control group (bottom).

In Vitro Cellular Experiments
The 125 I-PSMA-7 uptake in 22Rv1 and PC3 is shown in Figure 8. The 125 I-PSMA-7 uptake of 22Rv1 increased with time from 2.88 ± 0.14 IA%/10 6 at 5 min to 61.98 ± 3.43 IA%/10 6 at 24 h. The uptake of 22Rv1 was markedly blocked at 0.10 ± 0.00 IA%/10 6 by coincubation with ZJ43 for 24 h. The PC3 uptake of 125 I-PSMA-7 ranged from 0.34 ± 0.08 IA%/10 6 at 5 min to 1.60 ± 0.15 IA%/10 6 at 24 h, which was low compared with that of 22Rv1. The rate of 22Rv1 cell internalization was 46.1% at 1 h and 88.06% at 24 h ( Figure 9). Results of acute toxicity tests on mice. The investigated tissues included the heart, liver, spleen, lung, kidney, bladder, brain, salivary glands, and small intestine. Histopathological analysis of mice by HE staining after a high-dose injection of 125 I-PSMA-7 (top) and normal saline for the control group (bottom). The 125 I-PSMA-7 uptake in 22Rv1 and PC3 is shown in Figure 8. The 125 I-PSMA-7 up take of 22Rv1 increased with time from 2.88 ± 0.14 IA%/10 6 at 5 min to 61.98 ± 3.43 IA%/10 at 24 h. The uptake of 22Rv1 was markedly blocked at 0.10 ± 0.00 IA%/10 6 by coincubatio with ZJ43 for 24 h. The PC3 uptake of 125 I-PSMA-7 ranged from 0.34 ± 0.08 IA%/10 6 at min to 1.60 ± 0.15 IA%/10 6 at 24 h, which was low compared with that of 22Rv1. The rat of 22Rv1 cell internalization was 46.1% at 1 h and 88.06% at 24 h ( Figure 9).

Pharmacokinetics
WinNonlin was used to plot the time-activity curves for the average blood pharmacokinetics, which are shown in Figure 10. According to the curve type, the distribution and metabolism of the radiopharmaceuticals in the ICR mice was determined to be a twocompartment model. The elimination half-lives of 125 I-PSMA-7 were 1.04 min (Alpha_HL) and 29.24 min (Beta_HL).  Figure 11 shows that over 7 d, the uptake of 125 I-PSMA-7 in tumors decreased from 9.02 ± 0.30 ID%/g at 1 h to 4.11 ± 1.04 ID%/g at 7 d, whereas the tumor-to-muscle ratio

Pharmacokinetics
WinNonlin was used to plot the time-activity curves for the average blood pharmacokinetics, which are shown in Figure 10. According to the curve type, the distribution and metabolism of the radiopharmaceuticals in the ICR mice was determined to be a twocompartment model. The elimination half-lives of 125 I-PSMA-7 were 1.04 min (Alpha_HL) and 29.24 min (Beta_HL). Figure 11 shows that over 7 d, the uptake of 125 I-PSMA-7 in tumors decreased from 9.02 ± 0.30 ID%/g at 1 h to 4.11 ± 1.04 ID%/g at 7 d, whereas the tumor-to-muscle ratio increased from 5.88 at 1 h to 68.42 at 7 d. The organs with the highest uptake were the kidneys, in which the 125 I-PSMA-7 level was always high. The uptake in the spleen and salivary glands was also high, whereas low radioactivity accumulation was found for other organs. In addition, Table 1 showed that we used γ-counter to measure cpm per milligram of tumor and muscle on days 4 and 7. The background on day 4 is 42 cpm and the tumor is 1739 cpm/mg and the muscle is 45 cpm/mg, and the background on day 7 is 74 cpm and the tumor is 1404 cpm/mg and the muscle is 32 cpm/mg. The difference between tumor and muscle is striking.

Biodistribution
WinNonlin was used to plot the time-activity curves for the average blood cokinetics, which are shown in Figure 10. According to the curve type, the di and metabolism of the radiopharmaceuticals in the ICR mice was determined to compartment model. The elimination half-lives of 125 I-PSMA-7 were 1.04 min (A and 29.24 min (Beta_HL).  Figure 11 shows that over 7 d, the uptake of 125 I-PSMA-7 in tumors decrea 9.02 ± 0.30 ID%/g at 1 h to 4.11 ± 1.04 ID%/g at 7 d, whereas the tumor-to-mu increased from 5.88 at 1 h to 68.42 at 7 d. The organs with the highest uptake kidneys, in which the 125 I-PSMA-7 level was always high. The uptake in the sp salivary glands was also high, whereas low radioactivity accumulation was other organs. In addition, Table 1 showed that we used γ-counter to measure milligram of tumor and muscle on days 4 and 7. The background on day 4 is 42 the tumor is 1739 cpm/mg and the muscle is 45 cpm/mg, and the background o 74 cpm and the tumor is 1404 cpm/mg and the muscle is 32 cpm/mg. The diffe tween tumor and muscle is striking.

SPECT Imaging
Images of 125 I-PSMA-7 in 22Rv1 and PC3 and 22Rv1-blocking mice are shown in Figures 12 and 13. The 22Rv1 tumor-bearing mice were observed at 1, 2, 4, and 8 h and 1, 3, 5, and 7 d after injection. As the figures show, the tumor and kidneys were always clearly outlined, and the intensity of the background decreased gradually. Low radioactivity was observed for other normal tissues. The images were consistent with the biodistribution data. The tumor and kidneys of the 22Rv1 tumor-bearing mice coinjected with ZJ43 could not be visualized. Although the kidneys of the negative-contrast PC3 tumor-bearing mice could be observed, the tumor was not discernible.

SPECT Imaging
Images of 125 I-PSMA-7 in 22Rv1 and PC3 and 22Rv1-blocking mice are shown in Figures 12 and 13. The 22Rv1 tumor-bearing mice were observed at 1, 2, 4, and 8 h and 1, 3, 5, and 7 d after injection. As the figures show, the tumor and kidneys were always clearly outlined, and the intensity of the background decreased gradually. Low radioactivity was observed for other normal tissues. The images were consistent with the biodistribution data. The tumor and kidneys of the 22Rv1 tumor-bearing mice coinjected with ZJ43 could not be visualized. Although the kidneys of the negative-contrast PC3 tumor-bearing mice could be observed, the tumor was not discernible.

Discussion
In primary PCa patients, accurate staging and histologic grading are crucial for guiding treatment decisions. PSMA PET outperforms other imaging techniques for localizing the foci of prostate cancer tumors [15,16]. The potential of PSMA-PET for PCa puncture has been evaluated in a few studies. PET and ultrasound images have been fused in some studies, proving that PET-ultrasound fusion targeted biopsy can effectively detect csPCa [17,18]. In other studies, traditional transrectal puncture has been modified: representa-

Discussion
In primary PCa patients, accurate staging and histologic grading are crucial for guiding treatment decisions. PSMA PET outperforms other imaging techniques for localizing the foci of prostate cancer tumors [15,16]. The potential of PSMA-PET for PCa puncture has been evaluated in a few studies. PET and ultrasound images have been fused in some studies, proving that PET-ultrasound fusion targeted biopsy can effectively detect csPCa [17,18]. In other studies, traditional transrectal puncture has been modified: representa-

Discussion
In primary PCa patients, accurate staging and histologic grading are crucial for guiding treatment decisions. PSMA PET outperforms other imaging techniques for localizing the foci of prostate cancer tumors [15,16]. The potential of PSMA-PET for PCa puncture has been Pharmaceuticals 2022, 15, 1252 9 of 15 evaluated in a few studies. PET and ultrasound images have been fused in some studies, proving that PET-ultrasound fusion targeted biopsy can effectively detect csPCa [17,18]. In other studies, traditional transrectal puncture has been modified: representative procedures are a PSMA target biopsy using a single-puncture percutaneous transgluteal approach and a PSMA-PET-guided robotic-assisted transgluteal prostatic biopsy [19,20]. Robot-assisted radical prostatectomy (RARP) specimens and PCa localization on PET have been found to be highly consistent; thus, a PSMA-guided targeted-prostate biopsy can potentially be an accurate biopsy [19]. However, real-time puncture has been achieved in a few studies. Iodine-labeled radiopharmaceuticals are promising due to a low molecular weight and fast pharmacokinetics [21][22][23][24]. Therefore, we synthesized a novel high-affinity radiopharmaceutical, 125 I-PSMA-7 to guide real-time puncture.
We used a liquid-phase synthesis method to prepare 125 I-PSMA-7 with a purity of up to 99.6%. The stability of 125 I-PSMA-7 in solutions of PBS and 5% BSA was found to be higher than 95% over seven days. The NAALADase method was used to determine the in vitro affinity of 125 I-PSMA-7. The affinity was on the micromolar scale, indicating that 125 I-PSMA-7 has a high affinity for PSMA recombinant protein. The results of in vitro cell uptake tests showed a high uptake of 125 I-PSMA-7 by 22Rv1 cells, where an upward trend was maintained for over 24 h. Blocked 22Rv1 cells took up little 125 I-PSMA-7. The uptake of 125 I-PSMA-7 by PC3 cells was also very low. This result demonstrates the targeting specificity of 125 I-PSMA-7. The internalization rate of 22Rv1 cells was found to be as high as 88% at 24 h, indicating that the agent can be stored in cells for a long time. The short clearance half-life of 125 I-PSMA-7 of 29.24 min is due to the low molecular weight of the agent. In in vitro distribution experiments on 22Rv1 tumor-bearing mice, tumor uptake remained high up to the seventh day of the experiment. Other organs had a low uptake of 125 I-PSMA-7, with almost negligible radioactivity on Day 7, whereas the kidney uptake remained high because of the presence of PSMA receptors on the proximal convoluted tubules of the kidney. The results of the tumor-muscle ratio in live animals were also very impressive and increased with time, suggesting that tumors and muscle tissue can be effectively distinguished based on radioactivity. In animal imaging experiments, after injection of 125 I-PSMA-7 via the tail vein, 22Rv1 tumor-bearing mice showed rapid tumor targeting at 60 min, and tumor uptake was significantly reduced after blockade with ZJ43. By contrast, the tumor uptake of 125 I-PSMA-7 was very low in PC3 tumor-bearing mice in the control group. The tumor could be clearly imaged over 7 days. Based on these results, 125 I-PSMA-7 showed a high degree of targeting specificity against prostate cancer in vivo in animals, which was consistent with the results of in vitro experiments. Acute toxicity tests were conducted to assess the safety of the 125 I-PSMA-7 probe: no tissue or organ damage in mice was observed after a single high-dose injection, demonstrating the low toxicity and safety of 125 I-PSMA-7. In conclusion, 125 I-PSMA-7 has a high specificity and can be used for targeted puncture in prostate cancer.
As PSMA-1007 is excreted through the liver and a small fraction of PSMA-1007 is excreted through the urinary system, PSMA-1007 was used in a previous study to guide the puncture. This metabolic pathway rules out the interference of urine in the bladder and can more effectively identify the primary focus of prostate cancer. 18 F was used to detect radioactivity in tissues instead of 68 Ga because of a longer half-life [25]. However, 18 F has a half-life of just under 109.8 min. Due to the limitation of the half-life, the target tissue of a patient can only be punctured approximately 3 h after examination to detect radioactivity, making patient cooperation imperative. Thus, this method for guiding patient puncture is not practical. Therefore, we recommend the use of 125 I, which has a longer half-life, to ensure that the radionuclide is excreted through the urinary system as late as possible after puncture to avoid contamination; thus, 125 I with a long half-life can provide good guidance for both first or repeat punctures. In addition, the energy of 125 I is low, which can ensure the radiation safety of the operator. Additional γ-counter for measuring radioactivity counts is available in the operating room. All these ensure the feasibility of 125 I-PSMA-7 for prostate-cancer puncture. 125 I-PSMA-7 cannot be used for SPECT imaging because of its low energy, but we will replace the radionuclide later and label it with 123 I for clinical application. Iodine-123 has a short half-life and narrow puncture time window after drug injection, while iodine-125 has a long half-life and can be punctured at any time point 34 days after drug injection, so it is more likely to achieve puncture with iodine-125.

General Materials
All chemicals, solvents, and reagents (analytical grade) used for synthesis and analysis were purchased from Maclin Biochemical Technology Co., Ltd. (Shanghai, China). The materials used for cell experiments, such as 24-well plates and the RPMI-1640 medium, were obtained from Gibco Life Technologies (Grand Island, NY, USA). BALB/c male nude mice were purchased from Charles River Laboratories (Beijing, China).

Cell Lines and Mouse Models
The 22Rv1 and PC3 cell lines were obtained from GuYan Biotech Co., Ltd. (Shanghai, China), cultured in the RPMI-1640 medium (Gibco Life Technologies, Grand Island, NY, USA), and supplemented with 1% P/S and 10% fetal bovine serum (FBS). 22Rv1 and PC3 were cultivated in an incubator containing 5% CO 2 at 37 • C. All cells were grown to 80% to 90% confluence before trypsinization.
All animal experiments conformed to the protocol approved by the Animal Care and Use Committee of the PLA General Hospital. BALB/c male nude mice were purchased from Charles River Laboratories (Beijing, China). The mice were approximately 3-4 weeks old and weighed 13-15 g. Approximately 5 × 10 6 cells were implanted into the right shoulder of each mouse. Mice were imaged or used in biodistribution assays when the tumor volume reached 200-300 mm 3 . The same implantation method was used for PC3 as for 22Rv1.

Synthesis of Compound 1
Dimethyl 5-bromoisophthalate (549 mg, 2.0 mM) was dissolved in 1,4-dioxane by the successive addition of hexabutyldistannane (2325 mg, 4.0 mM, 2 equiv) and bistriphenylphosphine palladium dichloride (1405 mg, 0.2 mM, 0.1 equiv), followed by reaction overnight at 120 • C under nitrogen protection. The insoluble product was removed by suction and filtration. The crude material was obtained using a rotary evaporator. The crude material was separated and purified by silica column chromatography. Compound 1 (565 mg) was obtained as a colorless liquid in a 58.5% yield. 1

Synthesis of Compound 2
Compound 1 (329 mg, 0.68 mM) was dissolved in a methanol solution; lithium hydroxide aqueous solution was added to the resulting mixture, which was reacted overnight at 50 • C. After the reaction was complete, 1 M hydrochloric acid solution was used to neutralize the reaction solution. Methanol was removed using a rotary evaporator. Product 2 (213 mg) was obtained by extraction and filtration as a white solid in a 68.7% yield. 1

Synthesis of Compound 4
Di-tert-butyl (((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl) carbamoyl)-L-glutamate and triethylamine were added to a solution of 3 (284 mg, 0.38 mmol) in CH 2 Cl 2 solution. The resulting mixture was stirred for 8 h at room temperature, and the solvent was then evaporated off using a rotary evaporator. The crude product was purified on a silica column to afford 417 mg (80.7%) of 4 as a white solid. 1

Synthesis of Compound 5
Compound 5 was prepared using the same method used to prepare Compound 3 and was obtained as an off-white solid in a 90.3% yield. 1 H NMR (600 MHz, chloroform-d) data:

Synthesis of Compound 7
A volume of 3 mL of TFA was added to a solution of 6 (101 mg, 0.082 mM) in CH 2 Cl 2 (7 mL) under stirring. The mixture was reacted at room temperature overnight, the solvent was evaporated off using a rotary evaporator, and ethyl acetate was then added to precipitate a white solid. This solid was washed three times with ethyl acetate, washed three times with petroleum ether, and dried to obtain 72 mg of Compound 7 as a white solid in a 98.2% yield. 1

Determination of Radiochemical Purity by HPLC and MS Analysis
The purity of 125 I-PSMA-7 was determined by radio-HPLC. The flow rate was set to 1 mL/min, and the mobile phases were 75% solvent A (H 2 O 2 ) and 25% solvent B (acetonitrile + 0.1%TFA). Mass spectrometry was used to analyze and determine the molecular weight of the product. The mixture was homogenized in a microplate reader, transferred to a shaker incubator, and incubated at 37 • C for 1 h. Then, 100 µL of OPA was added to each well, which was then protected from light for 3 min. The fluorescence intensity of the resulting mixture was measured at Ex/Em = 350/450 nm. The Cheng-Prusoff equation was used to convert the IC 50 values to Ki values.

In Vitro Stability
An 125 I-PSMA-7 solution was added to 5% phosphate-buffered saline (PBS) or a bovine serum albumin (BSA) solution. After 1 h, 6 h, 12 h, 1 d, 3 d, 5 d, and 7 d at room temperature or 37 • C, radio-HPLC was used to identify the radiochemical purity of 125 I-PSMA-7. The experiment was repeated three times.

Partition Coefficient
A volume of 100 µL of an 125 I-PSMA-7 solution was added to a mixture of 2.9 mL of PBS (0.1 M, pH = 7.4) and 3 mL of n-octanol. The mixture was swirled in a vortex mixer for 2 min at room temperature and then centrifuged at 3000 rpm for 5 min. The resulting mixture was allowed to stand for an adequate period of time to enable separation into layers, a 1000 mL × 3 solution was taken from the top layer, and a 100 µL × 3 solution was taken from the bottom layer. A γ-counter was used to detect the radioactivity of the solutions. The experiment was repeated three times.

Acute Toxicity Test
The PSMA probe was injected through the tail veins of ICR male mice that were 3-4 weeks old and weighed 20-25 g. Based on the specific activity of the iodine compounds of 8.14 × 10 13 Bq/mM, we calculated the injection dose to be 10 mg/100 kg which was 1000 times compared to the normal dose. The same volume of normal saline was injected into the control group. Following injection, the diet, activity, mental state, skin, and other indicators of the two groups were observed daily. On the 14th day after injection, the mice were sacrificed, and the main organs were removed to observe whether there were significant differences between the two groups in terms of color, shape, texture, and other aspects. The organs were stained with HE.

In Vitro Cellular Experiments
22Rv1 cells and PC3 cells were inoculated in 24-well plates and placed in an incubator containing 5% CO 2 at 37 • C overnight. The RPMI-1640 medium in the well plates was changed 2 h before performing the cell uptake experiment. First, the solution was removed and rinsed with PBS. Second, 125 I-PSMA-7 (1.48 × 10 4 Bq, 100 µL) was added to each well, supplemented with RPMI-1640 medium to 1 mL and placed in an incubator containing 5% CO 2 at 37 • C. Then, the solution was abandoned at 5, 10, 15, and 30 min and 1, 2, 4, 8, 12, and 24 h, respectively. The cells were washed with 0.5 mL of ice PBS buffer containing 0.2% BSA twice. An NaOH (0.5 M, 0.5 mL) solution was used to lyse the cells in the pore plate, which were then collected in an EP tube. The radioactivity of the cells in each well was measured by a γ-counter and corrected to (%IA)/10 6 . Five parallel wells were set at each time point, and the experiment was repeated three times. 22Rv1 cells were added to an 125 I-PSMA-7 solution (1.48 × 10 4 Bq, 100 µL) and incubated for 1 h and 24 h. The cells were washed twice in PBS containing 0.2% BSA; then glycine-HCl (pH = 2.9) buffer solution was added to the cells, which were incubated at 37 • C for 5 min. The cells were washed with ice PBS buffer containing 0.2% BSA, and the radioactivity of the cells was counted using a γ-counter.
A blocking experiment was performed by treating 22Rv1 and PC3 cells with ZJ43 [28,29] for 15 min, followed by the same steps as for the cell uptake experiment.

Ex Vivo Biodistribution and Imaging
Male ICR mice that were 5-6 weeks old and weighed 32-34 g were injected with a 125 I-PSMA-7 solution (1.48 × 10 6 Bq, 50 µL) through the tail vein. Blood samples were collected from the tail (5 µL × 3) at 1, 3,5,7,9,15,20,40,60, and 120 min after injection. The radioactive activity of the blood was measured by a γ-counter. The time-blood radioactivity data of mice were analyzed using a two-compartment model in the Pharmacokinetic software WinNonlin (Delaware, America. 2018-06.). Then, the clearance half-life parameters of the radiopharmaceuticals in the mice were obtained.
To evaluate the distribution of 125 I-PSMA-7 in tumors and major tissues and organs, biological distribution experiments were carried out in 22Rv1 tumor-bearing mice. Twentyone 22Rv1 tumor-bearing mice were randomly divided into seven groups (n = 3/group). An 125 I-PSMA-7 solution (5.5 × 10 5 Bq, 100µL) was injected into the mice through the tail vein at 1 h, 6 h, 12 h, 1 d, 3 d, 5 d, and 7 d. Three mice were sacrificed at a prescribed time, and the tumors and major tissues and organs (blood, urine, heart, lung, liver, spleen, kidney, bladder, tumor, brain, muscle, bone, salivary-glands, and small intestine) were collected. The tissues and organs were washed and dried with normal saline and weighed using an electronic balance. The radioactive count of the tissues and organs was measured by a γ-counter, and the corresponding radioactive value was calculated after time attenuation correction (%ID/g). In addition, we used γ-counter to measure cpm per milligram of tumor and muscle on days 4 and 7.
22Rv1 tumor-bearing mice and PC3 tumor-bearing mice were used in SPECT (3D whole-body scan, MMP919 collimator) imaging studies. SPECT/CT images were reconstructed by HiSPECT software (California, America. 2016-03.) and analyzed by Vivoquant 2.5 software (Osaka, Japanese. 2015-11.). Three 22Rv1 tumor-bearing mice were randomly selected for injection of 125 I-PSMA-7 (1.665 × 10 6 Bq, 100 µL). The mice were placed in an anesthesia box at a prescribed time, and a 3.0% isoflurane/air gas mixture was diffused into the box. The mice exhibited no voluntary activity after 5 min and were placed in a prone position on the scanning bed, during which time anesthesia was maintained using a 1.0% isoflurane/air gas mixture.
In blocking experiments, imaging was performed 60 min after coinjection of ZJ43 (50 mg/kg) with 125 I-PSMA-7 (1.665 × 10 6 Bq, 100 µL). SPECT scanning images of the PC3 tumor-bearing mice were used as negative controls. 2018-10.) were used to perform a statistical analysis, where p < 0.05 is considered to being statistically significant. Data that conformed to a normal distribution were expressed as the mean ± standard deviation, and other data were expressed using median and quartile spacing. An independent-samples T test was performed on the normally distributed data, and the homogeneity of variance and Wilcoxon rank sum tests were performed on the data inconsistent with a normal distribution.

Conclusions
In this study, we synthesized a novel radiopharmaceutical 125 I-labeled PSMA ligand. 125 I-PSMA-7 has high specificity and sensitivity for PSMA (+) PCa. SPECT/CT images demonstrated that tumors could be clearly detected and 125 I-PSMA-7 was retained for a long time. Rapid targeting and a high tumor-muscle ratio show that 125 I-PSMA-7 is a promising radiotracer that can facilitate prostate puncture. Thus, 125 I-PSMA-7 could be applied to targeted biopsy and reduce the need for saturation biopsy.
Author Contributions: X.L. and H.Z. contributed equally to this work. Corresponding authors: B.X. and J.Z.; X.L. conceptualization, methodology, software, data curation, formal analysis, writingoriginal draft. H.Z. and K.C.: software, formal analysis. Y.C. and M.C.: pharmaceutical synthesis. X.Z. and X.X.: data curation. J.Z.: writing-review and editing. B.X.: writing-review and editing, supervision. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement:
The animal study protocol was approved by the Ethics Committee of the Animal Care and Use Committee of the PLA General (protocol code 2022-576, approved on 2022-9-26).