A Novel PSMA-Targeted Theragnostic Agent: Preclinical Validation of Dual-Modality Imaging and Targeted Radionuclide Therapy

A Novel PSMA-Targeted Theragnostic Agent: Preclinical Validation of Dual-Modality Imaging and Targeted Radionuclide Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Novel PSMA-Targeted Theragnostic Agent: Preclinical Validation of Dual-Modality Imaging and Targeted Radionuclide Therapy Fengyuan Zhang, Bohua Xu, Huiping Guo, Shuzhe Wang, Tian Qin, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8083698/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : Targeted radionuclide therapy (TRT) has remarkable potential in the diagnosis and treatment of tumors. Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein that is markedly overexpressed in prostate cancer cells, which renders it a premier target for theragnostic applications. This study sought to develop a new anti-PSMA small molecule TM-1labeled with 68 Ga or 177 Lu and assess the biodistribution via Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography Imaging (SPECT). Furthermore, the antitumor efficacy of TM-1 in tumor-bearing mice was extensively evaluated. Result : The radiotracer exhibited high radiochemical purity (RCP>95%) and stability in PBS. In vitro , a significant reduction in the uptake rate was observed between the experimental wells and block wells (0.31±0.01 vs 0.08±0.01, p<0.05), confirming PSMA-specific binding. Biodistribution data showed specific uptake in PSMA-positive tumor (9.41±3.11% ID/g for 177 Lu-TM-1 after 1h). Both PET and SPECT imaging showed rapid and sustained accumulation of the radiotracer at tumor sites (4.92±2.00% ID/g for PET and 6.56%±1.09% ID/g for SPECT after 1h), and the tumor tissues were clearly characterized. In therapeutic efficacy studies, the tumor volume of mice in the high-dose group significantly decreased compared to control (430.00±145.99 vs 1115.75±134.16 mm 3 , p<0.01). Conclusion ：A new PSMA tracer, TM-1, was successfully synthesized and radiolabeled in our study. Both in vitro and in vivo experiments demonstrated its ability to specifically target and treat PSMA-positive tumors. Targeted radionuclide therapy Tumor PSMA Small molecules PET SEPCT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Despite significant advancements in oncological diagnostic and therapeutic over recent decades, cancer continues to be one of the most critical health challenges facing humanity. The incidence and mortality of some malignant tumors are steadily increasing every year. For instance, between 2015 and 2019, the annual incidence rates increased by 0.6%–1% for breast cancer while prostate and liver cancers exhibited even steeper increases of 2%–3% per annum(1). According to the report from the National Cancer Center of China, malignant tumors are the predominant cause of mortality, accounting for 24.09% of all deaths among residents(2). In clinical practice, histopathology analysis and surgical intervention remain the gold standard for diagnostic and therapeutic. However, tumor biopsy, the cornerstone of histopathology, is fraught with limitations, including invasive risk of surgery, sampling difficulties, and the challenges in accurately assessing heterogeneous tumors(3). Therefore, substantial researches are being invested in pioneering methods of tumor diagnostic and therapeutic. In the past decades, Targeted radionuclide therapy (TRT) has emerged as a highly promising approach in oncology. This strategy utilizes radiolabeled ligands designed to bind with high specificity to receptors overexpressed on malignant cells, enabling the localized accumulation of radionuclide within tumor tissues. Such a mechanism not only enables precise tumor therapeutic but also embodies theragnostic integration. Compared to conventional radiological imaging techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI), TRT offers several distinct advantages, including high sensitivity, molecular targeting precision, and the ability for quantitative pharmacokinetic analysis(4–6). These benefits are attributed to the use of radiotracers, which can be detected at nanomolar concentrations by Positron Emission Tomography (PET) or Single-Photon Emission Computed Tomography (SPECT)(7). Furthermore, both PET and SPECT are non-invasive, whole-body imaging techniques, allowing for comprehensive assessment of radiotracer biodistribution and metabolic activity in real time(8). The radiopharmaceutical 18F-fluorodeoxyglucose (18F-FDG) performed by PET, has been widely used in the diagnosis of malignant tumors and cardiovascular inflammation(9, 10). Recent years have witnessed substantial progress in the development of novel radiotracers, using diverse targeting moieties such as monoclonal antibodies and bioactive peptides to enhance tumor specificity and therapeutic efficacy(11). 177 Lu-DOTATATE, a radiotracer based on peptide that targets the somatostatin receptor, has been approved by the U.S. Food and Drug Administration and plays an important role in therapeutic of neuroendocrine tumors(12–14). Similarly, PSMA-617, a small peptide directed against prostate-specific membrane antigen (PSMA), has revolutionized the diagnosis and therapeutic of castration-resistant prostate cancer(15). PSMA is a type II transmembrane glycoprotein which is overexpressed in prostate cancer cells(16). It promotes the oncogenic signaling pathway by activating glutamate receptors and the PI3K-AKT pathway, thereby promoting tumor progression and metastatic dissemination(17). Due to the critical role in prostate cancer pathogenesis and cell-surface accessibility, it’s regarded as the key molecular target for tumor targeted therapy. Despite the proven efficacy of 177 Lu-PSMA-617 in metastatic castration-resistant prostate cancer, its clinical impact is limited by treatment resistance due to heterogeneous PSMA expression and dose-limiting toxicities, primarily xerostomia and myelosuppression(18, 19). These limitations necessitate the development of novel PSMA-targeting agents with improved efficacy and safety profiles. In the present study, we developed a radiolabeling strategy utilizing either 68 Ga for diagnostic imaging and 177 Lu for therapeutic applications, based on TM-1, a novel small molecule targeting PSMA. Through comprehensive in vivo evaluation using LNCaP tumor-bearing models, we systematically assessed both the diagnostic efficacy and therapeutic potential of TM-1, laying the foundation for future research. Methods Material The novel small molecule TM-1 was synthesized by Shanghai Medicilon Ltd. (Shanghai, China). Unless otherwise specified, all chemical reagents used in the synthesis were purchased from Shanghai Bepharm Science & Technology Ltd. (Shanghai, China), with analytical grade purity. All reagents used in the radiolabeling procedures were purchased from Sinopharm Chemical Reagent Ltd. (Shanghai, China). Detailed information on the chemical reagents can be obtained in the Supplementary Information (Supplementary Table S1). Synthesis of Chemical Compounds TM-1 was synthesized according to the pathway outlined in Fig. 1 (see Results section). For the first step, benzyl alcohol (BnOH, 134 g, 1.24 mol) was added dropwise to a solution of L-2-Aminoadipic acid (20 g, 124 mmol) in concentrated HCl (10 mL) at room temperature. The resulting mixture was heated at 100 °C for 1 hour. The crude product was washed with diethyl ether (500 mL × 4). The white solid that precipitated was collected, further washed with diethyl ether (100 mL × 3), and dried to afford intermediate 2 (23 g, crude). Next, a solution of triphosgene (0.47 g, 1.58 mmol) in dichloromethane (DCM, 5 mL) was added to a mixture of ditert-butyl 2-isocyanatopentanedioate (1.36 g, 5.26 mmol) and triethylamine (TEA, 2.93 mL, 21.0 mmol) in DCM (15 mL) under an argon atmosphere at -78 °C. Intermediate 2 was added to this reaction mixture and reacted at room temperature for 1 hour. The reaction solution was quenched with water and extracted with dichloromethane. The residue was purified by HPLC (Acetonitrile/water: 10-90%, 0.1% TFA) to obtain intermediate 3 (1.5 g). Subsequently, 2-Tert-butyl-1,3-diisopropylisourea (1.14 g, 5.70 mmol) was added to a solution of intermediate 4 (1.5 g) in DCM (10 mL). The reaction mixture was then stirred under an argon atmosphere at 40 °C for 16 hours. Following the mentioned purification procedure, intermediate 4 was obtained (300 mg). To a solution of intermediate 4 (300 mg) in tetrahydrofuran (THF, 10 mL) was added 10% palladium on carbon (Pd/C, 110 mg) at room temperature. The reaction mixture was then stirred under a hydrogen atmosphere at room temperature for 16 h to complete the hydrogenation. The resulting solution was filtered and concentrated, yielding intermediate 5 (250 mg). Intermediate 5 (1 g) and benzyl N-(3-(phenylamino) propyl) carbamate (566 mg, 1.99 mmol) were dissolved in DCM (10 mL), followed by the addition of pyridine (1.57 g, 19.9 mmol). The reaction mixture, after the addition of phosphorus oxychloride (0.890 g, 5.82 mmol) below 0 °C, was maintained at 0 °C for 0.5 hours. Stirring was then continued at room temperature overnight. After extraction and washing with saturated aqueous NaHCO₃, the crude product was purified via C18 chromatography (Acetonitrile/water: 70/30), yielding the intermediate 6 (0.6 g). A mixture of intermediate 6 (600 mg, 0.78 mmol) and 10% Pd/C (346 mg) in isopropanol (IPA, 5 mL) was stirred under a hydrogen atmosphere at room temperature for 16 hours, until hydrogenation was complete. followed by concentration to obtain intermediate 7 (600 mg). Finally, a mixture of intermediate 7 and the 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligand was dissolved in acetonitrile. To this solution were added N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH, 318 mg, 1.14 mmol) and N-Methylimidazole (NMI, 186 mg, 2.25 mmol). The reaction mixture was stirred at room temperature overnight to obtain intermediate 8. After concentration, intermediate 8 was purified by RP-HPLC (Acetonitrile/water: 10-90%, 0.1% TFA). The resulting product was subsequently treated with trifluoroacetic acid at room temperature for 6 hours to remove the Boc-protecting group, affording the final compound TM-1, as a white solid (MW: 992.4, purity: 97.58%). Additional details, including the synthetic procedure for the DOTA ligand and quality control of TM-1, are provided in the Supporting Information (Figure S1, Figure S2-S5). Cell Culture and Animal Model The LNCaP prostate cancer cell line was purchased from Nanjing Cobioer Biosciences Co., Ltd (Nanjing, China). Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 15% (v/v) fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% (v/v) Penicillin-Streptomycin solution (Thermo Fisher Scientific, Waltham, MA, USA). The cultivation condition was set at 37°C with 5% CO 2 . All animal experiments and animal care procedures were approved by the Committee for the Care and Use of Laboratory Animals, Innostar Nantong (Approval IACUC number, IACUC-2024-m-293). B-NDG mice (6–8 weeks of age) were purchased from Zhejiang Vital River Laboratory Animal Technology Co. Ltd. To construct subcutaneous tumor model, 1×10 7 LNCaP cells were suspended in the serum-free medium and then injected into the right lateral abdomen of each mouse. Tumor growth was monitored regularly, and when the tumor reached a size of 200~300 mm 3 , these mice were used for subsequent experiments. For the euthanasia of experimental mice utilized in biodistribution experiment, carbon dioxide (CO₂) asphyxiation was employed in accordance with established guidelines for humane endpoints. This method was selected due to constraints imposed by the experimental conditions. Before dissection, the euthanasia chamber was pre-filled with CO₂ for 30 minutes to ensure uniform gas distribution and stabilization of the internal atmosphere. For PER/MR and SPECT/CT experiments, mice were anesthetized with 3% (v/v) isoflurane for 30 minutes, followed by maintenance of anesthesia throughout the imaging session. After the experiment, euthanasia was performed using the same method as described above. Radio-labeling TM-1 (0.029mg) was dissolved in the ultrapure water (20 μL) to achieve a final concentration of 1 mM. To this solution, we added 600 μL of freshly eluted [68Ga]GaCl 3 (300-400 MBq) obtained from a [68Ge]Ge/[68Ga]Ga generator (Eckert & Ziegler, Berlin, Germany) and 600 μL of 1 M sodium acetate buffer (pH 4.5). After incubation for 15 min at 95°C and 600 rpm, the solution was diluted in 2 mL of ultrapure water and purified on a Sep-Pak C18 column (Waters, Milford, MA, USA) pre-equilibrated with 5 ml anhydrous ethanol and 10 ml ultrapure water. The radiolabeled productions were eluted by 2 mL of ethanol/water (1/1, v/v) mixture and collected in four sequential fractions of 0.5 ml. The first fraction exhibited the highest radio activity which would be used for subsequent analysis and experiments. For 177 Lu radiolabeling, TM-1 were incubated with [ 177 Lu] LuCl 3 (185-259 MBq) and 0.5M sodium acetate for 15 minutes at 90°C. Upon the mixture was cooled to room temperature, the radiochemical yield was quantitatively analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). No further purification was required due to the high efficiency of the radiolabeling process. Quality Control The radiochemical purity (RCP) of the radiolabeled compounds was assessed by RP-HPLC and instantaneous thin layer chromatography (ITLC). RP-HPLC analysis (Waters ACQUITY I-Class) was performed on a C18 column (ACE Excel 3 C18, 150×4.6mm, Avantor, Radnor, USA) with the following gradient (A: 0.1% trifluoroacetic acid in water; B: 0.1% trifluoroacetic acid in acetonitrile): 0−2 min, 10% B; 2−12 min, 10%−70% B; 12−15 min, 70%−10% B; at a flow rate of 1 mL/min. ITLC was performed on silica-gel impregnated glass fiber sheets (ITLC-SG, Agilent) with 0.1M ethylenediaminetetraacetic acid (EDTA) solution as the mobile phase. All samples were filtered by 0.22μm membrane filters. To evaluate in vitro stability, the radiolabeled compounds were incubated in phosphate-buffered saline (PBS, pH 7.4) and mouse plasma at room temperature (RT). Aliquots were withdrawn at 1, 2, and 3 h time points, and the RCP was determined by RP-HPLC analysis under identical chromatographic conditions as described above. Cell Binding Assay LNCaP cells were seeded in 24-well plates at a density of 1 × 10⁵ cells per well and incubated overnight to allow attachment. Before experiment, cells were starved in serum-free medium for 2 h. 177 Lu-TM-1 were diluted to a final concentration of 10 nM in 0.5 mL RPMI 1640 medium and incubated with cells for 2h at 37°C under 5% CO 2 . In order to assess the non-specific binding, 1 mM 2-(phosphonomethyl) pentanedioic acid (2-PMPA), a specific PSMA inhibitor, was added into the Block well, which was 100-fold excess of radiolabeled TM-1. After incubation, cells were washed for two times with 1mL ice-cold PBS to remove unbound tracer and subsequently incubated with 1ml 1M glycine hydrochloride buffer (pH 2.8) for 10 minutes at 4°C to remove membrane-bound activity. After an additional PBS wash, internalized radioactivity was recovered by incubating cells with 0.5 mL NaOH (1 M) for 10 min at room temperature to facilitate cell detachment and lysis. After cells detached, washed one last time. All washing solution and supernatant of each step after acid hydrolysis were collected separately and determined by a gamma counter (3470 Automatic Gamma Counter; PerkinElmer). All experiments were performed in triplicate and data was expressed as percentage of radio activity. PET/MR Imaging PET/MR imaging were performed on a Micro PET/CT system (PET/MR 3T, Bruker BioSpin, Billerica, MA, USA). Before the injection, the mice were anesthetized with 3% (v/v) isoflurane for 30 mins, followed by maintenance of anesthesia throughout the imaging session. 68 Ga-TM-1 (1mci/ml, 3.7 MBq) was injected into the Cell-derived xenograft (CDX) B-NDG mice via the tail vein. To assess binding specificity, a blocking control group (n=3) were pretreated with the 2-PMPA in advance. The scanning program was set to dynamical scanning for 2h, and static scanning for 3h. MR imaging was performed simultaneously for anatomical co-registration and. The data was analyzed by PMOD (version 4.1; PMOD Technologies LLC, Switzerland) software and expressed as percentage of injection dose per gram of tissue (%ID/g). SPECT Imaging Micro-SPECT/CT was performed on the U-SPECT-II/CT (MILabs, Utrecht, The Netherlands). LNCaP tumor-bearing mice were scanned for 10 min using the 1.0 mm diameter pinhole mouse high sensitivity collimator tube. While SPECT acquisition, whole-body CT scans were obtained for anatomical co-registration, utilizing the following parameters: 55 kVp tube voltage, 615 μA current, and 160 μm spatial resolution. SPECT data were reconstructed by the MILabs Reconstruction Software (version 10.02). Image analysis was performed by PMOD software for quantitative assessment of radiotracer biodistribution. In vitro Biodistribution Study LNCaP subcutaneous tumor bearing mice were randomly divided into 5 groups (n=6 per group) and received a tail vein injection of 177 Lu-TM-1. After injection, mice were euthanized at 1, 4, 24, 48 and 72 h. Immediately following euthanasia, comprehensive tissue sampling was performed, including mice plasma, urine, and major organs. All collected samples were weighed and measured for radioactivity by a gamma counter. Radiotracer uptake was quantified as percentage of injected dose per gram of tissue (%ID/g), with correction of radioactive decay. In Vivo Pharmacodynamic Study Forty LNCaP tumor bearing mice were randomly divided into four groups (n=8 per group): Low-dose group, Medium-dose group, High-dose group and blank group. The specific dosage for each group is detailed in Table 1. The body weight, tumor volume of the mice was monitored weekly. The survival time of each mouse was recorded until the end of the study. Table 1. Drug Injection of Tumor-Bearing Mice Tumor model Group Radioactive dose(mci/unit) N umber of Animals LNCaP Low-dose 0.5 8 male Medium-dose 1 8 male High-dose 1.5 8 male blank NA 8 male Statistical analysis All statistical analyses were performed using Prism version 9.5 (GraphPad Software). All data were presented as mean ± standard deviation. Multiple t-tests confirmed the statistical significance of %ID/g between groups, with a p-value<0.05 indicating statistical significance. The original data and supplementary images are available in the supplementary information. Results Chemical Synthesis of TM-1 The novel small molecular TM-1 was successfully prepared via a multi-step synthetic sequence as illustrated in Fig 1. The sequence commenced with the acid-catalyzed esterification of L-2-Aminoadipic acid with BnOH to obtain intermediate 2. Subsequent triphosgene-mediated reaction with di-tert-butyl 2-isocyanatopentanedioate introduced a urea linkage via nucleophilic addition, yielded intermediate 3. To temporarily mask the carboxylic acid functionality, intermediate 3 was converted to its tert-butyl ester employing 2-tert-butyl-1,3-diisopropylisourea. This was followed by a palladium-catalyzed hydro genation step to cleave the benzyl protecting group, revealing the free acid. The synthesis continued with the introduction of the aniline-bearing side chain. Intermediate 5 with carboxylic acid was subjected to amide coupling with Benzyl N-(3-(phenylamino)propyl)carbamate under POCl₃ and pyridine conditions, delivering intermediate 6. Another hydrogenation removed Carbobenzoxy group, revealing a free amine in intermediate 7. To conjugate the amine intermediate with the DOTA ligand, amide coupling was performed using TCFH/NMI, followed by global deprotection with TFA to cleave the tert-butyl groups, affording the final product TM-1. The non-radioactive reference complexes, natGa-TM-1 and natLu-TM-1, were prepared via metal chelation under mild acidic conditions and were subjected to LC/MS quality control (Figure S6-S13). Radio Synthesis and Quality Control As illustrated in the Fig 2, the radiolabeled TM-1 were analyzed by RP-HPLC and ITLC. The conditions for radioactive labeling are shown in the Fig 2a and 2b. The chemical identity of the radiolabeled products was confirmed by RP-HPLC analysis through comparing with non-radioactive TM-1 (Fig 2c). After purification, both radiotracers exhibited excellent RCP more than 95% in RP-HPLC. The retention times of 68 Ga-TM-1 and 177 Lu-TM-1 were 6.651 min and 6.255 min, respectively (Fig 2d and 2e). Furthermore, comparative HPLC analysis performed via separate injections confirmed that the TM-1 radiolabeling product exhibited a retention time identical to its non-radioactive references, with natGa-TM-1 at 6.617 min and natLu-TM-1 at 6.203 min (Figure S14). The results confirmed identical retention times (natGa-TM-1: 6.617 min, natLu-TM-1: 6.203 min). The stability profiles of the radiolabeled compounds were evaluated by incubating with PBS and murine plasma for 1, 2 and 3 h at 37°C to simulate in vivo environments. Aliquots were analyzed at each timepoints using the previously established RP-HPLC method. As shown in the Fig 2f, 68 Ga-TM-1 and 177 Lu-TM-1 proved to be stable in PBS throughout the 3 h observation period, with RCP values exceeding 95%. Plasma stability studies revealed similar result, with RCP>95% at all timepoints, indicating sufficient resistance to enzymatic degradation and protein binding. Cell Characterization To evaluate the binding affinity and specificity of TM-1 for PSMA antigen, we conducted competitive cell binding assays on PSMA-positive LNCaP cells. Due to considerations of radioactive decay, 177 Lu-TM-1 was utilized in this experiment to ensure reliable quantification. The results showed that the binding of 177 Lu-TM-1 was receptor-mediated. There was a significant reduction in the uptake rate of block wells compared to the experimental wells (0.31±0.01 vs 0.08±0.01, p<0.001) (Fig 2g). Of the total radioactivity detected in each well, distinct subcellular distribution patterns were observed that 71.91% was attributed to the membrane-bound fraction in the experimental wells, whereas 58.07% in the block wells. Additional details, including a representative image (Figure S15) is available in the Supplementary Material. ITLC analysis The results of ITLC indicated that the radiolabeled products migrated to distinct positions compared to free radioactivity, as shown in Fig 3, which further proved the remarkable RCP of the radiolabeled products. These results collectively confirmed the successful formation of stable radiotracers with minimal impurities. PET/MR Imaging In order to investigate the tumor-targeting capability of TM-1, we used PET/MR to measure the tissue distribution and metabolism of radioactive tracers in vivo . The tissue distribution of 68 Ga-TM-1 in normal mice (C57BL/6J) can be found in the supplementary file (Figure S16). The LNCaP tumor bearing mice (n=6) were randomly divided into 2 groups, and separately injected with 68 Ga-TM-1 (Group1), 68 Ga-TM-1+ 2-PMPA (Group2). As demonstrated in Fig 4a and 4b, PET/MR whole-body imaging revealed distinct biodistribution patterns between the groups. After 1 h of injection, Group1 exhibited significant radioactive tracer accumulation in the tumor sites (7.66 ± 3.05 %ID/g), which made tumor tissues can be clearly characterized. In contrast, the group co-administered with 2-PMPA showed significantly reduced tumor uptake (3.27 ± 0.80 %ID/g), while a significant difference was observed in standardized uptake values (SUV) between two groups (1.42 ± 0.48 for Group1 vs 0.59 ± 0.09 for Group2, p<0.05), according to Fig 4c, confirming the specificity of TM-1 for PSMA receptor. The uptake of the heart (23.73 ± 5.26% ID/g), liver (14.45 ± 4.99% ID/g), and lungs (11.85 ± 1.86% ID/g) reaches its peak 1 min after injection, followed by swift clearance. Radioactivity gradually accumulated in spleen during the first 20 minutes (4.24 to 7.11 %ID/g) with subsequent rapid metabolic clearance. Prolonged tumor retention was observed, with uptake values (7.05 ± 2.88 %ID/g at 3 hours) surpassing all other organs except the bladder. For further details, please refer to the supplementary information: whole-body organ radioactive uptake is shown in Figure S17, and the SUV and AUC analyses are shown in Figures S18-S20. Based on the region of interest analysis using PMOD software, as shown in Fig 4d, the tumor-to-muscle (T/M) ratio in Group 1 was 28.70±20.78 at 3h post-injection, whereas in Group 2, the T/M ratio was significantly lower at 2.78±1.36 (**, p < 0.01, analyzed by Two-way ANOVA). For the systemic T/N ratios for both groups of animals are provided in the Figure S21 and S22. Fig 4e and 4f present the whole-body radioactive distribution in the two animal groups. Except for the kidney, no significant uptake in other organs was observed. In addition, there were no statistically difference in the uptake of radiotracer among other organs. Due to the renal metabolic pathway of animals, the highest uptake was observed in the kidneys, followed by tumors. Notably, static scanning at 3h post-injection confirmed persistent tumor retention in Group 1, with detectable radioactive uptake maintained in tumor tissue and remarkable tumor-to-background contrast. Overall, 68 Ga-TM-1 exhibits high target specificity, prolonged tumor retention and excellent diagnostic potential with high tumor-to-background ratio. SPECT/CT imaging To longitudinally profile the tissue distribution of TM-1, we conducted serial Micro-SPECT/CT imaging at 1, 4, 24, 48, 72, and 96 hours post-injection. The distribution profile of ¹⁷⁷Lu-TM-1 was found to be similar to that of ⁶⁸Ga-TM-1 (Fig 5a). Representative SPECT images of ¹⁷⁷Lu-PSMA-617 are displayed in Fig 5b. After 1h of injection, the radioactive uptake of tumors peaked at 6.56±1.09 % ID/g and subsequently declined over time. The uptake rate of tissues such as liver (0.73±0.19% ID/g), lungs (0.96±0.18% ID/g), etc. was very low except for the kidneys (4.67±1.52% ID/g). Notably, significant uptake was still observed at 48 hours (1.93% ID/g) and 96 hours (1.13% ID/g), demonstrating the prolonged retention of TM-1 in tumor tissues. In a word, TM-1 demonstrated remarkable targeting abilities and sustained retention in PSMA-expressing tumors. As shown in Fig 5c and 5d, 177 Lu-PSMA-617 and 177 Lu-TM-1 exhibited comparable radioactive tissue distribution profiles. However, PSMA-617 demonstrated higher radioactive uptake in tumors, with 3.71±0.95 % ID/g at 72 h and 2.71±0.43% ID/g at 96 h. In contrast, TM-1 showed more rapid metabolism clearance, as evidenced by the absence of visible radioactive accumulation in the bladder at 4 h. As shown in Fig 6, Tumor-to-background ratios after 1h of injection were 18.89 (tumor to muscle) and 17.12 (tumor to bone), suggesting excellent target specificity of TM-1. Further SPECT data analysis is provided in Figures S23-S25. In vitro Biodistribution The biodistribution data of 177 Lu-TM-1 in LNCaP tumor-bearing mice are summarized in the Table 2. The results of radio-distribution were consistent with PET/MR and SPECT/CT imaging. After 1h of injection, the highest radioactive uptake was observed in the kidneys (13.37 ± 3.93 %ID/g), as expected, followed by tumor tissue (9.41 ± 3.11 ID%/g). In contrast, the radioactivity of other organs was relatively negligible (<1 ID%/g). Notably, after 4 h of injection, renal uptake had precipitously declined by over 50%, while tumor tissue demonstrated remarkably sustained retention. Moreover, a slower clearance rate in tumor tissue compared to other organs was observed. This divergent clearance pattern became even more pronounced at the 72 h timepoint, with tumors maintaining radioactivity (1.75 ± 0.52 %ID/g), contrast to complete clearance in all non-target organs (below detection limit). The sustained tumor accumulation (>1.5 %ID/g for 72 h) exceeds the minimum therapeutic threshold or lutetium-177, strongly supporting its potential in tumor therapy. In Vivo Pharmacodynamic Study The changes in body weight and tumor volume across treatment groups are illustrated in the Fig 7. Compared with the blank group (20.61±0.53 g), there was no significant difference in body weight among the low- (20.96±1.54 g), medium- (20.97±1.45 g), high-dose group (20.18±0.93 g). At day 31 post-treatment, compared with the blank group, the tumor volume of mice in the low and medium dose groups showed no significant reduction. In contrast, the tumor volume of mice in the high-dose group decreased moderately (415.00±145.99 vs 1166.75±434.16 mm 3 ; **, p<0.01), representing a 61.5% reduction in tumor size. On the other hand, the positive control group injected with 177 Lu-PSMA-617 via tail vein showed more significant tumor growth inhibition (107±61 mm 3 vs 1166.75±434.16 mm 3 ; ****, P<0.0001) Table 2. Biodistribution Data of 177 Lu-TM-1 i Organ 177 Lu-TM-1 (0.2 mci/ml, 0.01ml) 1 h 4 h 24 h 48 h 72 h brain 0.03 ± 0.02 0.01 ± 0.00 0.01 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 heart 0.12 ± 0.05 0.01 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 liver 0.23 ± 0.14 0.06 ± 0.01 0.03 ± 0.00 0.03 ± 0.01 0.02 ± 0.00 lung 0.33 ± 0.11 0.04 ± 0.02 0.02 ± 0.00 0.01 ± 0.01 0.00 ± 0.00 kidney 13.37 ± 3.93 0.95 ± 0.41 0.14 ± 0.04 0.11 ± 0.04 0.09 ± 0.00 muscle 0.12 ± 0.06 0.01 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 spleen 0.61 ± 0.36 0.04 ± 0.03 0.01 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 bone 0.19 ± 0.13 0.03 ± 0.04 0.00 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 pancreas 0.17 ± 0.08 0.01 ± 0.01 0.00 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 stomach 0.16 ± 0.03 0.03 ± 0.02 0.02 ± 0.01 0.01 ± 0.01 0.00 ± 0.00 tumor 9.41 ± 3.11 6.41 ± 1.45 2.59 ± 0.91 1.70 ± 0.50 1.75 ± 0.52 i. Data are expressed as mean %ID/g ± SD (n=6) Discussion Prostate cancer is a significant contributor to male mortality worldwide. The diagnosis of prostate cancer typically involves a combination of methods, including tissue biopsy, MRI, and prostate-specific antigen testing. However, the inherent heterogeneity of prostate cancer significantly limits the diagnostic efficacy of these conventional methods, particularly in patients with advanced, or metastatic lesions(20). Traditional tracers, such as 18 F-FDG, are also limited in the early stages of tumor metastasis(21). Consequently, the development of an alternative targeted tracer for prostate cancer has great potential for improving diagnosis and treatment. In this study, we established a method for radiolabeling TM-1 and conducted quality control of radiolabeled products by RP-HPLC and ITLC. The result showed that the radiolabeled product exhibited excellent radiochemical purity and stability, with no detectable radiolytic byproducts. The discrepancy of retention times between 68 Ga-TM-1 and 177 Lu-TM-1 chromatograms can be attributed to the change in molecular polarity induced by radiolabeling, which subsequently affected the interaction with the stationary phase in RP-HPLC. Secondly, we confirmed the specific binding ability of radioactive tracers to PSMA-positive LNCaP cells in vitro . In this experiment, we introduced the experimental step of acid hydrolysis of the cell membrane, due to the expression of PSMA on the cell surface. The detected radioactivity was further distinguished between membrane-bound and intracellular radioactivity through acid hydrolysis, thereby further analyzing the specific binding of the radiotracer. Thirdly, we performed in vivo imaging of xenograft mice using PET/MR and SPECT/CT. As anticipated, high specific tumor uptake was observed in imaging of mice bearing PSMA-positive LNCaP xenografts. Consistent with previous findings, the highest radiation dose was found in the kidneys and bladder, due to the metabolism of radiotracers in vivo (22, 23). For this reason, we conducted the SPECT/CT imaging and biodistribution analysis time points. After 4h of injection, a significant reduction in radioactivity was observed in the kidneys and bladder (0.52 ± 0.03% ID/g), which was surpassed by tumor tissues (4.61±1.07% ID/g). In PET/MR imaging, compared with the block group, significant changes in tumor imaging and SUV were observed (1.42 ± 0.48 for Group1 vs 0.59 ± 0.09 for Group2, p<0.05). In addition, tumor uptake rapidly increases within 10-20 minutes, peaked at 1.5 hour, and then slowly decreases, with a residual uptake of 7.04 ± 2.87% ID/g at the end of the scan. Finally, in pharmacodynamic experiment, a significant antitumor effect was observed at a dose of 1.5 mCi per mouse. In contrast, no significant differences were observed between lower dose groups and the blank control. Several limitations of this study are worth discussing. On one hand, this study was unable to establish a positive control group for comparison with previous research findings, such as PSMA-617 or PSMA-11. On the other hand, while the tumor imaging results were promising, the pharmacological outcomes did not fully align with our expectations, indicating room for improvement in therapeutic efficacy. In future research, we will continue to optimize experimental design and attempt to improve anti-tumor efficacy from a mechanistic perspective. Conclusion In this study, radiolabeled TM-1 were successfully synthesized and evaluated both in vitro and in vivo . The specificity and high affinity for targeting PSMA of radiotracers based on small molecule TM-1 were observed. Tumor tissues were clearly imaged by PET/MR and SPECT in tumor model. Furthermore, 177 Lu-TM-1 significantly inhibited the growth of tumor after injection of single dose. These data suggest that TM-1 is a potential powerful probe for PSMA, providing a foundation for subsequent research. Abbreviations expanded form abbreviations Targeted Radionuclide Therapy TRT Prostate-Specific Membrane Antigen PSMA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DOTA Positron Emission Tmography PET Magnetic Resonance MR Single-Photon Emission Computed Tomography SPECT Computed Tmography CT 18F-fluorodeoxyglucose 18 F-FDG benzyl alcohol BnOH dichloromethane DCM triethylamine TEA tetrahydrofuran THF Radiochemical Purity RCP Reverse Phase High-performance Liquid Chromatography RP-HPLC Instantaneous Thin Layer Chromatography ITLC Cell-derived Xenograft CDX palladium on carbon Pd/C isopropanol IPA 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid DOTA N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate TCFH N-Methylimidazole NMI phosphate-buffered saline PBS 2-(phosphonomethyl) pentanedioic acid 2-PMPA percentage of injection dose per gram of tissue %ID/g standardized uptake values SUV Room temperature RT Declarations Ethics approval and consent to participate All animal experiments and animal care procedures were approved by the Committee for the Care and Use of Laboratory Animals, Innostar Nantong (Approval IACUC number, IACUC-2024-m-293). This study does not involve human participants or human data. Consent for publication Not applicable Availability of data and materials Partial data generated or analyzed during this study are included in this published article and its supplementary information files. All datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding This work was supported by National Natural Science Foundation of China (32570936, 32170800 and 31871490), the Fundamental Research Funds for the Central Universities. Grant Nos. 22120250374）and Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai Authors' contributions JX, YQ, FZ and BX designed the study. JX and YQ administrated the study. FZ, BX, HG, SW, TQ, YL, LC, MG, YC and HG conducted information research. FZ set the animal model. FZ, TQ and MG synthesized the radiotracers. FZ performed the animal experiments. FZ and SW analyzed the data. FZ, BX, JX and YQ wrote and edited the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors thank Yifei Shao, Yangyang Fang and Ziyuan Li for valuable technical assistance in imaging experiments. Statement on the syntheses of the compounds This work involved the synthesis of three main compounds (DOTA-TM-1, natGa-TM-1 and natLu-TM-1). The identity and purity (≥95%) of all target compounds were unequivocally confirmed by a combination of ¹H NMR, RP-HPLC, and Liquid Chromatograph Mass Spectrometer (LC/MS). Purity was determined by analytical HPLC (DAD detection) with UV detection at 214 nm. References Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. 2024;74(1):12-49. Zheng RS, Zhang SW, Sun KX, Chen R, Wang SM, Li L, et al. [Cancer statistics in China, 2016]. Zhonghua zhong liu za zhi [Chinese journal of oncology]. 2023;45(3):212-20. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8083698","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":555179174,"identity":"4b966f79-bb76-4dcf-a98c-a4ecc42e183a","order_by":0,"name":"Fengyuan Zhang","email":"","orcid":"","institution":"Yangzhi Affiliated Rehabilitation Hospital of Tongji University: Shanghai Sunshine Rehabilitation Center","correspondingAuthor":false,"prefix":"","firstName":"Fengyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":555179175,"identity":"1022525c-197d-4336-985a-da86224f7393","order_by":1,"name":"Bohua 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(\u003cstrong\u003ef\u003c/strong\u003e) H2, Pd/C, IPA (\u003cstrong\u003eg\u003c/strong\u003e) TCFH, NMI, CAN, rt, 16 h (\u003cstrong\u003eh\u003c/strong\u003e) TFA, rt, 6 h\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8083698/v1/c7caaff288b69f1968c9f01e.png"},{"id":97704569,"identity":"d5f8fbf1-90a3-4ba3-8cf7-5151c354f1d2","added_by":"auto","created_at":"2025-12-08 12:49:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6471877,"visible":true,"origin":"","legend":"\u003cp\u003eRadio labeling procedure and Quantities Control of TM-1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e\u0026nbsp;68\u003c/sup\u003eGa labeling reaction \u003cstrong\u003e(b)\u003c/strong\u003e \u003csup\u003e177\u003c/sup\u003eLu labeling reaction \u003cstrong\u003e(c)\u003c/strong\u003e RP-HPLC of TM-1 at 254 nm, T\u003csub\u003eR \u003c/sub\u003e=6.403 min \u003cstrong\u003e(d)\u003c/strong\u003e Radioactive spectrum of \u003csup\u003e68\u003c/sup\u003eGa-TM-1, T\u003csub\u003eR \u003c/sub\u003e=6.651min \u003cstrong\u003e(e) \u003c/strong\u003eRadioactive spectrum of \u003csup\u003e177\u003c/sup\u003eLu-TM-1, T\u003csub\u003eR \u003c/sub\u003e=6.257min \u003cstrong\u003e(f) \u003c/strong\u003eStability of radio products\u003cstrong\u003e (g)\u003c/strong\u003e Cell uptake of \u003csup\u003e177\u003c/sup\u003eLu-TM-1 in LNCaP cell. Block with 2-PMPA. ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003ea. The raw data for Fig 2f and 2g can be found in the Additional File (Raw Quantitative Data for main Figs.xlsx), Sheet \"Fig 2f and 2g\".\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8083698/v1/2cf0dd85e3b7e328c6b7af86.png"},{"id":97704571,"identity":"4a0a710e-6ed4-4e36-b028-9931ae17f5e3","added_by":"auto","created_at":"2025-12-08 12:49:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3116229,"visible":true,"origin":"","legend":"\u003cp\u003eITLC analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e \u003csup\u003e68\u003c/sup\u003eGaCl\u003csub\u003e3\u003c/sub\u003e in 0.1M EDTA \u003cstrong\u003e(b)\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e68\u003c/sup\u003eGa-TM-1 in 0.1M EDTA \u003cstrong\u003e(c)\u003c/strong\u003e \u003csup\u003e177\u003c/sup\u003eLu in 0.1M EDTA \u003cstrong\u003e(d)\u003c/strong\u003e \u003csup\u003e177\u003c/sup\u003eLu-TM-1 in 0.1M EDTA\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8083698/v1/115bfe2f2248a309bf2d5935.png"},{"id":97895426,"identity":"d7c93ec3-5856-46b1-ac8a-7ff955dc2b9e","added_by":"auto","created_at":"2025-12-10 15:34:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21229928,"visible":true,"origin":"","legend":"\u003cp\u003eMicro-PET/MR images and biodistribution of tumors\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e PET/MR imaging of \u003csup\u003e68\u003c/sup\u003eGa-TM-1 at 1,2,3 hours, Bar:0-20 \u003cstrong\u003e(b)\u003c/strong\u003e PET/MR imaging of \u003csup\u003e68\u003c/sup\u003eGa-TM-1 (left) and \u003csup\u003e68\u003c/sup\u003eGa-TM-1 with 2-PMPA (right) at 1 hour\u003cstrong\u003e (c)\u003c/strong\u003e The SUV of tumor tissues of two groups. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 \u003cstrong\u003e(d)\u003c/strong\u003e Tumor-to-muscle ratios for two groups, after 1h of injection. **, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01\u003cstrong\u003e (e)\u003c/strong\u003e The radio uptake of Group1 (\u003csup\u003e68\u003c/sup\u003eGa-TM-1)\u003cstrong\u003e (f)\u003c/strong\u003e The radio uptake of Group2 (\u003csup\u003e68\u003c/sup\u003eGa-TM-1+ 2-PMPA)\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\n\u003c/p\u003e\n\u003cp\u003eb. \u0026nbsp;Data is presented as mean value ± SD. (n=3)\u003c/p\u003e\n\u003cp\u003ec. \u0026nbsp;The raw data for Fig 4c-f can be found in the Additional File (Raw Quantitative Data for main Figs.xlsx), Sheet Fig 4c-4f.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8083698/v1/ddd46d78630559bcdbcf2432.png"},{"id":97894550,"identity":"7a429592-bbc7-4798-892f-45b10565bca4","added_by":"auto","created_at":"2025-12-10 15:32:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16667411,"visible":true,"origin":"","legend":"\u003cp\u003eThe images of SPECT and biodistribution\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e SPECT/CT imaging of \u003csup\u003e177\u003c/sup\u003eLu-TM-1 \u003cstrong\u003e(b)\u003c/strong\u003e SPECT/CT imaging of \u003csup\u003e177\u003c/sup\u003eLu-PSMA-617\u003cstrong\u003e \u003c/strong\u003e(Bar:1-15 for 1, 4 h, Bar: 2-4 for 24, 48, 72, 96 h) (\u003cstrong\u003ec\u003c/strong\u003e) Biodistribution at 1, 4, 24, 72 and 96 h after the injection of \u003csup\u003e177\u003c/sup\u003eLu-TM-1 (\u003cstrong\u003ed\u003c/strong\u003e) Biodistribution at 1, 4, 24, 72 and 96 h after the injection of \u003csup\u003e177\u003c/sup\u003eLu-PSMA-617\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\n\u003c/p\u003e\n\u003cp\u003ed. Data is presented as mean value ± SD. (n=3)\u003c/p\u003e\n\u003cp\u003ee. The raw data for Fig 5c and 5d can be found in the Additional File (Raw Quantitative Data for main Figs.xlsx), Sheet \"Fig 5c\" and \"Fig 5d\".\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8083698/v1/b9b5edd1998eba50f7333def.png"},{"id":97893216,"identity":"cd6941b0-2b25-433f-bd67-914137dbd9de","added_by":"auto","created_at":"2025-12-10 15:28:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":586170,"visible":true,"origin":"","legend":"\u003cp\u003eRatios of Tumor/muscle and Tumor/Boneof TM-1 from SPECT/CT imaging\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003ef. The raw data for Fig 6 can be found in the Additional File (Raw Quantitative Data for main Figs.xlsx), Sheet \"Fig 6\".\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8083698/v1/7054f68493d0c18d118ec607.png"},{"id":97892826,"identity":"c3f75147-c5a7-49b8-942c-fcfe83c1aba3","added_by":"auto","created_at":"2025-12-10 15:22:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3269239,"visible":true,"origin":"","legend":"\u003cp\u003ePharmacodynamic evaluation of \u003csup\u003e177\u003c/sup\u003eLu-TM-1\u003csup\u003egh\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eChanges of tumor volume in each group. **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001\u003cstrong\u003e (b)\u003c/strong\u003e Changes of body weight in each group.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\n\u003c/p\u003e\n\u003cp\u003eg. Data is presented as mean value ± SD. (n=8)\u003c/p\u003e\n\u003cp\u003eh. The raw data for Fig 7 can be found in the Additional File (Raw Quantitative Data for main Figs.xlsx), Sheet \"Fig 7a\" and Sheet \"Fig 7b\"\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8083698/v1/7abc416f4b1d5e0a713c3c42.png"},{"id":98797876,"identity":"be3bf0df-48e9-4772-9749-87347ea2c906","added_by":"auto","created_at":"2025-12-22 14:00:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":51161735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8083698/v1/d1c8f017-5e24-4174-adad-9b88eab714d4.pdf"},{"id":97704566,"identity":"106be458-4634-4855-9641-267d6f136946","added_by":"auto","created_at":"2025-12-08 12:49:08","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":40367,"visible":true,"origin":"","legend":"","description":"","filename":"RawQuantitativeDataformainFigs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8083698/v1/0b7f1c5e43e338ecb6f4ba10.xlsx"},{"id":97704585,"identity":"cd92c559-f09c-4448-b73b-e22ea9aa82bd","added_by":"auto","created_at":"2025-12-08 12:49:08","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4970260,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigsandTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-8083698/v1/d4a06a8f934f0791bfda1cb7.docx"}],"financialInterests":"","formattedTitle":"A Novel PSMA-Targeted Theragnostic Agent: Preclinical Validation of Dual-Modality Imaging and Targeted Radionuclide Therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDespite significant advancements in oncological diagnostic and therapeutic over recent decades, cancer continues to be one of the most critical health challenges facing humanity. The incidence and mortality of some malignant tumors are steadily increasing every year. For instance, between 2015 and 2019, the annual incidence rates increased by 0.6%\u0026ndash;1% for breast cancer while prostate and liver cancers exhibited even steeper increases of 2%\u0026ndash;3% per annum(1). According to the report from the National Cancer Center of China, malignant tumors are the predominant cause of mortality, accounting for 24.09% of all deaths among residents(2). In clinical practice, histopathology analysis and surgical intervention remain the gold standard for diagnostic and therapeutic. However, tumor biopsy, the cornerstone of histopathology, is fraught with limitations, including invasive risk of surgery, sampling difficulties, and the challenges in accurately assessing heterogeneous tumors(3). Therefore, substantial researches are being invested in pioneering methods of tumor diagnostic and therapeutic.\u003c/p\u003e\u003cp\u003eIn the past decades, Targeted radionuclide therapy (TRT) has emerged as a highly promising approach in oncology. This strategy utilizes radiolabeled ligands designed to bind with high specificity to receptors overexpressed on malignant cells, enabling the localized accumulation of radionuclide within tumor tissues. Such a mechanism not only enables precise tumor therapeutic but also embodies theragnostic integration. Compared to conventional radiological imaging techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI), TRT offers several distinct advantages, including high sensitivity, molecular targeting precision, and the ability for quantitative pharmacokinetic analysis(4\u0026ndash;6). These benefits are attributed to the use of radiotracers, which can be detected at nanomolar concentrations by Positron Emission Tomography (PET) or Single-Photon Emission Computed Tomography (SPECT)(7). Furthermore, both PET and SPECT are non-invasive, whole-body imaging techniques, allowing for comprehensive assessment of radiotracer biodistribution and metabolic activity in real time(8).\u003c/p\u003e\u003cp\u003eThe radiopharmaceutical 18F-fluorodeoxyglucose (18F-FDG) performed by PET, has been widely used in the diagnosis of malignant tumors and cardiovascular inflammation(9, 10). Recent years have witnessed substantial progress in the development of novel radiotracers, using diverse targeting moieties such as monoclonal antibodies and bioactive peptides to enhance tumor specificity and therapeutic efficacy(11). \u003csup\u003e177\u003c/sup\u003eLu-DOTATATE, a radiotracer based on peptide that targets the somatostatin receptor, has been approved by the U.S. Food and Drug Administration and plays an important role in therapeutic of neuroendocrine tumors(12\u0026ndash;14). Similarly, PSMA-617, a small peptide directed against prostate-specific membrane antigen (PSMA), has revolutionized the diagnosis and therapeutic of castration-resistant prostate cancer(15).\u003c/p\u003e\u003cp\u003ePSMA is a type II transmembrane glycoprotein which is overexpressed in prostate cancer cells(16). It promotes the oncogenic signaling pathway by activating glutamate receptors and the PI3K-AKT pathway, thereby promoting tumor progression and metastatic dissemination(17). Due to the critical role in prostate cancer pathogenesis and cell-surface accessibility, it\u0026rsquo;s regarded as the key molecular target for tumor targeted therapy. Despite the proven efficacy of \u003csup\u003e177\u003c/sup\u003eLu-PSMA-617 in metastatic castration-resistant prostate cancer, its clinical impact is limited by treatment resistance due to heterogeneous PSMA expression and dose-limiting toxicities, primarily xerostomia and myelosuppression(18, 19). These limitations necessitate the development of novel PSMA-targeting agents with improved efficacy and safety profiles.\u003c/p\u003e\u003cp\u003eIn the present study, we developed a radiolabeling strategy utilizing either \u003csup\u003e68\u003c/sup\u003eGa for diagnostic imaging and \u003csup\u003e177\u003c/sup\u003eLu for therapeutic applications, based on TM-1, a novel small molecule targeting PSMA. Through comprehensive \u003cem\u003ein vivo\u003c/em\u003e evaluation using LNCaP tumor-bearing models, we systematically assessed both the diagnostic efficacy and therapeutic potential of TM-1, laying the foundation for future research.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch3\u003e\u003cstrong\u003eMaterial\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe novel small molecule TM-1 was synthesized by Shanghai Medicilon Ltd. (Shanghai, China). Unless otherwise specified, all chemical reagents used in the synthesis were purchased from Shanghai Bepharm Science \u0026amp; Technology Ltd. (Shanghai, China), with analytical grade purity. All reagents used in the radiolabeling procedures were purchased from Sinopharm Chemical Reagent Ltd. (Shanghai, China). Detailed information on the chemical reagents can be obtained in the Supplementary Information (Supplementary Table S1).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eSynthesis of Chemical Compounds\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTM-1 was synthesized according to the pathway outlined in Fig. 1 (see Results section). For the first step, benzyl alcohol (BnOH, 134 g, 1.24 mol) was added dropwise to a solution of L-2-Aminoadipic acid (20 g, 124 mmol) in concentrated HCl (10 mL) at room temperature. The resulting mixture was heated at 100 \u0026deg;C for 1 hour. The crude product was washed with diethyl ether (500 mL \u0026times; 4). The white solid that precipitated was collected, further washed with diethyl ether (100 mL \u0026times; 3), and dried to afford intermediate 2 (23 g, crude).\u003c/p\u003e\n\u003cp\u003eNext, a solution of triphosgene (0.47 g, 1.58 mmol) in dichloromethane (DCM, 5 mL) was added to a mixture of ditert-butyl 2-isocyanatopentanedioate (1.36 g, 5.26 mmol) and triethylamine (TEA, 2.93 mL, 21.0 mmol) in DCM (15 mL) under an argon atmosphere at -78 \u0026deg;C. Intermediate 2 was added to this reaction mixture and reacted at room temperature for 1 hour. The reaction solution was quenched with water and extracted with dichloromethane. The residue was purified by HPLC\u0026nbsp;(Acetonitrile/water: 10-90%, 0.1% TFA) to obtain intermediate\u0026nbsp;3 (1.5 g).\u003c/p\u003e\n\u003cp\u003eSubsequently, 2-Tert-butyl-1,3-diisopropylisourea (1.14 g, 5.70 mmol) was added to a solution of intermediate 4 (1.5 g) in DCM (10 mL). The reaction mixture was then stirred under an argon atmosphere at 40 \u0026deg;C for 16 hours. Following the mentioned purification procedure, intermediate 4 was obtained (300 mg).\u003c/p\u003e\n\u003cp\u003eTo a solution of intermediate 4 (300 mg) in tetrahydrofuran (THF, 10 mL) was added 10% palladium on carbon (Pd/C, 110 mg) at room temperature. The reaction mixture was then stirred under a hydrogen atmosphere at room temperature for 16 h to complete the hydrogenation.\u0026nbsp;The resulting solution was filtered and concentrated, yielding intermediate 5 (250 mg).\u003c/p\u003e\n\u003cp\u003eIntermediate 5 (1 g) and benzyl N-(3-(phenylamino) propyl) carbamate (566 mg, 1.99 mmol) were dissolved in DCM (10 mL), followed by the addition of pyridine (1.57 g, 19.9 mmol). The reaction mixture, after the addition of phosphorus oxychloride\u0026nbsp;(0.890 g, 5.82 mmol) below 0 \u0026deg;C, was maintained at 0 \u0026deg;C for 0.5 hours. Stirring was then continued at room temperature overnight. After extraction and washing with saturated aqueous NaHCO₃, the crude product was purified via C18 chromatography (Acetonitrile/water: 70/30), yielding the intermediate 6 (0.6 g).\u003c/p\u003e\n\u003cp\u003eA mixture of intermediate 6 (600 mg, 0.78 mmol) and 10% Pd/C (346 mg) in isopropanol (IPA, 5 mL) was stirred under a hydrogen atmosphere at room temperature for 16 hours, until hydrogenation was complete. followed by concentration to obtain intermediate 7 (600 mg).\u003c/p\u003e\n\u003cp\u003eFinally, a mixture of intermediate 7 and the 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligand was dissolved in acetonitrile. To this solution were added N,N,N\u0026apos;,N\u0026apos;-tetramethylchloroformamidinium hexafluorophosphate (TCFH, 318 mg, 1.14 mmol) and N-Methylimidazole (NMI, 186 mg, 2.25 mmol). The reaction mixture was stirred at room temperature overnight to obtain intermediate 8. After concentration, intermediate 8 was purified by RP-HPLC (Acetonitrile/water: 10-90%, 0.1% TFA). The resulting product was subsequently treated with trifluoroacetic acid at room temperature for 6 hours to remove the Boc-protecting group, affording the final compound TM-1, as a white solid (MW: 992.4, purity: 97.58%). Additional details, including the synthetic procedure for the DOTA ligand and quality control of TM-1, are provided in the Supporting Information (Figure S1, Figure S2-S5).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eCell Culture and Animal Model\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe LNCaP prostate cancer cell line was purchased from Nanjing Cobioer Biosciences Co., Ltd (Nanjing, China). Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 15%\u0026nbsp;(v/v) fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% (v/v) Penicillin-Streptomycin solution (Thermo Fisher Scientific, Waltham, MA, USA). The cultivation condition was set at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eAll animal experiments and animal care procedures were approved by the Committee for the Care and Use of Laboratory Animals, Innostar Nantong (Approval IACUC number, IACUC-2024-m-293). B-NDG mice (6\u0026ndash;8 weeks of age) were purchased from Zhejiang Vital River Laboratory Animal Technology Co. Ltd. To construct subcutaneous tumor model, 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e LNCaP cells were suspended in the serum-free medium and then injected into the right lateral abdomen of each mouse. Tumor growth was monitored regularly,\u0026nbsp;and when the tumor reached a size of 200~300 mm\u003csup\u003e3\u003c/sup\u003e, these mice were used for subsequent experiments. For the euthanasia of experimental mice utilized in biodistribution experiment, carbon dioxide (CO₂) asphyxiation was employed in accordance with established guidelines for humane endpoints. This method was selected due to constraints imposed by the experimental conditions. Before dissection, the euthanasia chamber was pre-filled with CO₂ for 30 minutes to ensure uniform gas distribution and stabilization of the internal atmosphere. For PER/MR and SPECT/CT experiments, mice were anesthetized with 3% (v/v) isoflurane for 30 minutes, followed by maintenance of anesthesia throughout the imaging session. After the experiment, euthanasia was performed using the same method as described above.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eRadio-labeling\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTM-1 (0.029mg) was dissolved in the ultrapure water (20 \u0026mu;L) to achieve a final concentration of 1 mM. To this solution, we added 600 \u0026mu;L of freshly eluted [68Ga]GaCl\u003csub\u003e3\u003c/sub\u003e (300-400 MBq) obtained from a [68Ge]Ge/[68Ga]Ga generator (Eckert \u0026amp; Ziegler, Berlin, Germany) and 600 \u0026mu;L of 1 M sodium acetate buffer (pH 4.5). After incubation for 15 min at 95\u0026deg;C and 600 rpm, the solution was diluted in 2 mL of ultrapure water and purified on a Sep-Pak C18 column (Waters, Milford, MA, USA) pre-equilibrated with 5 ml anhydrous ethanol and 10 ml ultrapure water. The radiolabeled productions were eluted by 2 mL of ethanol/water (1/1, v/v) mixture and collected in four sequential fractions of 0.5 ml. The first fraction exhibited the highest radio activity which would be used for subsequent analysis and experiments.\u003c/p\u003e\n\u003cp\u003eFor \u003csup\u003e177\u003c/sup\u003eLu radiolabeling, TM-1 were incubated with [\u003csup\u003e177\u003c/sup\u003eLu] LuCl\u003csub\u003e3\u0026nbsp;\u003c/sub\u003e(185-259 MBq) and 0.5M sodium acetate for 15 minutes at 90\u0026deg;C. Upon the mixture was cooled to room temperature, the radiochemical yield was quantitatively analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). No further purification was required due to the high efficiency of the radiolabeling process.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eQuality Control\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe radiochemical purity (RCP) of the radiolabeled compounds was assessed by RP-HPLC and instantaneous thin layer chromatography (ITLC). RP-HPLC analysis (Waters ACQUITY I-Class) was performed on a C18 column (ACE Excel 3 C18, 150\u0026times;4.6mm,\u0026nbsp;Avantor, Radnor, USA) with the following gradient (A: 0.1% trifluoroacetic acid in water; B: 0.1% trifluoroacetic acid in acetonitrile): 0\u0026minus;2 min, 10% B; 2\u0026minus;12 min, 10%\u0026minus;70% B; 12\u0026minus;15 min, 70%\u0026minus;10% B; at a flow rate of 1 mL/min. ITLC was performed on silica-gel impregnated glass fiber sheets (ITLC-SG, Agilent) with 0.1M ethylenediaminetetraacetic acid (EDTA) solution as the mobile phase. All samples were filtered by 0.22\u0026mu;m membrane filters.\u003c/p\u003e\n\u003cp\u003eTo evaluate \u003cem\u003ein vitro\u003c/em\u003e stability, the radiolabeled compounds were incubated in phosphate-buffered saline (PBS, pH 7.4) and mouse plasma at room temperature (RT). Aliquots were withdrawn at 1, 2, and 3 h time points, and the RCP was determined by RP-HPLC analysis under identical chromatographic conditions as described above.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eCell Binding Assay\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eLNCaP cells were seeded in 24-well plates at a density of 1 \u0026times; 10⁵ cells per well and incubated overnight to allow attachment. Before experiment, cells were starved in serum-free medium for 2 h. \u003csup\u003e177\u003c/sup\u003eLu-TM-1 were diluted to a final concentration of 10 nM in 0.5 mL RPMI 1640 medium and incubated with cells for 2h at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. In order to assess the non-specific binding, 1 mM 2-(phosphonomethyl) pentanedioic acid (2-PMPA), a specific PSMA inhibitor, was added into the Block well, which was 100-fold excess of radiolabeled TM-1. After incubation, cells were washed for two times with 1mL ice-cold PBS to remove unbound tracer and subsequently incubated with 1ml 1M glycine hydrochloride buffer (pH 2.8) for 10 minutes at 4\u0026deg;C to remove membrane-bound activity. After an additional PBS wash, internalized radioactivity was recovered by incubating cells with 0.5 mL NaOH (1 M) for 10 min at room temperature to facilitate cell detachment and lysis. After cells detached, washed one last time. All washing solution and supernatant of each step after acid hydrolysis were collected separately and determined by a gamma counter (3470 Automatic Gamma Counter; PerkinElmer). All experiments were performed in triplicate and data was expressed as percentage of radio activity.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003ePET/MR Imaging\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003ePET/MR imaging were performed on a Micro PET/CT system (PET/MR 3T, Bruker BioSpin, Billerica, MA, USA). Before the injection, the mice were anesthetized with 3% (v/v) isoflurane for 30 mins, followed by maintenance of anesthesia throughout the imaging session. \u003csup\u003e68\u003c/sup\u003eGa-TM-1 (1mci/ml, 3.7 MBq) was injected into the Cell-derived xenograft (CDX) B-NDG mice via the tail vein. To assess binding specificity, a blocking control group (n=3) were pretreated with the 2-PMPA in advance. The scanning program was set to dynamical scanning for 2h, and static scanning for 3h. MR imaging was performed simultaneously for anatomical co-registration and. The data was analyzed by PMOD (version 4.1; PMOD Technologies LLC, Switzerland)\u0026nbsp;software and expressed as percentage of injection dose per gram of tissue (%ID/g).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eSPECT Imaging\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eMicro-SPECT/CT was performed on the U-SPECT-II/CT (MILabs, Utrecht, The Netherlands). LNCaP tumor-bearing mice were scanned for 10 min using the 1.0 mm diameter pinhole mouse high sensitivity collimator tube. While SPECT acquisition, whole-body CT scans were obtained for anatomical co-registration, utilizing the following parameters: 55 kVp tube voltage, 615 \u0026mu;A current, and 160 \u0026mu;m spatial resolution. SPECT data were reconstructed by the MILabs Reconstruction Software (version 10.02). Image analysis was performed by PMOD software for quantitative assessment of radiotracer biodistribution.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Biodistribution Study\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eLNCaP subcutaneous tumor bearing mice were randomly divided into 5 groups (n=6 per group) and received a tail vein injection of \u003csup\u003e177\u003c/sup\u003eLu-TM-1. After injection, mice were euthanized at 1, 4, 24, 48 and 72 h.\u0026nbsp;Immediately following euthanasia, comprehensive tissue sampling was performed, including mice plasma, urine, and major organs. All collected samples were weighed and measured for radioactivity by a gamma counter. Radiotracer uptake was quantified as percentage of injected dose per gram of tissue (%ID/g), with correction of radioactive decay.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e\u003cem\u003eIn Vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Pharmacodynamic Study\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eForty LNCaP tumor bearing mice were randomly divided into four groups (n=8 per group): Low-dose group, Medium-dose group, High-dose group and blank\u0026nbsp;group. The specific dosage for each group is detailed in Table 1. The body weight, tumor volume of the mice was monitored weekly. The survival time of each mouse was recorded until the end of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eDrug Injection of Tumor-Bearing Mice\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor model\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRadioactive dose(mci/unit)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003cstrong\u003eumber of Animals\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 104px;\"\u003e\n \u003cp\u003eLNCaP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eLow-dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e8 male\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eMedium-dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e8 male\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eHigh-dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e8 male\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eblank\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e8 male\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch3\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/h3\u003e\n\u003ch3\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll statistical analyses were performed using Prism version 9.5 (GraphPad Software). All data were presented as mean \u0026plusmn; standard deviation. Multiple t-tests confirmed the statistical significance of %ID/g between groups, with a p-value\u0026lt;0.05 indicating statistical significance. The original data and supplementary images are available in the supplementary information.\u003c/p\u003e"},{"header":"Results","content":"\u003ch3\u003e\u003cstrong\u003eChemical Synthesis of TM-1\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe novel small molecular TM-1 was successfully prepared via a multi-step synthetic sequence as illustrated in Fig 1. The sequence commenced with the acid-catalyzed esterification of L-2-Aminoadipic acid with BnOH to obtain intermediate 2. Subsequent triphosgene-mediated reaction with di-tert-butyl 2-isocyanatopentanedioate introduced a urea linkage via nucleophilic addition, yielded intermediate 3. To temporarily mask the carboxylic acid functionality, intermediate 3 was converted to its tert-butyl ester employing 2-tert-butyl-1,3-diisopropylisourea. This was followed by a palladium-catalyzed hydro genation step to cleave the benzyl protecting group, revealing the free acid. The synthesis continued with the introduction of the aniline-bearing side chain. Intermediate 5 with carboxylic acid was subjected to amide coupling with Benzyl N-(3-(phenylamino)propyl)carbamate under POCl₃ and pyridine conditions, delivering intermediate 6. Another hydrogenation removed Carbobenzoxy group, revealing a free amine in intermediate 7. To conjugate the amine intermediate with the DOTA ligand, amide coupling was performed using TCFH/NMI, followed by global deprotection with TFA to cleave the tert-butyl groups, affording the final product TM-1. The non-radioactive reference complexes, natGa-TM-1 and natLu-TM-1, were prepared via metal chelation under mild acidic conditions and were subjected to LC/MS quality control (Figure S6-S13).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eRadio Synthesis and Quality Control\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAs illustrated in the Fig 2, the radiolabeled TM-1 were analyzed by RP-HPLC and ITLC. The conditions for radioactive labeling are shown in the Fig 2a and 2b.\u003c/p\u003e\n\u003cp\u003eThe chemical identity of the radiolabeled products was confirmed by RP-HPLC analysis through comparing with non-radioactive TM-1 (Fig 2c). After purification, both radiotracers exhibited excellent RCP more than 95% in RP-HPLC. The retention times of \u003csup\u003e68\u003c/sup\u003eGa-TM-1 and \u003csup\u003e177\u003c/sup\u003eLu-TM-1 were 6.651 min and 6.255 min, respectively (Fig 2d and 2e). Furthermore, comparative HPLC analysis performed via separate injections confirmed that the TM-1 radiolabeling product exhibited a retention time identical to its non-radioactive references, with natGa-TM-1 at 6.617 min and natLu-TM-1 at 6.203 min (Figure S14). The results confirmed identical retention times (natGa-TM-1: 6.617 min, natLu-TM-1: 6.203 min). The stability profiles of the radiolabeled compounds were evaluated by incubating with PBS and murine plasma for 1, 2 and 3 h at 37\u0026deg;C to simulate \u003cem\u003ein vivo\u003c/em\u003e environments. Aliquots were analyzed at each timepoints using the previously established RP-HPLC method. As shown in the Fig 2f, \u003csup\u003e68\u003c/sup\u003eGa-TM-1 and \u003csup\u003e177\u003c/sup\u003eLu-TM-1 proved to be stable in PBS throughout the 3 h observation period, with RCP values exceeding 95%. Plasma stability studies revealed similar result, with RCP\u0026gt;95% at all timepoints, indicating sufficient resistance to enzymatic degradation and protein binding.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eCell Characterization\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTo evaluate the binding affinity and specificity of TM-1 for PSMA antigen, we conducted competitive cell binding assays on PSMA-positive LNCaP cells. Due to considerations of radioactive decay, \u003csup\u003e177\u003c/sup\u003eLu-TM-1 was utilized in this experiment to ensure reliable quantification. The results showed that the binding of \u003csup\u003e177\u003c/sup\u003eLu-TM-1 was receptor-mediated. There was a significant reduction in the uptake rate of block wells compared to the experimental wells (0.31\u0026plusmn;0.01 vs 0.08\u0026plusmn;0.01, p\u0026lt;0.001) (Fig 2g). Of the total radioactivity detected in each well, distinct subcellular distribution patterns were observed that 71.91% was attributed to the membrane-bound fraction in the experimental wells, whereas 58.07% in the block wells.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAdditional details, including a representative image (Figure S15) is available in the Supplementary Material.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eITLC analysis\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe results of ITLC indicated that the radiolabeled products migrated to distinct positions compared to free radioactivity, as shown in Fig 3, which further proved the remarkable RCP of the radiolabeled products. These results collectively confirmed the successful formation of stable radiotracers with minimal impurities.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003ePET/MR Imaging\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eIn order to investigate the tumor-targeting capability of TM-1, we used PET/MR to measure the tissue distribution and metabolism of radioactive tracers \u003cem\u003ein vivo\u003c/em\u003e. The tissue distribution of \u003csup\u003e68\u003c/sup\u003eGa-TM-1 in normal mice (C57BL/6J) can be found in the supplementary file (Figure S16).\u003c/p\u003e\n\u003cp\u003eThe LNCaP tumor bearing mice (n=6) were randomly divided into 2 groups, and separately injected with \u003csup\u003e68\u003c/sup\u003eGa-TM-1 (Group1), \u003csup\u003e68\u003c/sup\u003eGa-TM-1+ 2-PMPA (Group2). As demonstrated in Fig 4a and 4b, PET/MR whole-body imaging revealed distinct biodistribution patterns between the groups. After 1 h of injection, Group1 exhibited significant radioactive tracer accumulation in the tumor sites (7.66 \u0026plusmn; 3.05 %ID/g), which made tumor tissues can be clearly characterized. In contrast, the group co-administered with 2-PMPA showed significantly reduced tumor uptake (3.27 \u0026plusmn; 0.80 %ID/g), while a significant difference was observed in standardized uptake values (SUV) between two groups (1.42 \u0026plusmn; 0.48 for Group1 vs 0.59 \u0026plusmn; 0.09 for Group2, p\u0026lt;0.05), according to Fig 4c, confirming the specificity of TM-1 for PSMA receptor. The uptake of the heart (23.73 \u0026plusmn; 5.26% ID/g), liver (14.45 \u0026plusmn; 4.99% ID/g), and lungs (11.85 \u0026plusmn; 1.86% ID/g) reaches its peak 1 min after injection, followed by swift clearance. Radioactivity gradually accumulated in spleen during the first 20 minutes (4.24 to 7.11 %ID/g) with subsequent rapid metabolic clearance. Prolonged tumor retention was observed, with uptake values (7.05 \u0026plusmn; 2.88 %ID/g at 3 hours) surpassing all other organs except the bladder. For further details, please refer to the supplementary information: whole-body organ radioactive uptake is shown in Figure S17, and the SUV and AUC analyses are shown in Figures S18-S20.\u003c/p\u003e\n\u003cp\u003eBased on the region of interest analysis using PMOD software, as shown in Fig 4d, the tumor-to-muscle (T/M) ratio in Group 1 was 28.70\u0026plusmn;20.78 at 3h post-injection, whereas in Group 2, the T/M ratio was significantly lower at 2.78\u0026plusmn;1.36 (**, p \u0026lt; 0.01, analyzed by Two-way ANOVA). For the systemic T/N ratios for both groups of animals are provided in the Figure S21 and S22.\u003c/p\u003e\n\u003cp\u003eFig 4e and 4f present the whole-body radioactive distribution in the two animal groups. Except for the kidney, no significant uptake in other organs was observed. In addition, there were no statistically difference in the uptake of radiotracer among other organs. Due to the renal metabolic pathway of animals, the highest uptake was observed in the kidneys, followed by tumors. Notably, static scanning at 3h post-injection confirmed persistent tumor retention in Group 1, with detectable radioactive uptake maintained in tumor tissue and remarkable tumor-to-background contrast. Overall, \u003csup\u003e68\u003c/sup\u003eGa-TM-1 exhibits high target specificity, prolonged tumor retention and excellent diagnostic potential with high tumor-to-background ratio.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eSPECT/CT imaging\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTo longitudinally profile the tissue distribution of TM-1, we conducted serial Micro-SPECT/CT imaging at 1, 4, 24, 48, 72, and 96 hours post-injection. The distribution profile of \u0026sup1;⁷⁷Lu-TM-1 was found to be similar to that of ⁶⁸Ga-TM-1 (Fig 5a). Representative SPECT images of \u0026sup1;⁷⁷Lu-PSMA-617 are displayed in Fig 5b. After 1h of injection, the radioactive uptake of tumors peaked at 6.56\u0026plusmn;1.09 % ID/g and subsequently declined over time. The uptake rate of tissues such as liver (0.73\u0026plusmn;0.19% ID/g), lungs (0.96\u0026plusmn;0.18% ID/g), etc. was very low except for the kidneys (4.67\u0026plusmn;1.52% ID/g). Notably, significant uptake was still observed at 48 hours (1.93% ID/g) and 96 hours (1.13% ID/g), demonstrating the prolonged retention of TM-1 in tumor tissues. In a word, TM-1 demonstrated remarkable targeting abilities and sustained retention in PSMA-expressing tumors. As shown in Fig 5c and 5d, \u003csup\u003e177\u003c/sup\u003eLu-PSMA-617 and \u003csup\u003e177\u003c/sup\u003eLu-TM-1 exhibited comparable radioactive tissue distribution profiles. However, PSMA-617 demonstrated higher radioactive uptake in tumors, with 3.71\u0026plusmn;0.95 % ID/g at 72 h and 2.71\u0026plusmn;0.43% ID/g\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eat 96 h. In contrast, TM-1 showed more rapid metabolism clearance, as evidenced by the absence of visible radioactive accumulation in the bladder at 4 h. As shown in Fig 6, Tumor-to-background ratios after 1h of injection were 18.89 (tumor to muscle) and 17.12 (tumor to bone), suggesting excellent target specificity of TM-1. Further SPECT data analysis is provided in Figures S23-S25.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Biodistribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biodistribution data of \u003csup\u003e177\u003c/sup\u003eLu-TM-1 in LNCaP tumor-bearing mice are summarized in the Table 2. The results of radio-distribution were consistent with PET/MR and SPECT/CT imaging. After 1h of injection, the highest radioactive uptake was observed in the kidneys (13.37 \u0026plusmn; 3.93 %ID/g), as expected, followed by tumor tissue (9.41 \u0026plusmn; 3.11 ID%/g). In contrast, the radioactivity of other organs was relatively negligible (\u0026lt;1 ID%/g). Notably, after 4 h of injection, renal uptake had precipitously declined by over 50%, while tumor tissue demonstrated remarkably sustained retention. Moreover, a slower clearance rate in tumor tissue compared to other organs was observed. This divergent clearance pattern became even more pronounced at the 72 h timepoint, with tumors maintaining radioactivity (1.75 \u0026plusmn; 0.52 %ID/g), contrast to complete clearance in all non-target organs (below detection limit). The sustained tumor accumulation (\u0026gt;1.5 %ID/g for 72 h) exceeds the minimum therapeutic threshold or lutetium-177, strongly supporting its potential in tumor therapy.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e\u003cem\u003eIn Vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Pharmacodynamic Study\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe changes in body weight and tumor volume across treatment groups are illustrated in the Fig 7. Compared with the blank group (20.61\u0026plusmn;0.53 g), there was no significant difference in body weight among the low- (20.96\u0026plusmn;1.54 g), medium- (20.97\u0026plusmn;1.45 g), high-dose group (20.18\u0026plusmn;0.93 g). At day 31 post-treatment, compared with the blank group, the tumor volume of mice in the low and medium dose groups showed no significant reduction. In contrast, the tumor volume of mice in the high-dose group decreased moderately (415.00\u0026plusmn;145.99 vs 1166.75\u0026plusmn;434.16 mm\u003csup\u003e3\u003c/sup\u003e; **, p\u0026lt;0.01), representing a 61.5% reduction in tumor size. On the other hand, the positive control group injected with \u003csup\u003e177\u003c/sup\u003eLu-PSMA-617 via tail vein showed more significant tumor growth inhibition (107\u0026plusmn;61 mm\u003csup\u003e3\u0026nbsp;\u003c/sup\u003evs 1166.75\u0026plusmn;434.16 mm\u003csup\u003e3\u003c/sup\u003e; ****, P\u0026lt;0.0001)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Biodistribution Data of \u003csup\u003e177\u003c/sup\u003eLu-TM-1\u003c/strong\u003e\u003cstrong\u003e\u003cstrong\u003e\u003csup\u003ei\u003c/sup\u003e\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrgan\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 487px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csup\u003e177\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003eLu-TM-1\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(0.2 mci/ml, 0.01ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e1 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e4 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e24 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e48 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ebrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eheart\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e0.12 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eliver\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e0.23 \u0026plusmn; 0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.06 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.02 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003elung\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e0.33 \u0026plusmn; 0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.04 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.02 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ekidney\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e13.37 \u0026plusmn; 3.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.95 \u0026plusmn; 0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.14 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.11 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.09 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emuscle\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e0.12 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003espleen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e0.61 \u0026plusmn; 0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.04 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ebone\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e0.19 \u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epancreas\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e0.17 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003estomach\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.02 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003etumor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e9.41 \u0026plusmn; 3.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e6.41 \u0026plusmn; 1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e2.59 \u0026plusmn; 0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e1.70 \u0026plusmn; 0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e1.75 \u0026plusmn; 0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ei. Data are expressed as mean %ID/g \u0026plusmn; SD (n=6)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eProstate cancer is a significant contributor to male mortality worldwide. The diagnosis of prostate cancer typically involves a combination of methods, including tissue biopsy, MRI, and prostate-specific antigen testing. However, the inherent heterogeneity of prostate cancer significantly limits the diagnostic efficacy of these conventional methods, particularly in patients with advanced, or metastatic lesions(20). Traditional tracers, such as \u003csup\u003e18\u003c/sup\u003eF-FDG, are also limited in the early stages of tumor metastasis(21). Consequently, the development of an alternative targeted tracer for prostate cancer has great potential for improving diagnosis and treatment.\u003c/p\u003e\n\u003cp\u003eIn this study, we established a method for radiolabeling TM-1 and conducted quality control of radiolabeled products by RP-HPLC and ITLC. The result showed that the radiolabeled product exhibited excellent radiochemical purity and stability, with no detectable radiolytic byproducts. The discrepancy of retention times between \u003csup\u003e68\u003c/sup\u003eGa-TM-1 and\u003csup\u003e\u0026nbsp;177\u003c/sup\u003eLu-TM-1 chromatograms can be attributed to the change in molecular polarity induced by radiolabeling, which subsequently affected the interaction with the stationary phase in RP-HPLC.\u003c/p\u003e\n\u003cp\u003eSecondly, we confirmed the specific binding ability of radioactive tracers to PSMA-positive LNCaP cells \u003cem\u003ein vitro\u003c/em\u003e. In this experiment, we introduced the experimental step of acid hydrolysis of the cell membrane, due to the expression of PSMA on the cell surface. The detected radioactivity was further distinguished between membrane-bound and intracellular radioactivity through acid hydrolysis, thereby further analyzing the specific binding of the radiotracer.\u003c/p\u003e\n\u003cp\u003eThirdly, we performed \u003cem\u003ein vivo\u003c/em\u003e imaging of xenograft mice using PET/MR and SPECT/CT. As anticipated, high specific tumor uptake was observed in imaging of mice bearing PSMA-positive LNCaP xenografts. Consistent with previous findings, the highest radiation dose was found in the kidneys and bladder, due to the metabolism of radiotracers \u003cem\u003ein vivo\u003c/em\u003e(22, 23). For this reason, we conducted the SPECT/CT imaging and biodistribution analysis time points. After 4h of injection, a significant reduction in radioactivity was observed in the kidneys and bladder (0.52 \u0026plusmn; 0.03% ID/g), which was surpassed by tumor tissues (4.61\u0026plusmn;1.07% ID/g). In PET/MR imaging, compared with the block group,\u0026nbsp;significant changes in tumor imaging and SUV were observed (1.42 \u0026plusmn; 0.48 for Group1 vs 0.59 \u0026plusmn; 0.09 for Group2, p\u0026lt;0.05). In addition, tumor uptake rapidly increases within 10-20 minutes, peaked at 1.5 hour, and then slowly decreases, with a residual uptake of 7.04 \u0026plusmn; 2.87% ID/g at the end of the scan.\u003c/p\u003e\n\u003cp\u003eFinally, in pharmacodynamic experiment, a significant antitumor effect was observed at a dose of 1.5 mCi per mouse. In contrast, no significant differences were observed between lower dose groups and the blank control.\u003c/p\u003e\n\u003cp\u003eSeveral limitations of this study are worth discussing. On one hand, this study was unable to establish a positive control group for comparison with previous research findings, such as PSMA-617 or PSMA-11. On the other hand, while the tumor imaging results were promising, the pharmacological outcomes did not fully align with our expectations, indicating room for improvement in therapeutic efficacy. In future research, we will continue to optimize experimental design and attempt to improve anti-tumor efficacy from a mechanistic perspective.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, radiolabeled TM-1 were successfully synthesized and evaluated both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The specificity and high affinity for targeting PSMA of radiotracers based on small molecule TM-1 were observed. Tumor tissues were clearly imaged by PET/MR and SPECT in tumor model. Furthermore, \u003csup\u003e177\u003c/sup\u003eLu-TM-1 significantly inhibited the growth of tumor after injection of single dose. These data suggest that TM-1 is a potential powerful probe for PSMA, providing a foundation for subsequent research.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eexpanded form\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eabbreviations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eTargeted Radionuclide Therapy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eTRT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eProstate-Specific Membrane Antigen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003ePSMA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003e1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eDOTA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003ePositron Emission Tmography\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003ePET\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eMagnetic Resonance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eMR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eSingle-Photon Emission Computed Tomography\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eSPECT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eComputed Tmography\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003e18F-fluorodeoxyglucose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003csup\u003e18\u003c/sup\u003eF-FDG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003ebenzyl alcohol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eBnOH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003edichloromethane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eDCM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003etriethylamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eTEA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003etetrahydrofuran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eTHF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eRadiochemical Purity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eRCP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eReverse Phase High-performance Liquid Chromatography\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eRP-HPLC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eInstantaneous Thin Layer Chromatography\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eITLC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eCell-derived Xenograft\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eCDX\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003epalladium on carbon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003ePd/C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eisopropanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eIPA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003e1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eDOTA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eN,N,N\u0026apos;,N\u0026apos;-tetramethylchloroformamidinium hexafluorophosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eTCFH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eN-Methylimidazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eNMI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003ephosphate-buffered saline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003e2-(phosphonomethyl) pentanedioic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003e2-PMPA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003epercentage of injection dose per gram of tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003e%ID/g\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003estandardized uptake values\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eSUV\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 425px;\"\u003e\n \u003cp\u003eRoom temperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments and animal care procedures were approved by the Committee for the Care and Use of Laboratory Animals, Innostar Nantong (Approval IACUC number, IACUC-2024-m-293). This study does not involve human participants or human data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePartial data generated or analyzed during this study are included in this published article and its supplementary information files. All datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (32570936, 32170800 and 31871490), the Fundamental Research Funds for the Central Universities. Grant Nos. 22120250374）and Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJX, YQ, FZ and BX designed the study. JX and YQ administrated the study. FZ, BX, HG, SW, TQ, YL, LC, MG, YC and HG conducted information research. FZ set the animal model. FZ, TQ and MG synthesized the radiotracers. FZ performed the animal experiments. FZ and SW analyzed the data. FZ, BX, JX and YQ wrote and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Yifei Shao, Yangyang Fang and Ziyuan Li for valuable technical assistance in imaging experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement on the syntheses of the compounds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work involved the synthesis of three main compounds (DOTA-TM-1, natGa-TM-1 and natLu-TM-1). The identity and purity (\u0026ge;95%) of all target compounds were unequivocally confirmed by a combination of \u0026sup1;H NMR, RP-HPLC, and Liquid Chromatograph Mass Spectrometer (LC/MS). Purity was determined by analytical HPLC (DAD detection) with UV detection at 214 nm.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. 2024;74(1):12-49.\u003c/li\u003e\n\u003cli\u003eZheng RS, Zhang SW, Sun KX, Chen R, Wang SM, Li L, et al. [Cancer statistics in China, 2016]. Zhonghua zhong liu za zhi [Chinese journal of oncology]. 2023;45(3):212-20.\u003c/li\u003e\n\u003cli\u003eOliva J, Anastay V, Baboudjian M, Roumiguie M, Peltier A, Dariane C, et al. Active surveillance for low-risk prostate cancer with high tumor burden at biopsy: lessons learned from a contemporary radical prostatectomy cohort. World J Urol. 2024;42(1):513.\u003c/li\u003e\n\u003cli\u003eDuan H, Iagaru A, Aparici CM. Radiotheranostics - Precision Medicine in Nuclear Medicine and Molecular Imaging. Nanotheranostics. 2022;6(1):103-17.\u003c/li\u003e\n\u003cli\u003eGomes Marin JF, Nunes RF, Coutinho AM, Zaniboni EC, Costa LB, Barbosa FG, et al. Theranostics in Nuclear Medicine: Emerging and Re-emerging Integrated Imaging and Therapies in the Era of Precision Oncology. 2020;40(6):1715-40.\u003c/li\u003e\n\u003cli\u003eVahidfar N, Eppard E, Farzanehfar S, Yordanova A, Fallahpoor M, Ahmadzadehfar H. An Impressive Approach in Nuclear Medicine: Theranostics. PET clinics. 2021;16(3):327-40.\u003c/li\u003e\n\u003cli\u003eYang Y, Wang J, Zhong Y, Tian M, Zhang H. Advances in Radionuclide-Labeled Biological Carriers for Tumor Imaging and Treatment. ACS applied materials \u0026amp; interfaces. 2025;17(3):4316-36.\u003c/li\u003e\n\u003cli\u003eFruhwirth GO, Kneilling M, de Vries IJM, Weigelin B, Srinivas M, Aarntzen E. The Potential of In Vivo Imaging for Optimization of Molecular and Cellular Anti-cancer Immunotherapies. Molecular imaging and biology. 2018;20(5):696-704.\u003c/li\u003e\n\u003cli\u003eTrivedi SJ, Bourque JM. Innovations in Imaging: (18)F-Fluorodeoxyglucose PET/CT for Assessment of Cardiovascular Infection and Inflammation. Current cardiology reports. 2024;26(12):1413-25.\u003c/li\u003e\n\u003cli\u003eSoekojo C, Cheng LTJ, Peh WM, de Mel S, Ooi M, Nai YH, et al. Clinical utility of PET/MRI in multiple myeloma. Annals of the Academy of Medicine, Singapore. 2023;52(11):590-600.\u003c/li\u003e\n\u003cli\u003eArtigas C, Mileva M, Flamen P, Karfis I. Targeted radionuclide therapy: an emerging field in solid tumours. Current opinion in oncology. 2021;33(5):493-9.\u003c/li\u003e\n\u003cli\u003eKwekkeboom DJ, Bakker WH, Kam BL, Teunissen JJ, Kooij PP, de Herder WW, et al. Treatment of patients with gastro-entero-pancreatic (GEP) tumours with the novel radiolabelled somatostatin analogue [177Lu-DOTA(0),Tyr3]octreotate. European journal of nuclear medicine and molecular imaging. 2003;30(3):417-22.\u003c/li\u003e\n\u003cli\u003eStrosberg J, El-Haddad G, Wolin E, Hendifar A, Yao J, Chasen B, et al. Phase 3 Trial of (177)Lu-Dotatate for Midgut Neuroendocrine Tumors. The New England journal of medicine. 2017;376(2):125-35.\u003c/li\u003e\n\u003cli\u003eShin Y, Moon BH, Ryoo BY, Chang HM, Kim KP, Hong YS, et al. Efficacy and Safety of Lu-177 DOTATATE Peptide Receptor Radionuclide Therapy in Patients with Unresectable or Metastatic Neuroendocrine Tumors in Korea. Targeted oncology. 2024;19(1):41-9.\u003c/li\u003e\n\u003cli\u003eSartor O, de Bono J, Chi KN, Fizazi K, Herrmann K, Rahbar K, et al. Lutetium-177-PSMA-617 for Metastatic Castration-Resistant Prostate Cancer. The New England journal of medicine. 2021;385(12):1091-103.\u003c/li\u003e\n\u003cli\u003eDavis MI, Bennett MJ, Thomas LM, Bjorkman PJ. Crystal structure of prostate-specific membrane antigen, a tumor marker and peptidase. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(17):5981-6.\u003c/li\u003e\n\u003cli\u003eBakht MK, Yamada Y, Ku SY, Venkadakrishnan VB, Korsen JA, Kalidindi TM, et al. Landscape of prostate-specific membrane antigen heterogeneity and regulation in AR-positive and AR-negative metastatic prostate cancer. Nature cancer. 2023;4(5):699-715.\u003c/li\u003e\n\u003cli\u003eKratochwil C, Giesel FL, Stefanova M, Bene\u0026scaron;ov\u0026aacute; M, Bronzel M, Afshar-Oromieh A, et al. PSMA-Targeted Radionuclide Therapy of Metastatic Castration-Resistant Prostate Cancer with \u0026lt;sup\u0026gt;177\u0026lt;/sup\u0026gt;Lu-Labeled PSMA-617. 2016;57(8):1170-6.\u003c/li\u003e\n\u003cli\u003eRahbar K, Ahmadzadehfar H, Kratochwil C, Haberkorn U, Sch\u0026auml;fers M, Essler M, et al. German Multicenter Study Investigating \u0026lt;sup\u0026gt;177\u0026lt;/sup\u0026gt;Lu-PSMA-617 Radioligand Therapy in Advanced Prostate Cancer Patients. 2017;58(1):85-90.\u003c/li\u003e\n\u003cli\u003eEapen RS, Nzenza TC, Murphy DG, Hofman MS, Cooperberg M, Lawrentschuk N. PSMA PET applications in the prostate cancer journey: from diagnosis to theranostics. World J Urol. 2019;37(7):1255-61.\u003c/li\u003e\n\u003cli\u003eToner L, Papa N, Perera M, Katelaris N, Weerakoon M, Chin K, et al. Multiparametric magnetic resonance imaging for prostate cancer-a comparative study including radical prostatectomy specimens. World J Urol. 2017;35(6):935-41.\u003c/li\u003e\n\u003cli\u003eBene\u0026scaron;ov\u0026aacute; M, Sch\u0026auml;fer M, Bauder-W\u0026uuml;st U, Afshar-Oromieh A, Kratochwil C, Mier W, et al. Preclinical Evaluation of a Tailor-Made DOTA-Conjugated PSMA Inhibitor with Optimized Linker Moiety for Imaging and Endoradiotherapy of Prostate Cancer. Journal of nuclear medicine : official publication, Society of Nuclear Medicine. 2015;56(6):914-20.\u003c/li\u003e\n\u003cli\u003eWeineisen M, Schottelius M, Simecek J, Baum RP, Yildiz A, Beykan S, et al. 68Ga- and 177Lu-Labeled PSMA I\u0026amp;T: Optimization of a PSMA-Targeted Theranostic Concept and First Proof-of-Concept Human Studies. Journal of nuclear medicine : official publication, Society of Nuclear Medicine. 2015;56(8):1169-76.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Targeted radionuclide therapy, Tumor, PSMA, Small molecules, PET, SEPCT","lastPublishedDoi":"10.21203/rs.3.rs-8083698/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8083698/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Targeted radionuclide therapy (TRT) has remarkable potential in the diagnosis and treatment of tumors. Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein that is markedly overexpressed in prostate cancer cells, which renders it a premier target for theragnostic applications. This study sought to develop a new anti-PSMA small molecule TM-1labeled with \u003csup\u003e68\u003c/sup\u003eGa or \u003csup\u003e177\u003c/sup\u003eLu and assess the biodistribution via Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography Imaging (SPECT). Furthermore, the antitumor efficacy of TM-1 in tumor-bearing mice was extensively evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult\u003c/strong\u003e: The radiotracer exhibited high radiochemical purity (RCP\u0026gt;95%) and stability in PBS. \u003cem\u003eIn vitro\u003c/em\u003e, a significant reduction in the uptake rate was observed between the experimental wells and block wells (0.31±0.01 vs 0.08±0.01, p\u0026lt;0.05), confirming PSMA-specific binding. Biodistribution data showed specific uptake in PSMA-positive tumor (9.41±3.11% ID/g for \u003csup\u003e177\u003c/sup\u003eLu-TM-1 after 1h). Both PET and SPECT imaging showed rapid and sustained accumulation of the radiotracer at tumor sites (4.92±2.00% ID/g for PET and 6.56%±1.09% ID/g for SPECT after 1h), and the tumor tissues were clearly characterized. In therapeutic efficacy studies, the tumor volume of mice in the high-dose group significantly decreased compared to control (430.00±145.99 vs 1115.75±134.16 mm\u003csup\u003e3\u003c/sup\u003e, p\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e：A new PSMA tracer, TM-1, was successfully synthesized and radiolabeled in our study. Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments demonstrated its ability to specifically target and treat PSMA-positive tumors.\u003c/p\u003e","manuscriptTitle":"A Novel PSMA-Targeted Theragnostic Agent: Preclinical Validation of Dual-Modality Imaging and Targeted Radionuclide Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 12:49:03","doi":"10.21203/rs.3.rs-8083698/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7cfc86b5-1207-41e5-8597-7b28a15b5ed1","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T12:51:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 12:49:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8083698","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8083698","identity":"rs-8083698","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}