Materials and equipment

The Gd standard solution, with a concentration of 1000 μg/ml, was procured from the Beijing Nonferrous Metals Research Institute, Beijing, China. All additional chemicals, solvents, and reagents were supplied by Sigma-Aldrich, St. Louis, MO, USA. Key equipment and biochemicals used in this study included a Germanium gallium generator from New Radiomedicine Technology Co., Ltd., Chengdu, China, and antibodies such as PSMA, γ-H2AX, Bax, Bcl-2, PCNA, and p53, which were sourced from Abcam and Cell Signaling Technology (USA). Human prostate cancer cell lines, 22Rv1 (RRID: CVCL1045) and PC-3 (RRID: CVCLE2RM), were obtained from Wuhan Pricella Biotechnology Co., Ltd., Wuhan, China. All human cell lines have been authenticated using STR profiling, and it has been confirmed that they were derived from mycoplasma-free cells. Biochemical reagents, including RPMI 1640 medium, phosphate-buffered saline, and fetal bovine serum, were supplied by Shanghai Basalmedia Technologies Co., Ltd., Shanghai, China. Analytical equipment included a 9.4 T animal MRI scanner from United Imaging Healthcare, Shanghai, China, an animal PET/CT scanner from RAYSOLUTION Healthcare Co., Ltd, Hefei, China, a TOF-ICP-MS system from TOFWERK AG, Thun, Switzerland, an ICP-MS system from Thermo Fisher Scientific, Waltham, USA, a High-Resolution Mass Spectrometry (HR-MS) system and High Performance Liquid Chromatography (HPLC) from SHIMADZU, Kyoto, Japan.

Male SPF-grade nude mice (Six-week-old), were purchased from Ziyuan Experimental Animal Technology Co., Ltd., Hangzhou, China and housed in the Animal Experiment Center of Our University. A thermal-neutron beam was produced at the Institute of Energy, Hefei Comprehensive National Science Center, using a deuterium-deuterium (D-D) neutron generator, which produces 2.5 MeV neutrons at a rate of 2.5 × 108 neutrons per second. The generator operated at an accelerator voltage of 80 kV and a current of 9.3 mA. Neutrons were moderated to thermal neutrons, with a flux of approximately 4.1–4.4 × 108 neutrons/cm2. After anesthesia, the tumor-bearing mice were fixed on a self-made 3D printed cell irradiation device. This device was designed to expose mouse tumors to neutrons as much as possible to minimize the exposure of surrounding tissues to neutron radiation. Neutron irradiation lasted for about 2–3 h.

All experimental procedures were conducted in accordance with the guidelines of the Institutional Animal Experimentation Ethics Committee of Anhui Medical University under Grant No. LLSC20232209.

Synthesis of 157Gd -DOTA-PSMA and molecular docking

The DOTA-PSMA Peptides were synthesized by Anhui Guoping Pharmaceutical Co., Ltd., Hefei, China, using standard solid-phase peptide synthesis (SPPS) techniques. We designed a DOTA-PSMA ligand in our paper, similar to PSMA-617. We design DOTA-PSMA in the form of direct chain to minimize the intake of salivary glands and other important organs.The synthesis begins with soaking 2-CL resin in a vertical reactor, followed by the coupling of Fmoc-Arg(Pbf)-OH and 1-Hydroxybenzotriazole (HOBT) under nitrogen gas. Fmoc groups are then cleaved using a 20% piperidine/DMF solution. The remaining Fmoc-protected amino acids are sequentially coupled, and DOTA is added to the C-terminus of the peptide chain. The peptide is cleaved from the resin, precipitated with anhydrous ether, and purified by HPLC, achieving a final purity of about 95%, confirming the successful synthesis of DOTA-PSMA.

The synthesis of 157Gd-DOTA-PSMA involved dissolving the DOTA-PSMA peptide in a 30% acetonitrile–water solution, followed by the addition of an excess Gd standard solution(1000ug/ml), The ratio of DOTA-PSMA to Gd solution is 1 mg: 0.3 ml. The pH of the reaction mixture was adjusted to approximately 7 using ammonium bicarbonate, and the solution was stirred overnight. The resulting 157Gd-DOTA-PSMA complex was successfully synthesized and subsequently underwent further purification. Chromatographic separation was achieved using a 20 mm × 250 mm column packed with Diagsol stationary phase (8 μm particle size), with a gradient elution ranging from 10 to 65% over a period of 45 min.

Molecular docking studies were conducted to investigate covalent interactions between the carboxyl group of DOTA and the amino group of the PSMA protein side chains, resulting in a stable covalent structure. Further molecular docking with Gd-ions was performed, and the binding free energies for both docking interactions were calculated independently to assess the stability and affinity of the synthesized complexes.

Preparation of 68Ga-DOTA-PSMA

A total of 50 μg of DOTA-PSMA peptide was dissolved in 0.5 mol/l sodium acetate buffer to create an appropriate environment for labeling. 0.1 mol/l Hydrochloric acid was utilized to elute the germanium-gallium generator and obtain the 68Ga radionuclide. The labeling reaction was performed at 95 °C in a sodium acetate buffer with a pH range of 3 to 4, and maintained for 15 min to ensure optimal binding between the 68Ga radionuclide and the DOTA.

Monitoring of Biodistribution Dynamics.

Normal mice(n = 15),22Rv1(n = 12) and PC-3(n = 12) xenograft models were administered intravenous (i.v.) injections of 68Ga-DOTA-PSMA (3.7 MBq, 100 μl) via the tail vein, three rats were included at each observation time point. Following the injections, the mice were euthanized through cervical dislocation, and their organs were carefully excised and weighed. The radioactivity of each organ was measured using a γ-counter to calculate the percentage of the injected dose per gram of tissue (%ID/g), providing insights into the distribution of the radiotracer. To gain a more comprehensive understanding of the biodistribution over time, PET/CT imaging was conducted at multiple time points after the administration of 68Ga-DOTA-PSMA. This imaging technique allowed for real-time visualization of the radiotracer's dynamic distribution, facilitating the observation of how its accumulation in both the tumor and surrounding organs changed over time.

Biodistribution analysis of 157Gd-DOTA-PSMA

The biodistribution of 157Gd-DOTA-PSMA was assessed using 9.4 T animal MRI scanner, ICP-TOF–MS imaging, and ICP-MS quantification, guided by dynamic time windows derived from previous nuclear medicine imaging and biodistribution studies. The 9.4 T animal MRI imaging employed Gd as a T1 contrast agent to evaluate T1 enhancement in 22Rv1 tumor-bearing mice (n = 3). MRI Imaging was performed both before and after the administration of 157Gd-DOTA-PSMA (13.4 mg [157Gd] / kg, 100 μl) to visualize changes in gadolinium accumulation in the tumors and various organs.

For ICP-TOF–MS imaging, tissue samples were collected from 22Rv1 xenograft models (n = 3) following the injection of 157Gd-DOTA-PSMA (13.4 mg [157Gd] / kg, 100 μl). The mice were euthanized, and their organs were carefully excised, snap-frozen, and sectioned for further analysis. To visualize the localization of Gd in the harvested tissues, a combination of matrix-assisted laser desorption ionization (MALDI) with laser ablation and ICP-TOF–MS imaging techniques was employed. For Gd quantification via ICP-MS, freeze-dried tissue samples were weighed and placed in cleaned Polytetrafluoroethylene (PTFE) digestion vessels. A mixture of 1 ml of nitric acid, 0.5 ml of hydrochloric acid, and 2 drops of hydrofluoric acid was added to each vessel. The vessels were then sealed and subjected to a microwave digestion program to ensure the complete dissolution of the samples. Following digestion, the resulting solutions were diluted with ultrapure water before being analyzed using ICP-MS for accurate quantification of Gd content.

Biological effects of Gd-NCT in vitro assays

To evaluate the effects of 157Gd-DOTA-PSMA on cell viability and radiosensitivity, we implemented a series of in vitro assays using the CCK-8 methodology and colony formation assays. 22Rv1 Cells were treated with varying concentrations (0–500 µg/ml) of 157Gd-DOTA-PSMA to establish a concentration–response relationship regarding cytotoxic effects. Following treatment, 22Rv1 cells were subjected to thermal neutron irradiation to assess the compound's ability (0–100 µg/ml) to enhance cell death and inhibit growth. During this process, 22Rv1 cells in the experimental group were incubated with 157Gd-DOTA-PSMA for 2 h before undergoing neutron irradiation. Following the incubation period, the cell culture medium was replaced with fresh medium to eliminate any potential influence of extracellular factors on the experimental results. We performed colony formation assays to quantify the long-term effects of combined treatment with 157Gd-DOTA-PSMA and neutron irradiation on 22Rv1 cell proliferation. We established both irradiated and non-irradiated control groups, further dividing them into subgroups based on treatment administration: NCT + Gd + , NCT + Gd − , NCT − Gd − , and NCT − Gd + . To investigate DNA damage resulting from the treatments, γ-H2AX immunofluorescence staining was conducted, enabling the visualization of DNA double-strand breaks in the treated 22Rv1 cells.

Biological effects of Gd -NCT in vivo experiments

The study focused on the therapeutic potential of combining 157Gd-DOTA-PSMA with neutron irradiation in xenograft models. We established both irradiated and non-irradiated control groups and divided them into subgroups based on treatment administration (NCT + Gd + , NCT + Gd − , NCT − Gd − , NCT − Gd +). In the NCT + Gd + group, 22Rv1 xenograft models (n = 3) were administered 157Gd-DOTA-PSMA (13.4 mg [157Gd] / kg, 100 μl) via tail vein injection before undergoing 2 h of targeted thermal neutron irradiation using a 3D-printed acrylic frame.

Subsequent experiments assessed biological markers, including γ-H2AX to quantify DNA double-strand breaks, Bcl-2 and Bax to evaluate the balance between anti-apoptotic and pro-apoptotic signaling, as well as p53 and PCNA as indicators of cell proliferation and growth. These markers were analyzed using Wb analysis to determine their expression levels in treated versus control groups, providing insight into the efficacy of the combined NCT and 157Gd-DOTA-PSMA treatment. Tumor sections were processed and stained using a TUNEL kit, enabling the detection of DNA fragmentation indicative of apoptosis.

Statistical analysis

Data were analyzed using SPSS 23.0 and GraphPad Prism 9.3.0. Results were expressed as mean ± standard deviation \(\left( \pm SD} \right)\). An independent sample t-test or a paired t-test was employed for data analysis. A significance level of p < 0.05 was considered statistically significant.