An Albumin-Binding PSMA Ligand with Higher Tumor Accumulation for PET Imaging of Prostate Cancer

Prostate-specific membrane antigen (PSMA) is an ideal target for the diagnosis and treatment of prostate cancer. Due to the short half-life in blood, small molecules/peptides are rapidly cleared by the circulatory system. Prolonging the half-life of PSMA probes has been considered as an effective strategy to improve the tumor detection. Herein, we reported a 64Cu-labeled PSMA tracer conjugating with maleimidopropionic acid (MPA), 64Cu-PSMA-CM, which showed an excellent ability to detect PSMA-overexpressing tumors in delayed time. Cell experiments in PSMA-positive 22Rv1 cells, human serum albumin binding affinity, and micro-PET imaging studies in 22Rv1 model were performed to investigate the albumin binding capacity and PSMA specificity. Comparisons with 64Cu-PSMA-BCH were performed to explore the influence of MPA on the biological properties. 64Cu-PSMA-CM could be quickly prepared within 30 min. The uptake of 64Cu-PSMA-CM in 22Rv1 cells increased over time and it could bind to HSA with a high protein binding ratio (67.8 ± 1.5%). When compared to 64Cu-PSMA-BCH, 64Cu-PSMA-CM demonstrated higher and prolonged accumulation in 22Rv1 tumors, contributing to high tumor-to-organ ratios. These results showed that 64Cu-PSMA-CM was PSMA specific with a higher tumor uptake, which demonstrated that MPA is an optional strategy for improving the radioactivity concentration in PSMA-expressing tumors and for developing the ligands for PSMA radioligand therapy.


In Vitro Stability
The in vitro stability of 64 Cu-PSMA-CM was tested in 5% human serum albumin (HSA) and saline at 37.0 °C. As shown in Figure 2, the radiochemical purity (RCP) of 64 Cu-PSMA-CM was over 95% after more than 48 h incubation in saline and 5% HSA, indicating that 64 Cu-PSMA-CM was quite stable and can be applied for further study.

Partition Coefficient
The partition coefficient of 64 Cu-PSMA-CM was calculated, and the log p-value was −2.25 ± 0.05, indicating that 64 Cu-PSMA-CM was highly hydrophilic.

Western Blotting and In Vitro Cell Experiments
The expression of PSMA in 22Rv1 and PC3 cells were measured by a Western blot assay. The 22Rv1 cells showed significantly higher expression level of PSMA than PC3 cells with relative values of 1.23 ± 0.07 and 0.283 ± 0.07 (p = 0.00018), which indicates the high expression of PSMA in 22Rv1 cells (Figure 3a,b).
Cell uptake experiments were performed on PSMA (+) 22Rv1 and PSMA (−) PC-3 cells, as shown in Figure 3c. The uptake of 64 Cu-PSMA-CM in 22Rv1 cells increased with

In Vitro Stability
The in vitro stability of 64 Cu-PSMA-CM was tested in 5% human serum albumin (HSA) and saline at 37.0 • C. As shown in Figure 2, the radiochemical purity (RCP) of 64 Cu-PSMA-CM was over 95% after more than 48 h incubation in saline and 5% HSA, indicating that 64 Cu-PSMA-CM was quite stable and can be applied for further study.

In Vitro Stability
The in vitro stability of 64 Cu-PSMA-CM was tested in 5% human serum albumin (HSA) and saline at 37.0 °C. As shown in Figure 2, the radiochemical purity (RCP) of 64 Cu-PSMA-CM was over 95% after more than 48 h incubation in saline and 5% HSA, indicating that 64 Cu-PSMA-CM was quite stable and can be applied for further study.

Partition Coefficient
The partition coefficient of 64 Cu-PSMA-CM was calculated, and the log p-value was −2.25 ± 0.05, indicating that 64 Cu-PSMA-CM was highly hydrophilic.

Western Blotting and In Vitro Cell Experiments
The expression of PSMA in 22Rv1 and PC3 cells were measured by a Western blot assay. The 22Rv1 cells showed significantly higher expression level of PSMA than PC3 cells with relative values of 1.23 ± 0.07 and 0.283 ± 0.07 (p = 0.00018), which indicates the high expression of PSMA in 22Rv1 cells (Figure 3a,b).
Cell uptake experiments were performed on PSMA (+) 22Rv1 and PSMA (−) PC-3 cells, as shown in Figure 3c. The uptake of 64 Cu-PSMA-CM in 22Rv1 cells increased with

Partition Coefficient
The partition coefficient of 64 Cu-PSMA-CM was calculated, and the log p-value was −2.25 ± 0.05, indicating that 64 Cu-PSMA-CM was highly hydrophilic.

Western Blotting and In Vitro Cell Experiments
The expression of PSMA in 22Rv1 and PC3 cells were measured by a Western blot assay. The 22Rv1 cells showed significantly higher expression level of PSMA than PC3 cells with relative values of 1.23 ± 0.07 and 0.283 ± 0.07 (p = 0.00018), which indicates the high expression of PSMA in 22Rv1 cells (Figure 3a,b). time, it was comparable to 64 Cu-PSMA-BCH (5.45 ± 0.42 %IA/10 6 cells vs. 6.31 ± 0.60 %IA/10 6 cells at 120 min, respectively). The uptake in 22Rv1 cells can be significantly blocked by ZJ-43 (blocked to 3.38 ± 0.04 IA%/10 6 cells, p = 0.02). However, the uptake values of 64 Cu-PSMA-CM in PC-3 cells were lower than that in 22Rv1 cells, and the uptake cannot be blocked by excess of ZJ-43 (p > 0.05) (Figure 3d).

Binding Affinity Assay
Human serum albumin binding assay was performed to test the albumin binding ability of 64 Cu-PSMA-CM. The protein binding ratio of 64 Cu-PSMA-BCH and 64 Cu-PSMA-CM were calculated as 55.9 ± 4.0% and 67.8 ± 1.5% (p = 0.003).

Binding Affinity Assay
Human serum albumin binding assay was performed to test the albumin binding ability of 64 Cu-PSMA-CM. The protein binding ratio of 64 Cu-PSMA-BCH and 64 Cu-PSMA-CM were calculated as 55.9 ± 4.0% and 67.8 ± 1.5% (p = 0.003).
The binding affinity of 64 Cu-PSMA-CM and 64 Cu-PSMA-BCH to PSMA were con-

Pharmacokinetics
The pharmacokinetics profile of 64 Cu-PSMA-CM in blood circulation was tested by injecting 3.7 MBq of 64 Cu-PSMA-CM into Balb/c male mice (n = 4). As shown in Figure 5, the blood activity-time profile of 64 Cu-PSMA-CM was: Ct = 7.21e −4.287t + 7.29e −0.176t . The half-life of the distribution phase and elimination phase were 0.16 h and 3.93 h, respectively, which significantly delayed the elimination phase compared to 64 Cu-PSMA-BCH (0.208 min) [29].

Biodistribution Studies
The biodistribution studies of 64 Cu-PSMA-CM were performed in 22Rv1-beared male mice. As shown in Figure 6a and Table S1, within 1 h, 64 Cu-PSMA-CM rapidly accumulated in the blood pool (16.95 ± 3.05 ID%/g), liver (5.34 ± 1.13 ID%/g), and kidneys (95.84 ± 3.33 %ID/g). Subsequently, the clearance in the kidneys was fast with uptake values of 8.92 ± 0.41 %ID/g at 24 h p.i. and was accompanied by the decline of radioactivity in other organs, such as heart, lung, spleen, and blood, demonstrating that

Pharmacokinetics
The pharmacokinetics profile of 64 Cu-PSMA-CM in blood circulation was tested by injecting 3.7 MBq of 64 Cu-PSMA-CM into Balb/c male mice (n = 4). As shown in Figure 5, the blood activity-time profile of 64 Cu-PSMA-CM was: Ct = 7.21e −4.287t + 7.29e −0.176t . The half-life of the distribution phase and elimination phase were 0.16 h and 3.93 h, respectively, which significantly delayed the elimination phase compared to 64 Cu-PSMA-BCH (0.208 min) [29].

Pharmacokinetics
The pharmacokinetics profile of 64 Cu-PSMA-CM in blood circulation was tested by injecting 3.7 MBq of 64 Cu-PSMA-CM into Balb/c male mice (n = 4). As shown in Figure 5, the blood activity-time profile of 64 Cu-PSMA-CM was: Ct = 7.21e −4.287t + 7.29e −0.176t . The half-life of the distribution phase and elimination phase were 0.16 h and 3.93 h, respectively, which significantly delayed the elimination phase compared to 64 Cu-PSMA-BCH (0.208 min) [29].

Biodistribution Studies
The biodistribution studies of 64 Cu-PSMA-CM were performed in 22Rv1-beared male mice. As shown in Figure 6a and Table S1, within 1 h, 64 Cu-PSMA-CM rapidly accumulated in the blood pool (16.95 ± 3.05 ID%/g), liver (5.34 ± 1.13 ID%/g), and kidneys (95.84 ± 3.33 %ID/g). Subsequently, the clearance in the kidneys was fast with uptake values of 8.92 ± 0.41 %ID/g at 24 h p.i. and was accompanied by the decline of radioactivity in other organs, such as heart, lung, spleen, and blood, demonstrating that

Biodistribution Studies
The biodistribution studies of 64 Cu-PSMA-CM were performed in 22Rv1-beared male mice. As shown in Figure 6a and Table S1, within 1 h, 64 Cu-PSMA-CM rapidly accumulated in the blood pool (16.95 ± 3.05 ID%/g), liver (5.34 ± 1.13 ID%/g), and kidneys (95.84 ± 3.33 %ID/g). Subsequently, the clearance in the kidneys was fast with uptake values of 8.92 ± 0.41 %ID/g at 24 h p.i. and was accompanied by the decline of radioactivity in other organs, such as heart, lung, spleen, and blood, demonstrating that 64 Cu-PSMA-CM was mainly excreted from the urinary system. The uptake in 22Rv1 tumor was increased within 24 h with a highest uptake value of 14.29 ± 1.44 %ID/g at 24 h p.i., and it decreased at 48 h p.i., advantageously providing a high tissue contrast at 24 h p.i. (Figure 6b).
Pharmaceuticals 2022, 15, x FOR PEER REVIEW 6 of 15 64 Cu-PSMA-CM was mainly excreted from the urinary system. The uptake in 22Rv1 tumor was increased within 24 h with a highest uptake value of 14.29 ± 1.44 %ID/g at 24 h p.i., and it decreased at 48 h p.i., advantageously providing a high tissue contrast at 24 h p.i. (Figure 6b).

Micro-PET Imaging
Micro-PET imaging of 64 Cu-PSMA-CM and a comparison with 64 Cu-PSMA-BCH were demonstrated on mice bearing 22Rv1 tumors. As shown in Figures 7a and S2a, the heart, liver, kidneys, salivary glands, and 22Rv1 tumors were observed. Kidneys had the highest uptake of 64 Cu-PSMA-CM within 12 h, followed by the heart and liver, and the radioactivity of these organs rapidly decreased over time. The accumulation in 22Rv1 tumors continuously increased until 24 h p.i. and then decreased at 48 h, which was similar to the results of previous biodistribution studies. When co-injected with excess ZJ-43 (50 μg), the uptake in tumors and kidneys significantly decreased (p < 0.001). Due to the fast clearance of 64 Cu-PSMA-CM from the nontumor organs, relatively selective tumor images become quite clear at 12 h, and good contrast images in tumors was obtained at 24 h. Generally, the tumor-to-non-target organ ratios of 64 Cu-PSMA-CM increased with time, and the excellent tumor-to-kidneys ratio (1.29 ± 0.05) and tumor-to-liver ratio (1.76 ± 0.08) were at 24 h p.i. ( Figure S3). When compared to 64 Cu-PSMA-BCH, a tracer without MPA, 64 Cu-PSMA-CM demonstrated prolonged circulation in vivo and significantly higher uptake in 22Rv1 tumor and organs, such as heart, liver, and kidneys (Figures 7a,c and S2). As previously mentioned, 64 Cu-PSMA-CM showed the highest accumulation in 22Rv1 tumor with an SUV mean value of 1.88 ± 0.04 at 24 h p.i., whereas the peak uptake of 64 Cu-PSMA-BCH was 0.84 ± 0.02 at 4 h p.i. ( Figure S2). In addition, due to the fast clearance of 64 Cu-PSMA-BCH from the kidneys, the tumor-to-kidney ratio was higher than 64 Cu-PSMA-CM, while the tumor-to-liver ratio of 64 Cu-PSMA-CM was superior to 64 Cu-PSMA-BCH ( Figure S3). Furthermore, immunohistochemical staining results indicated the positive expression of PSMA in 22Rv1 tumor tissue (Figure 7d), further confirming the targeted accumulation of the probe in 22Rv1 tumor.

Micro-PET Imaging
Micro-PET imaging of 64 Cu-PSMA-CM and a comparison with 64 Cu-PSMA-BCH were demonstrated on mice bearing 22Rv1 tumors. As shown in Figures 7a and S2a, the heart, liver, kidneys, salivary glands, and 22Rv1 tumors were observed. Kidneys had the highest uptake of 64 Cu-PSMA-CM within 12 h, followed by the heart and liver, and the radioactivity of these organs rapidly decreased over time. The accumulation in 22Rv1 tumors continuously increased until 24 h p.i. and then decreased at 48 h, which was similar to the results of previous biodistribution studies. When co-injected with excess ZJ-43 (50 µg), the uptake in tumors and kidneys significantly decreased (p < 0.001). Due to the fast clearance of 64 Cu-PSMA-CM from the nontumor organs, relatively selective tumor images become quite clear at 12 h, and good contrast images in tumors was obtained at 24 h. Generally, the tumor-to-non-target organ ratios of 64 Cu-PSMA-CM increased with time, and the excellent tumor-to-kidneys ratio (1.29 ± 0.05) and tumor-to-liver ratio (1.76 ± 0.08) were at 24 h p.i. (Figure S3). When compared to 64 Cu-PSMA-BCH, a tracer without MPA, 64 Cu-PSMA-CM demonstrated prolonged circulation in vivo and significantly higher uptake in 22Rv1 tumor and organs, such as heart, liver, and kidneys (Figures 7a,c and S2). As previously mentioned, 64 Cu-PSMA-CM showed the highest accumulation in 22Rv1 tumor with an SUV mean value of 1.88 ± 0.04 at 24 h p.i., whereas the peak uptake of 64 Cu-PSMA-BCH was 0.84 ± 0.02 at 4 h p.i. (Figure S2). In addition, due to the fast clearance of 64 Cu-PSMA-BCH from the kidneys, the tumor-to-kidney ratio was higher than 64 Cu-PSMA-CM, while the tumor-to-liver ratio of 64 Cu-PSMA-CM was superior to 64 Cu-PSMA-BCH ( Figure S3). Furthermore, immunohistochemical staining results indicated the positive expression of PSMA in 22Rv1 tumor tissue (Figure 7d), further confirming the targeted accumulation of the probe in 22Rv1 tumor.

Estimation of Radiation Dosimetry
Human organ radiation dosimetry was estimated based on the biodistribution data in mice bearing 22Rv1 tumors. As shown in Table 1, the liver received the highest absorbed dose (0.107 mGy/MBq), followed by the kidneys and gallbladder wall with absorbed doses of 0.0997 mGy/MBq and 0.0414 mGy/MBq, respectively. The effective dose

Estimation of Radiation Dosimetry
Human organ radiation dosimetry was estimated based on the biodistribution data in mice bearing 22Rv1 tumors. As shown in Table 1, the liver received the highest absorbed dose (0.107 mGy/MBq), followed by the kidneys and gallbladder wall with absorbed doses

Discussion
Due to the fast clearance in blood and particularly insufficient dose delivery to tumor of small molecule compounds 177 Lu/ 225 Ac-PSMA-617, there have been long-term efforts in developing a general strategy which can effectively prolong the compounds' half-life in vivo. Among the reported methods, the combination of pharmaceuticals and albuminbinding molecules is one of the most commonly used methods, which has high efficiency and few side effects [17]. In the past five years, many albumin-based PSMA probes have been developed, but most of the studies focused on 4-(p-iodophenyl)butyric acid and Evans blue derivatives, aiming to improve metabolic behavior in the body [22,[30][31][32]. The clinical studies of 177 Lu-EB-PSMA-617 had been performed and achieved promising results in the therapy of mCRPC [21,32]. We previously confirmed maleimidopropionic acid (MPA) modification, offering an another effective platform for binding albumin and conjugating with the PSMA tracer [27]. Here, benefiting from the moderate half-life of copper-64, we developed a 64 Cu-labeled MPA-modified PSMA tracer and evaluated the targeting capability of the PSMA-tumor. 64 Cu-PSMA-CM was prepared with high radiochemical yield, high radiochemical purity, and moderate specific activity. It was quite stable in vitro, which meets the criteria of quality control and can be used for further studies.
When compared with 64 Cu-PSMA-BCH, a PSMA probe without MPA moiety, 64 Cu-PSMA-CM showed a higher human serum albumin binding ratio and longer half-life. This indicated that the conjugation of MPA indeed enhanced the binding ability of the tracer to HSA and extended the circulation in blood, which was expected to offset the defect of the rapid clearance of small molecule inhibitors. Although the binding affinity of Cu-PSMA-CM to PSMA decreased, the Kd value was still at the level of nanomole, which was comparable with other reported PSMA probes. 64 Cu-PSMA-CM was specifically accumulated in PSMA (+) 22Rv1 cells and increased over time with the highest uptake value of 5.45 ± 0.42 IA%/10 6 cells at 120 min, which was significantly higher than that in PSMA (−) PC-3 cells (1.79 ± 0.20 IA%/10 6 cells). It can be specifically blocked to 3.38 ± 0.04 IA%/10 6 cells by PSMA inhibitor, ZJ-43, indicating the high specificity of 64 Cu-PSMA-CM to PSMA. The comparable uptake values of 64 Cu-PSMA-CM and 64 Cu-PSMA-BCH in PSMA (+) 22Rv1 cells indicated that the conjugation of MPA moiety had little effect on the specificity and binding affinity, or for the uptake in cells.
Both biodistribution and micro-PET imaging studies in 22Rv1-beared mice confirmed that 64 Cu-PSMA-CM was highly accumulated in PSMA (+) tumors and washed out from normal organs at delayed time points. This resulted in a continuous increase of tumorto-organ ratio, prospectively facilitating the observation of more tumor lesions. longer circulation in the blood resulted in longer retention of radioactivity in the kidneys and liver.
When compared with 64 Cu-PSMA-BCH, 22Rv1 tumors showed excellent permeability and retention of 64 Cu-PSMA-CM, which may improve the contrast of tumors to nontarget organs. The higher uptake in tumors and comparable uptake in cells indicated that the higher uptake in PSMA (+) positive tumors was due to the prolonged circulation of 64 Cu-PSMA-CM in the blood. In addition, the significantly higher tumor accumulation in delayed time demonstrated that the compound fulfilled the prerequisites for dosimetry in the course of therapy planning with 67 Cu. It also showed that modification with MPA is a good strategy to improve radioactivity accumulation in PSMA (+) tumors, which may effectively guide the development of 177 Lu or 225 Ac labeled tracers for PRLT.

In Vitro Stability
64 Cu-PSMA-CM was incubated in saline or 5% human serum albumin (HSA) at 37 • C and the radiochemical purity was determined by radio-HPLC at different points in time (1 h, 4 h, 12 h, 24 h, and 48 h). For the stability in 5% HSA, 15 µL of mixture was taken out and precipitated by ethanol, filtered, and the supernatant was analyzed.

Partition Coefficient
Amounts of 10 µL of 64 Cu-PSMA-CM in saline (0.37 MBq), 990 µL of phosphatebuffered saline (PBS, 0.1 M, pH 7.4), and 1mL of octanol were mixed in a tube. The mixture was vortexed for 3 min and centrifuged (3000 rpm × 5 min). Then, five samples (100 µL) from each phase were removed and the radioactivity was measured. The experiment was repeated three times. The partition coefficient value was calculated as below: P = (average of counts in octanol/average of counts in PBS), the value was expressed as log P ± SD.

Cell Culture and Animal Models
Human prostate cancer cell lines (22Rv1 and PC-3) were obtained from the Stem Cell Bank, Chinese Academy of Science. Both cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Biological Industries, Shanghai, China). The cells were cultured in a humidified incubator at 37 • C with 5% CO 2 .
Healthy male KM mice and BALB/c nude mice aged 6-8 weeks were acquired from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Healthy male KM mice were used for the pharmacokinetics experiment. About 2 × 10 6 cells of 22Rv1 in RPMI 1640 medium were subcutaneously injected into the front left of the BALB/c nude mice. When the tumors reached a size of approximately 0.5-1 cm 3 in volume, the mice were used for further animal studies including biodistribution and micro-PET imaging. All animal experiments were conducted with the approval of the Ethics Committee of Peking University Cancer Hospital.

Western Blotting and Cell Uptake
The Western blotting assay was conducted on 22Rv1 and PC3 cell lines to verify the expression of PSMA. The details are described in the supporting information.
The 22Rv1 and PC-3 cell lines were plated on 24-well plates (2 × 10 5 cells/well). After incubating for 24 h, the medium was removed and washed with PBS (phosphate buffer saline, 0.01 M). Then, 500 µL medium with 74 KBq of 64 Cu-PSMA-CM was added to the well plates and co-incubated with cells at 37 • C for 10 min, 30 min, 60 min, and 120 min. The medium was then removed and the cells were washed twice with 1 mL of cold PBS. After that, cells were lysed with 1M NaOH, and then the NaOH solution was collected for analysis. For the blocking experiment, cells were co-incubated with ZJ-43 (1 µg/well), a PSMA inhibitor, for 120 min. Five samples of 64 Cu-PSMA-CM were taken out and were thought to be standard. The result was expressed as %IA/10 6 cells. For comparison, the cell uptake experiment of 64 Cu-PSMA-BCH in 22Rv1 cells was performed.

Binding Affinity Assay
In order to test the HSA protein binding ratio, 10 µL of 64 Cu-PSMA-CM (0.37 MBq) was added to 0.2 mL of 20% HSA (n = 5) and incubated at 37 • C for 4 h. Then the protein was precipitated by 0.5 mL of ethanol, centrifuged (5000 rpm), washed by saline (2 × 0.2 mL), and centrifuged twice, then measured for the radioactivity of the precipitation and supernatant by a γ-counter. The result was presented as the percentage of precipitation counts/(precipitation counts + supernatant counts). The human serum albumin binding ability of 64 Cu-PSMA-BCH was measured with the same protocol.
The dissociation constant of 64 Cu-PSMA-CM was performed on 22Rv1 cells by adding different concentrations of 64 Cu-PSMA-CM (0.185-74 kBq/mL, 400 µL/well, 4 well/group) to the 24-well plate (2 × 10 5 cells/well). After incubation for 2 h at 37 • C, the medium was removed, the cells were washed twice with cold PBS and then lysed by 0.5 mL NaOH. The radioactivity of NaOH solution was measured and the dissociation constant was calculated using the one-site total model in Graph Pad software (Graph Pad prism 5). Similarly, the dissociation constant of 64 Cu-PSMA-CM was determined with the same protocol.

Pharmacokinetics in Blood
An amount of 3.7 MBq (250 µL) of 64 Cu-PSMA-CM was intravenously injected into Babl/c male mice (n = 4). Blood was collected from the ophthalmic artery at different time points, then the radioactivity of blood was weighed and measured by a γ-counter. An injection of 2.5 µL was taken out as standard. The results were expressed as the percentage of injected dose per gram (%ID/g).
A two-phase decay Equation (1) in Graph Pad software (Graph Pad prism 5) was used to serve as the two-compartment model which models biological process of the probe by fitting the ID%/g versus to time of 64 Cu-PMA-CM to describe blood pharmacokinetics.
where A and B are relevant constants, and α and β are rate constants which were used to calculate as ln(2)/α and ln(2)/β for the half-life of distribution phase and elimination phase, respectively.

Biodistribution
Twelve 22Rv1 tumor model mice were randomly divided into four groups and then 64 Cu-PSMA-CM (200 µL, 0.74 MBq/mouse) was injected via tail vein. After 1 h, 6 h, 24 h, and 48 h, the mice were sacrificed by cervical dislocation. Organs of interest including blood, heart, liver, spleen, lung, kidney, small intestine, large intestine, muscle, brain, and 22Rv1 tumor were collected. The organs were then weighed and measured for their radioactivity. Five samples of the 1% injection volume were taken out as a standard. The final uptake values of each organ were expressed as the percentage of injected dose per gram (%ID/g).

Micro-PET Imaging and Immunohistochemical Staining
An amount of 200 µL of 64 Cu-PSMA-CM (9.32 MBq) was injected into mice bearing 22Rv1 xenograft tumors via tail vein. For blocking, the mice were co-injected with 50 µg of ZJ-43. Imaging was performed on Super Nova PET/CT (PINGSENG, Shanghai, China). The mice were anaesthetized with 3% (v/v) isoflurane, then fixed on the bed at 4 h, 8 h, 12 h, 24 h, and 48 h p.i. Micro-PET scans were conducted with continuous 1% (v/v) isoflurane for 15 min. The images of 64 Cu-PSMA-CM were obtained at different time points and SUV mean values of regions of interest (ROIs) over the heart, kidney, muscle, liver, and tumor were collected. The ratio of tumor-to-non-target organs was then calculated at different time points. To better assess its anti-tumor efficiency in vivo, a comparison with 64 Cu-PSMA-BCH (5.55 MBq/mouse) of micro-PET imaging was performed in mice bearing 22Rv1 tumor models at 4 h p.i., 12 h p.i., and 24 h p.i. The mean standardized uptake value (SUVmean) in each organ was calculated according to following Formula (2) [7]. SUV = Activity VOI (MBq/mL)/Injected dose (MBq) × Body weight (g) After micro-PET imaging, the 22Rv1 mice were sacrificed by cervical dislocation, and the 22Rv1 tumor tissues were quickly collected and immersed in formalin solution for further immunohistochemical staining. The details are presented in the supporting information.

Estimation of Radiation Dosimetry in Human Organs
The fraction of radioactivity uptake in human tissues was calculated according to the biodistribution results in mice bearing 22Rv1 tumors. The time-activity curves of various organs and the whole body were obtained and then calculated the area under the curves (AUCs) of different organs with origin 2019b software (OriginLab, Northampton, MA, USA). The results were further analyzed with OLINDA/EXM software (version 2.2; HERMES Medical Solutions AB) to estimate the radiation dosimetry of each organ and effective dose.

Statistical Analysis
All quantified data were expressed as the mean ± SD. The significant differences of cell uptake and human serum albumin binding affinity were calculated by two-tailed, paired t-test. Quantified ROI data based on the results of micro-PET imaging were analyzed using two-way ANOVA. Statistical analysis was performed using the Microsoft Excel 2016 software program (Microsoft Corporation) or Graph Pad software (Graph Pad prism 5).
The p values less than 0.05 were considered statistically significant.

Conclusions
In this study, we successfully developed an albumin-based PSMA probe conjugated with MPA, 64 Cu-PSMA-CM. It exhibited good physicochemical and biological properties. It showed a longer half-life in blood, a high affinity to PSMA, and a higher uptake in PSMA (+) tumors-which effectively monitored the change of radioactivity accumulation of PSMA-targeted tracers in tumors. The probe offers a good strategy and was initially verified for the potential to develop radiotracers for PRLT for prostate cancer.