Imageological/Structural Study regarding the Improved Pharmacokinetics by 68Ga-Labeled PEGylated PSMA Multimer in Prostate Cancer

PMSA (prostate-specific membrane antigen) is currently the most significant target for diagnosing and treating PCa (prostate cancer). Herein, we reported a series 68Ga/177Lu-labeled multimer PSMA tracer conjugating with PEG chain, including [68Ga]Ga-DOTA-(1P-PEG4), [68Ga]Ga-DOTA-(2P-PEG0), [68Ga]Ga-DOTA-(2P-PEG4), and [68Ga]Ga/[177Lu]Lu-DOTA-(2P-PEG4)2, which showed an advantage of a multivalent effect and PEGylation to achieve higher tumor accumulation and faster kidney clearance. To figure out how structural optimizations based on a PSMA multimer and PEGylation influence the probe’s tumor-targeting ability, biodistribution, and metabolism, we examined PSMA molecular probes’ affinities to PC-3 PIP (PSMA-highly-expressed PC-3 cell line), and conducted pharmacokinetics analysis, biodistribution detection, small animal PET/CT, and SPECT/CT imaging. The results showed that PEG4 and PSMA dimer optimizations enhanced the probes’ tumor-targeting ability in PC-3 PIP tumor-bearing mice models. Compared with the PSMA monomer, the PEGylated PSMA dimer reduced the elimination half-life in the blood and increased uptake in the tumor, and the biodistribution results were consistent with PET/CT imaging results. [68Ga]Ga-DOTA-(2P-PEG4)2 exhibited higher tumor-to-organ ratios. When labeled by lutetium-177, relatively high accumulation of DOTA-(2P-PEG4)2 was still detected in PC-3 PIP tumor-bearing mice models after 48 h, indicating its prolonged tumor retention time. Given the superiority in imaging, simple synthetic processes, and structural stability, DOTA-(2P-PEG4)2 is expected to be a promising tumor-targeting diagnostic molecular probe in future clinical practice.


Introduction
According to the global statistical data regarding cancers issued by WHO in 2020, PCa (prostate cancer) has become the second largest threat to male health among malignant tumors [1], and ranks second only to lung cancer. In recent years, the incidence of PCa has been increasing rapidly. People with PCa are mostly diagnosed at the middle and advanced stages, severely impacting patients' prognosis and life quality. The key to treating PCa falls on early diagnosis. PSMA is a type II integrated membrane glycoprotein consisting of 750 amino acids [2]. Due to the significantly high expression in PCa, which is 100~1000 times higher than that in normal cells, PSMA has been regarded as an ideal biomarker for PCa diagnosis and therapy [3].
Researchers have proposed several optimization strategies regarding the pharmacokinetics of PSMA radioactive ligands to improve the tumor-targeting ability and settle the insufficiency of pharmacokinetics. Strategies included extending in vivo retention time by adding albumin-binding groups ((P-iodophenyl)butyric acid, ibuprofen, Evans Blue, etc. [15,[18][19][20]) to prolong the serum half-life of drugs, or reorganizing DOTA chelating agent to enhance the binding between ligand and receptor [21], and reduced toxicity towards non-targeting organs and tissues [22]. Hence, a drug-designing scheme that endows PSMA with improved tumor-targeting ability, reduced non-targeting accumulation, accelerated clearance, and lessened toxicity to normal tissues is urgently required.
According to previous research, polypeptides possess more effective ligand-binding force and superior pharmacokinetics due to their multivalent effect, which could improve tumor-targeting ability [23,24]. PEG has been ubiquitously applied to improve pharmacokinetics [16,[25][26][27]. To prolong tumor retention time and improve pharmacokinetics, based on the multivalent effect of PSMA, a series of PSMA small molecules with different valence states were successfully designed and synthesized, followed by structural optimization by PEG 4 , namely DOTA-(1P-PEG 4 ), DOTA-(2P-PEG 0 ), DOTA-(2P-PEG 4 ), and DOTA-(2P-PEG 4 ) 2 (Scheme 1). These molecular probes' corresponding tumor-targeting ability, pharmacokinetics, and tumor retention time were verified in vitro and in vivo by 68 Ga/ 177 Lu-labeling. In our preliminary research findings, the PSMA dimer showed a higher tumor accumulation rate in PCa than the PSMA monomer. The PEG 4 chain not only improved the tumor uptake of ligands by prolonging serum half-life, but also accelerated drug metabolism and kidney clearance. Although we have not entered into the research of DOTA-(2P-PEG 4 ) 2 -based PSMA TRT (targeting radionuclide therapy), [ 177 Lu]Lu-DOTA-(2P-PEG 4 ) 2 still shows high tumor accumulation after 48 h, showing great potential for therapeutic applications in PCa.

Chemistry and Radiochemistry
68 GaCl 3+ was eluted from a 68 Ge/ 68 Ga generator with 0.05M HCl (≈200 MBq/mL). A total of 20 nmol DOTA-(1P-PEG 4 ), DOTA-(2P-PEG 0 ), DOTA-(2P-PEG 4 ), or DOTA-(2P-PEG 4 ) 2 was added into 1 mL of the eluted 68 GaCl 3 solution, followed by regulating pH to 4.5-5 with sodium acetate solution (0.5 M). The reaction system was sealed after shaking evenly and heated at 95 • C for 15 min, after which 68 Ga-labeled DOTA-PSMA-PEG compounds were produced. The radiochemical purity and in vitro stability of the reaction products (20 µL) were examined by HPLC and thin-layer chromatography with the mobile phase of 0.1 M tri-sodium-citrate to a PH of 5.0. The radiolabeled ligands remained at the origin while the free gallium-68 moved with the solvent front by radio-TLC. The same labeling method and analysis method was applied to lutetium-177.

Cell Culture
Cellular experiments in this study were performed in the PC-3 PIP cell line. PC-3 PIP, a PCa cell line with stably high-expressed PSMA [28], was a gift from Professor Chen from the National University of Singapore. Cells were cultured in ATCC 1640 medium with 10% of FBS, L-glutamine, and antibiotic (37 • C, 5% CO 2 ).

In Vitro Internalization Assay
PC-3 PIP cells were cultured in ATCC 1640 medium containing 10% FBS. When cultured to 70-80% confluency, cells were collected and seeded in a 24-well plate. After culturing overnight (37 • C, 5% CO 2 ), the culture medium was replaced by serum-free ATCC 1640 medium (serum-starved cell culture) for 0.5 h. Then nuclide-labeled compounds were added into wells (50 µL, 1 µCi/well) with gentle shakes to ensure they were well distributed. After incubating for 30 min/60 min/120 min, cells at each time point were digested by NaOH and measured by the γ-counter to calculate the uptake rate (n ≥ 3). Cells were co-incubated with glycine (pH = 2.8) for 1 min, and then the procedure above was repeated after dissolving in 1 M NaOH (procedure for internalization). Cells were incubated with PSMA inhibitor (2-PMPA) for blocking assays for 15 min before adding the labeled ligands.

Lipophilicity Determination
Nuclide-labeled ligand (20 µL, ≈50 µCi), PBS (480 µL), and saturated n-octanol (500 µL) were added in a 1 mL EP tube, followed by vortex. Then the mixture was centrifuged at a low temperature (−5 • C) for 10 min until the mixture was separated into the upper and the lower layers. Liquids in the two phases were transferred into EP tubes and measured by the γ-counter.

In Vitro Determination of Albumin Binding
The albumin-binding rate of each ligand was measured by the γ-counter [16]. 68 Galabeled PSMA ligand (≈50 µCi) was added into the human albumin solution (20 mg/mL). The mixture was incubated in a water bath (37 • C) for 30 min and 120 min, followed by ultrafiltration centrifugation for 5 min (10 kD, 10,000 rpm). The compound activities of the uncombined albumin or those retained on the filter membrane were measured by the γ counter, according to which the percentage of serum albumin binding activity (retained on the filter membrane) to total activity was calculated.
The calculation was based on the following equation: (counting of albumin retained on membrane − background counting)/(counting of albumin retained on membrane + counting of filtered liquid − 2* background counting) × 100%, and the average of three sets of data was taken as the final result.

Determination of the IC 50 Values in PC3 PIP Cells
PC-3 PIP cells were seeded in 96-well plates in triplicate and cultured to a density of 5* × 10 5 cells per well. [ 68 Ga]Ga-PSMA-11 was added into wells after serum-starved cell culture (1 µCi/well, ≈171 nmol/L), after which different PBS-diluted competitive ligands were added in wells and cultured for another hour. Then cells were washed with cold PBS twice removing the supernatant. Cells were collected and transferred to test tubes for counting by the γ-counter. IC 50 values were analyzed and confirmed by nonlinear regression on GraphPad Prism Software.

Animal Tumor Model
PC-3 PIP cells at the logarithmic phase were collected after trypsinization and resuspended to 5 × 10 7 /mL with serum-free medium after removing the supernatant. Mice (6 weeks) were subcutaneously injected with 200 µL of cell suspension, and utilized for small animal PET/CT imaging, SPECT/CT imaging, and biodistribution analysis when tumor volume was 100 mm 3 . All animal experiments were conducted with the approval of The Southwest Medical University Animal Ethics Committee.

Pharmacokinetics and Biodistribution in Mice
Healthy male SD rats were intravenously injected with four 68 Ga-labeled ligands through the caudal vein. Blood was collected and weighed through the ocular venous plexus at different times. The radioactivity of the collected blood was measured by the γ counter, and the calculated results were presented as % ID/g. The blood pharmacokinetics of four ligands were evaluated using biventricular models. Blood metabolism models were established on the basis of Ct = A e −αt + Be −βt [29] after fitting ID%/g and time.
PC-3 PIP tumor-bearing mice (n > 3) were divided into four groups and respectively injected with 0. Mice were executed at 30 min/60 min/120 min after injection. Heart, blood, lung, liver, stomach, spleen, kidney, muscle, intestine, bone, salivary gland, and tumor were collected and detected by the γ-counter. The fitted curve was calculated on the basis of standard diluted samples of the initial dosage, and then the percentage of injected dose per gram of tissue (ID%/g) was calculated.

In Vivo PET/CT and SPECT/CT Imaging
Tumor-bearing mice (n = 3) were intravenously injected with 0.1 mL (80µCi) of freshly prepared 68 Ga/ 177 Lu-labeled PSMA ligands. PET/CT imaging was performed at 0.5/1/2/5 h after injection, and SPECT/CT imaging was performed at 6/24/48 h after injection. The acquired PET/CT and SPECT/CT data were re-constructed by OSEM (ordered subsets expectation maximization).

Statistical Analysis
Relevant measurement data were proceeded by Prism 8.0.1. The student's t-test was adopted to analyze measurement data. The difference was considered statistically significant when p < 0.05.

Radiochemical Synthesis and Quality Control
A total of four kinds of PSMA-related ligands were designed and synthesized. The radiochemical purity of these four 68 Ga/ 177 Lu-labeled compounds was detected by HPLC ( Figure 1) and thin-layer chromatography ( Figure S5). The purity of each 68 Ga-labeled compound was over 95% at a molar activity of up to 10 MBq/nmol ( Figure 1A

Partition Coefficient
The  Figure 2 illustrates that the PSMA monomer was hydrophilic, while the hydrophilic performance of the PSMA tetramer was significantly better with the augmentation of hydrophilia by polypeptide PEGylation.

Partition Coefficient
The  Figure 2 illustrates that the PSMA monomer was hydrophilic, while the hydrophilic performance of the PSMA tetramer was significantly better with the augmentation of hydrophilia by polypeptide PEGylation.

Imaging of Micro Animal PET/CT and SPECT/CT
The Micro Animal PET/CT and SPECT/CT imaging of mice injected with ligands are presented in Figure 6. The results indicated that all the radiotracers were absorbed by the tumor, and other organs demonstrated no obvious specific uptake except for the kidney and the bladder. The tumor uptake of DOTA-(1P-PEG4), DOTA-(2P-PEG0), and DOTA-(2P-PEG4) began to decline at 2 h after injection ( Figure 6A-C), while DOTA-(2P-PEG4)2

Imaging of Micro Animal PET/CT and SPECT/CT
The Micro Animal PET/CT and SPECT/CT imaging of mice injected with ligands are presented in Figure 6. The results indicated that all the radiotracers were absorbed by the tumor, and other organs demonstrated no obvious specific uptake except for the kidney and the bladder. The tumor uptake of DOTA-(1P-PEG 4 ), DOTA-(2P-PEG 0 ), and DOTA-(2P-PEG 4 ) began to decline at 2 h after injection ( Figure 6A-C), while DOTA-(2P-PEG 4 ) 2 remained stable and exhibited obvious uptake in the tumor at 5 h after injection ( Figure 6D). Furthermore, DOTA-(2P-PEG 4 ) 2 exhibited a favorable T/NT ratio at all time points, with a remarkably lower kidney uptake than [ 68 Ga]Ga-PSMA-11 ( Figure 6E). DOTA-(2P-PEG 4 ) 2 was further labeled by lutetium-177 and utilized for imaging. [ 177 Lu]Lu-DOTA-(2P-PEG 4 ) 2 showed better imaging capacity than [ 177 Lu]Lu-PSMA-617 ( Figure 6G), and the in vivo tumor uptake was still obvious at 48 h after injection ( Figure 6F), indicating that it possessed a longer detention time in vivo and a sound tumor-targeting ability. Conclusively, DOTA-(2P-PEG 4 ) 2 was a potential candidate drug for radionuclide therapy. In addition, the tumor uptake was apparently blocked in a 2-PMPA-blocking experiment ( Figure 6H), indicating that these ligands had great PSMA specificity ( Figure 6H).

Imaging of Micro Animal PET/CT and SPECT/CT
The Micro Animal PET/CT and SPECT/CT imaging of mice injected with ligands are presented in Figure 6. The results indicated that all the radiotracers were absorbed by the tumor, and other organs demonstrated no obvious specific uptake except for the kidney and the bladder. The tumor uptake of DOTA-(1P-PEG4), DOTA-(2P-PEG0), and DOTA-(2P-PEG4) began to decline at 2 h after injection ( Figure 6A-C), while DOTA-(2P-PEG4)2 remained stable and exhibited obvious uptake in the tumor at 5 h after injection ( Figure  6D). Furthermore, DOTA-(2P-PEG4)2 exhibited a favorable T/NT ratio at all time points, with a remarkably lower kidney uptake than [ 68 Ga]Ga-PSMA-11 ( Figure 6E). DOTA-(2P-PEG4)2 was further labeled by lutetium-177 and utilized for imaging. [ 177 Lu]Lu-DOTA-(2P-PEG4)2 showed better imaging capacity than [ 177 Lu]Lu-PSMA-617 ( Figure 6G), and the in vivo tumor uptake was still obvious at 48 h after injection ( Figure 6F), indicating that it possessed a longer detention time in vivo and a sound tumor-targeting ability. Conclusively, DOTA-(2P-PEG4)2 was a potential candidate drug for radionuclide therapy. In addition, the tumor uptake was apparently blocked in a 2-PMPA-blocking experiment (Figure 6H), indicating that these ligands had great PSMA specificity ( Figure 6H).

Discussion
Polymeric peptides constructed on the basis of the polyvalent effect can improve the tumor-targeting ability and prolong tumor retention time remarkably. Plenty of polymerized peptides have been reported previously, and polymerization can be performed between the same peptides, such as 2P(RGD)2 [33] and 2P(FAPI) [24], or between two kinds of peptides, for example, PSMA-FAPI [34] and RGD-FAPI [35]. According to current clinical data, based on polymerization strategy, 2P(RGD)2 and 2P(FAPI) have been constructed and testified to gain an increased tumor uptake rate and better tumor retention capacity. In this study, a polymeric modification was performed on PSMA, by which we obtained a series of polymerized molecules, namely DOTA-(1P-PEG4), DOTA-(2P-PEG0), DOTA-(2P-PEG4), and DOTA-(2P-PEG4)2, and conducted particular in vitro and in vivo

Discussion
Polymeric peptides constructed on the basis of the polyvalent effect can improve the tumor-targeting ability and prolong tumor retention time remarkably. Plenty of polymerized peptides have been reported previously, and polymerization can be performed between the same peptides, such as 2P(RGD) 2 [33] and 2P(FAPI) [24], or between two kinds of peptides, for example, PSMA-FAPI [34] and RGD-FAPI [35]. According to current clinical data, based on polymerization strategy, 2P(RGD) 2 and 2P(FAPI) have been constructed and testified to gain an increased tumor uptake rate and better tumor retention capacity. In this study, a polymeric modification was performed on PSMA, by which we obtained a series of polymerized molecules, namely DOTA-(1P-PEG 4 ), DOTA-(2P-PEG 0 ), DOTA-(2P-PEG 4 ), and DOTA-(2P-PEG 4 ) 2 , and conducted particular in vitro and in vivo assessments and examinations on these 68 Ga-labeled PSMA ligands. According to the results of the in vitro experiments, these newly synthesized radiotracers possessed sound stability, and the monomer, dimer, and tetramer all obtained superior binding capacity, among which the tetramer achieved the best effect. Phenomena observed by PET imaging and biodistribution analysis proved that these ligands could be metabolized rapidly in vivo, while signals of ligands in tumors were still detectable at 5 h after injection, where the uptake of the tetramer remained the highest.
The introduction of PEG chains is generally recognized as a way to optimize pharmacokinetics [36], which has been applied in designing many radiotracers. Previous studies have reported that replacing PEG 3 with PEG 3 + PEG 7 could improve tumor uptake, prolong tumor retention time, and reduce kidney uptake [16]. Nonetheless, some reports concluded that tumor uptake and biodistribution were not improved when PEG 4 had a modified structure, especially when the PEG chain was too long; for example, the modification of PEG 12 /PEG 24 markedly impeded the affinity of the compound [27,37], which could be caused by the "PEG dilemma" [38]. Other research reports that PEG 2 /PEG 4 modification can maintain the maximum affinity of PSMA [31]. We explored the effects of PEG 4 on the affinity of PSMA ligands. DOTA-(2P-PEG 4 ) was constructed by insert a PEG 4 chain into DOTA-2P, and it exhibited better tumor uptake according to imaging results, indicating that PEG 4 slightly facilitated the compound's affinity. In addition, DOTA-(2P-PEG 4 ) possessed better stability compared with DOTA-(2P-PEG 0 ). From the aspect of molecular structure, PEG 4 endowed the molecule with extra flexibility [38], which enabled the binding between ligand and binding spot, as well as maintaining biostability. Although the DOTA-(2P-PEG4) reduced kidney uptake to some extent, referring to our imaging data and cellular experiment results, improvement in the tumor-targeting ability of DO-TA-(2P-PEG4) was not acquired. Nevertheless, the effect of DOTA-(2P-PEG 4 ) was not as satisfactory as we expected; although kidney uptake was reduced to some extent, referring to our imaging data and cellular experiment results, improvement in the tumor-targeting ability of DOTA-(2P-PEG 4 ) was not acquired.
To address the low tumor uptake of the PSMA dimer, we designed DOTA-(2P-PEG 4 ) 2 , which again adopted a polymeric peptide strategy, aiming to enhance the tumor-targeting ability of the symmetric PSMA dimer. Meanwhile, the PEG 4 was doubled, which further accelerated kidney clearance. Some reports claim that the peptide tetramer and peptide octamer can increase tumor uptake, but, more significantly, the kidney uptake [39]. To the best of our knowledge, we have found that PEG chains could improve the pharmacokinetics, and have the potential to overcome this problem. Fortunately, imaging and cellular experiments successfully validated this deduction of our newly designed structures. The cellular affinity of DOTA-(2P-PEG 4 ) 2 was 4-5 times more than that of DOTA-(2P-PEG 4 ), and the uptake rate of DOTA-(2P-PEG 4 ) 2 was 2-3 times higher than that of DOTA-(2P-PEG 4 ). In the imaging experiment, the tumor uptake of DOTA-(2P-PEG 4 ) 2 was higher than that of DOTA-(2P-PEG 4 ) at each time point, and the kidney uptake of DOTA-(2P-PEG 4 ) 2 at 2 h was basically undetectable. We realized the 1 + 1 > 2 effect through this crafty design.
Serum retention time is also an essential factor in radiopharmaceuticals. In this study, we found that the albumin-binding rate could be promoted by simply increasing PSMA numbers, since the albumin-binding rate of DOTA-(2P-PEG 4 ) 2 reached 94.7%, which has not been reported by any other researchers before. This phenomenon was possibly caused by the fatty acid of glutamic acid in the PSMA molecule binding to the site2 in albumin [40]. The albumin-binding rate was improved with increasing glutamic acid numbers in the symmetric PSMA dimer. In the pharmacokinetics experiment, the serum retention time of the tetramer was short, which may have been due to the PEG 4 chain, considering that PEG 4 could significantly accelerate serum clearance and kidney clearance.

Conclusions
In this study, we firstly synthesized polymerized PSMA ligands, namely DOTA-(2P-PEG 0 ), DOTA-(2P-PEG 4 ), and DOTA-(2P-PEG 4 ) 2 . The designed symmetric PSMA dimer significantly improved the affinity to the PSMA ligand and prolonged the detention time in the tumor. With the modification of the PEG chain, not only were the compounds' stability, hydrophilia, and albumin-binding rate improved, but also the kidney uptake of ligands was significantly reduced. In conclusion, the modification strategies in this study may provide a fresh idea for optimizing PC-targeting PSMA molecular probes, and DOTA-(2P-PEG 4 ) 2 has shown potential in PSMA high expression tumor imaging and therapy.