Dopamine hydrochloride, MnCl₂, and ammonia aqueous solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). Amine-mPEG5000 (mPEG5000–NH₂, 5,000 Da) was acquired from Aladdin (Shanghai, China). Herceptin was obtained from Roche Pharmaceuticals (Shanghai, China) Ltd. 2-Mercaptoethanol was purchased from Sinopharm Chemical Reagent Co. Ltd. For the cell experiments, Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (USA); penicillin–streptomycin mixture double and anti-0.25% trypsin (including EDTA) were purchased from Invitrogen (USA). In the experiment, high nuclear pure simple ¹²⁴I was prepared by ¹²⁴Te solid target of HM-20 cyclotron at the Nuclear Medicine Department of Peking University Cancer Hospital. The nuclide was purified by ¹²⁴I special purification device and collected in 0.02 M NaOH solution (radioactive concentration 148 MBq/ml). The positron nuclide ⁶⁴Cu was prepared by bombarding a ⁶⁴Ni solid target with a medical cyclotron at the Nuclear Medicine Department of Peking University Cancer Hospital with 3.7 MBq/μl in 0.01 M HCl solution (32).

Dopamine–melanin nanoparticles were prepared according to the methods reported in previous literature (30). Briefly, ammonia aqueous solution (0.3 ml, NH₄OH, 28%–30%) was mixed with ethanol (4 ml) and deionized water (9 ml) under mild stirring at 40°C for 30 min. Fifty milligrams of dopamine hydrochloride was dissolved in water (1 ml), then injected into the above mixture solution, and stirred for 48 h. The color of this solution changes from light yellow to dark brown. The bright black solution was then centrifuged with a centrifugal filter (Amicon centrifugal filter device, MWCO = 50 kDa) and washed several times to remove the unpolymerized dopamine–melanin molecules. The reaction solution was taken and diluted to 5 ml ultrapure water by ultrasonication for 5 min, and the reaction system pH = 9 was adjusted by NH₄OH (25%–28%). Then, the solution was slowly dripped into the reaction bottle containing 25 mg diamino polyethylene glycol (NH₂–PEG₅₀₀₀–NH₂) and stirred at room temperature for 12 h. The unreacted NH₂–PEG₅₀₀₀–NH₂ was removed by a 50-kDa ultrafiltration centrifuge to obtain NH₂–PEG-dMNPs.

The amino-sulfhydryl cross-linking agent Sulfo-SMCC was used as a linker in the combination of Herceptin-SH and NH₂–PEG-dMNPs. Five milligrams of NH₂–PEG-dMNPs (0.5 × 10⁻⁴ mM) was fully dispersed in 1 ml phosphate buffered solution (PBS), and excess amino-sulfhydryl cross-linking agent Sulfo-SMCC (5 µM) was stirred at room temperature for 2 h. Then, the unreacted Sulfo-SMCC was separated by the PD-10 column. The reaction solution was concentrated to 0.6 ml by ultrafiltration and slowly added to Herceptin-SH (0.33 × 10⁻⁴ mM). After reacting for 2 h at room temperature with stirring, the unreacted sulfhydryl–Herceptin was removed using a PD-10 column (GE Healthcare, USA) with an eluent of PBS (pH = 7.4) and lyophilized to obtain a purified Her-PEG-dMNP. Two milligrams of Her-PEG-dMNPs and MnCl₂ (10 × 10⁻³ mM) were fully dispersed in 1 ml deionized water and stirred at room temperature for 2 h. The Mn-Her-PEG-dMNPs were obtained by centrifugation with an ultrafiltration centrifuge tube (MWCO = 10 kDa).

The morphology of Her-PEG-dMNPs was characterized by using a JEM-2100F transmission electron microscope with an accelerating voltage of 200 kV, and the sample was made by using a film of copper mesh. Dynamic light scattering (DLS) was used to detect the hydrodynamic diameter of the dMNPs, PEG-dMNPs, and Her-PEG-dMNPs with a laser light-scattering spectrometer (SLS-5022F, Germany) ¹H-NMR spectra of dMNPs and PEG-dMNPs were detected by a 600-MHz NMR (Bruker) spectrometer with D₂O as the solvent.

The viability and proliferation of HeLa and HepG2 cells were evaluated by a standard 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Typically, HeLa and HepG2 cells were incubated in the culture medium at 37°C in an atmosphere of 5% CO₂ and 95% air for 24 h. Then, the medium was removed. The cells were incubated in cultured medium containing Her-PEG-dMNPs and Mn-Her-PEG-dMNPs with different concentrations of 0.125, 0.25, 0.5, 1, and 2 mg/ml for 24 h and washed with the medium twice. The MTT solution (20 µl, 0.5 mg/ml) was added to each well, and the cells were incubated for 4 h without light at 37°C. After removal of the supernatant, the residues were lysed with 150 µl of dimethyl sulfoxide (DMSO). Finally, the light absorption (OD) values were measured at 490 nm with a microplate reader. The untreated cells were processed analogously, and their OD values were used as the reference to calculate 100% cellular viability.

⁶⁴Cu was diluted with hydrochloric acid (0.01 M, 300 µl), then a small amount of sodium acetate (0.1 mol/L) was added to adjust the pH to 5.5, and then Her-PEG-dMNP (2 mg/ml) was added and incubated at 40°C for 1 h. When the reaction was over, saturated EDTA solution (0.05 M) was added and mixed with the reaction liquid. ⁶⁴Cu-Her-PEG-dMNP was purified using PD-10 column in PBS (pH = 7.4). ¹²⁴I can label Her-PEG-dMNPs in situ using N-bromosuccinimide as the oxidant. The ¹²⁴I buffer was diluted with PB solution (0.1 M, 500 µl) before being added to the Mn-Her-PEG-MNPs. Then, 20 µl N-bromosuccinimide (NBS, 5.6 mM) was added to the above solution for 1 min. The radiolabeled reaction was then ended and the ¹²⁴I-Her-PEG-dMNP was achieved via the PD-10 column in PBS buffer, and radio-TLC was used to detect the radiolabeling rate of the ¹²⁴I-Her-PEG-dMNPs with acetone as the developer. The stability of the ⁶⁴Cu-HER-PEG-dMNPs and ¹²⁴I-Her-PEG-dMNPs in vitro was measured by incubations in PBS (pH = 7.4) and 5% HSA solution. Radio-TLC was used to analyze the radiochemical purity of the ¹²⁴I/⁶⁴Cu-Her-PEG-dMNPs at different time points.

One day before the study, human ovarian carcinoma SKOV-3 cells were seeded in 24-well plates at an average of 1 × 10⁵ cells per well and incubated overnight in 1 ml of DMEM medium. Prior to the study, the cells were washed twice with 1 ml of cold PBS, and after the culture solution was aspirated in each well, the cells were re-incubated with 500 μl of serum-free medium for 2 h at 37°C. Then, four wells in every dish were divided into one group. A total of 37 kBq of ¹²⁴I/⁶⁴Cu-Her-PEG-dMNPs in 500 µl serum-free medium was added to each well, and the cells were cultured at 37°C with 5% CO₂. To assess the specific targeting ability of ¹²⁴I/⁶⁴Cu-Her-PEG-dMNPs for targeting HER2, three groups of SKOV-3 cells (n = 4) were co-incubated with ¹²⁴I/⁶⁴Cu-HER-PEG-dMNPs and 20 µg of Her-PEG-dMNPs. After co-incubation with the radiotracer for 10, 30, 60, and 120 min, the cell culture media of each group was sequentially aspirated, and the cells were rinsed three times with 1 ml of cold PBS and lysed with 500 µl of 0.5 M NaOH. Cell lysates in each well were then collected and counted by a γ-counter.

All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Peking University (Beijing, China), and experiments were approved by the Animal Ethics Committee of the National Regulation of China for the Care and Use of Laboratory Animals and in compliance with the guidelines established by Peking University Cancer Hospital Animal Care and Use Committee. The PDX models were constructed as in reference (11). Fresh tumor tissue mass derived from patients was inoculated into the right groin area of NOD-SCID mice, and the tumor tissue was removed when the tumor grew to 1 cm in diameter. The tissue blocks were cut into 0.05 cm * 0.05 cm * 0.05 cm for subculture.

Healthy KM mice (18–20 g) were used to conduct the pharmacokinetics study. Mice were sealed off 3 days in advance with 0.5% potassium iodide (KI), and ¹²⁴I-Her-PEG-dMNP (3.7 MBq) was intravenously injected into each mouse (n = 5) at set intervals (1, 3, 5, 10, 15, 30, 60, and 120 min and 4, 8, 24, 48, and 72 h). Then, ⁶⁴Cu-Her-PEG-dMNP also was intravenously injected into each mouse (n = 5) at set intervals (1, 3, 5, 10, 15, 30, 60, and 120 min and 4, 8, 24, and 48 h) and into the blood through the orbital vein. The blood samples were weighed before counting using a γ-counter. The results were calculated as the percentage of injected dose per gram (%ID/g, mean ± SD), and the parameters of the pharmacokinetics were analyzed using the GraphPad Prism software.

Micro-PET/CT imaging studies were conducted by using PDX model NOD-SCID mice with a micro-PET/CT system (Super Nova PET/CT, PINGSENG, Shanghai, China). Tumor-bearing NOD-SCID mice were injected in situ with ¹²⁴I-Her-PEG-dMNPs and ¹²⁴I-PEG-dMNPs (1.11 MBq). Anesthesia for the animals was performed using a PINGSENG isoflurane gas anesthesia system. Axial and coronal PET/CT images of the tumor were collected at 2, 24, 48, and 72 h. The mice were anesthetized with isoflurane before imaging, and anesthesia was maintained during imaging (volume coefficient 1%). Micro-PET images were acquired for 15 min and reconstructed with attenuation correction based on the CT data (CT–AC reconstruction). Regions of interest (ROIs) were drawn on the CT images and further mapped on PET. The experiments were repeated three times at each time point.

For in-vivo MRI, PDX model NOD-SCID mice (n = 3) were first anesthetized by intraperitoneal injection of 4% chloral hydrate solution (50 mg/kg), when the tumors reached approximately 0.8 cm, followed by in-situ injection of Mn-Her-PEG-dMNPs (0.125 mM, 30 µl/mouse). Axial images of tumor locations were collected at 2, 24, and 72 h, using a clinical 1.5-T magnetic resonance scanner (Siemens) with animal microcoils. The MRI sequence was as follows: TR, 531 ms; TE, 9.1 ms; flip angle, 30°; FOV, 160 × 100 mm²; matrix, 256 × 256; and slice thickness, 3 mm. The analysis of the PET/MRI signal in the tumor images was conducted with the 3D Slicer software.

Mice were sacrificed after the last imaging point, and then the tumor tissue was immediately removed and fixed in formalin. For immunohistochemical staining, sections of PDX tumor tissue were placed in a beaker containing citrate antigenic repair solution. Intermittent heating was performed three times with microwaves. Then, the tissue was treated with 10% goat serum at 37°C for 30 min. After being rinsed three times with PBS, the samples were incubated overnight with anti-HER2 antibody (1:300) at 4°C, and then sections were incubated with biotinylated goat anti-rabbit (HRP) antibody (1:1,000) at 37°C for 30 min. The sections were then stained with DAB substrate kit and hematoxylin and differentiated with 0.25% hydrochloric alcohol. Sections were dehydrated and sealed with neutral gum and photographed for analysis.

Scheme 1 shows the reaction flowchart of the Her-PEG-dMNP imaging probe. Typical scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging showed that the synthesized dMNPs and Her-PEG-DMNPs had clear structures and uniform and regular shapes and sizes ( Figures 1A, B ). As shown in Figures 1C, D and Figure S1 , the hydrodynamic particle size of dMNPs is about 62.25 ± 6.12 nm, and PEG-dMNP was 101.28 ± 13.63 nm, which was slightly higher than before. To construct a targeted probe for tumors with high HER2 expression, trastuzumab with an active sulfhydryl group was bound to the surface of the nanoparticles by electrophilic substitution, and the size of Her-PEG-dMNPs increased to 148.16 ± 20.04 nm. The ¹H-NMR spectra of PEG-dMNPs in D₂O showed an emerging peak at 3.5 ppm, which represented the –OCH₂CH₂O– group of the PEG molecule ( Figures 1E, F ). In addition, both Her-PEG-dMNPs and Mn-Her-PEG-dMNPs showed high biocompatibility and low cytotoxicity in HepG2 and HeLa cells after being cultured for 24 h (compared with the non-toxic control). The results showed that the cell survival rate was above 95% when the concentration of both probes reached 2 mg/ml ( Figures 2A, B ).

The radiochemical yield of ⁶⁴Cu-Her-PEG-dMNPs was 85.1%–89.6% and the specific activity was 4.77–8.25 Bq/mg, while ¹²⁴I-Her-PEG-dMNPs had a radiochemical yield of 90%–93% and a specific activity of 7.98–13.30 MBq/mg. The final purity of both radiotracers was >98% ( Figure 2C and Figure S2 ). The in-vitro stability of ⁶⁴Cu/¹²⁴I-Her-PEG-dMNPs in PBS and 5% HSA was evaluated by radio-TLC. The PBS (pH = 7.4) and 5% HSA had more than 89% radioactive purity at 96 h ( Figure 2D and Figure S2 ).

The cell uptake of ¹²⁴I-Her-PEG-dMNPs in HER2-expressed human ovarian carcinoma SKOV-3 cell showed a higher uptake than that of ⁶⁴Cu-Her-PEG-dMNPs in cells at 10 min, 30 min, and 1 h incubation time periods ( Figure 2E and Figure S2 ). For SKOV-3 cells, the uptake of ¹²⁴I-Her-PEG-dMNPs reached 0.65% ± 0.31%, 1.56% ± 0.81%, and 3.61% ± 0.74% of the collected count to the total added count (% AD/10⁵ cells) at 30, 60, and 120 min, while the uptake of ⁶⁴Cu-Her-PEG-dMNPs was 1.55% ± 1.29%, 2.43% ± 0.51%, and 4.03% ± 0.55% AD/10⁵ cells, respectively. After blocking with the precursor, the uptake of ¹²⁴I-Her-PEG-dMNPs at 60 and 120 min was significantly reduced (1.56 ± 0.81 vs. 0.72 ± 0.06 at 1 h, P = 0.008; 3.61 ± 0.74 vs. 1.24 ± 0.43 for 2 h, P = 0.002) ( Figure 2E ). The uptake of ⁶⁴Cu-Her-PEG-dMNPs was significantly reduced at 1 and 2 h (2.43 ± 0.51 vs. 1.65 ± 0.41 for 1 h, P = 0.002; 4.03 ± 0.55 vs. 1.00 ± 0.12 for 2 h, P = 0.001) ( Figure S2 ). These results suggest that ¹²⁴I/⁶⁴Cu-Her-PEG-dMNPs have specific targeting ability against HER2-expressed tumor cells.

The profile of radioactivity in the blood after tail injection of ¹²⁴I/⁶⁴Cu-Her-PEG-dMNPs in KM healthy mice was delineated using GraphPad Prism software ( Figure 2F and Figure S2 ). Circulating pharmacokinetics of ¹²⁴I/⁶⁴Cu-Her-PEG-dMNPs were evaluated using a two-compartment model. The data represent mean values, representing the percentage (ID%/g) of normal (n = 5) KM mice per gram of blood injection dose. The model parameters were obtained by fitting the relationship between the percentage of injected dose per gram of blood (ID%/g) and post-injection time into the following equation: Ct (%ID/g) = SpanFast * exp(αt) + SpanSlow * exp(βt), where SpanFast and SpanSlow are the y-intercepts of the model, and α and β are the fast and slow rate constants, expressed as the reciprocal of the time axis. In this model, the equations of ¹²⁴I/⁶⁴Cu-Her-PEG-dMNPs in KM mice were as follows: Ct (%ID/g) = 4.507 + 406.566 * exp(−17.81t) + 319.96 * exp(−0.384t); Ct (%ID/g) = 2.588 + 207.73 * exp(−0.466t) + 52.48 * exp(−0.053t). The half-life of ¹²⁴I/⁶⁴Cu-Her-PEG-dMNP distribution stage was 1.485 and 0.03892 h, respectively, while the elimination stage was 12.87 and 1.803 h, respectively.

All animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of Peking University (Ethics Approval License: 2015KT08). HER2-positive patient-derived xenograft (PDX) models were built for multimodality imaging. When the tumor volume reached 0.5–1.0 cm in diameter, mice were randomly assigned. The represented immunohistochemistry results showed that the PDX tumor tissues exhibited high HER2 expression ( Figure 4C ).

We first used micro-PET/CT imaging to observe the accumulation of two radioactive tracers in HER2 highly expressing gastric cancer PDX model tumor site with ¹²⁴I/⁶⁴Cu-Her-PEG-dMNPs. In recent years, studies have shown that nanoprobes will be rapidly removed after in-vivo injection by human phagocytes, and their targeting function is also affected by the inevitable formation of protein crown. In-situ tumor injection, to some extent, can avoid such effects. The images of delayed acquisition are shown in Figure 3A and Figure S3 . Both ¹²⁴I and ⁶⁴Cu showed excellent retention effects in the PDX model (n = 3) by in-situ injection compared with the control of the non-targeted molecular probe. ROI-based micro-PET/CT quantification analysis of those probes is shown in Figures 3B , C . The maximum standardized uptake values (SUVmaxs) of the liver were 0.24 ± 0.03, 0.42 ± 0.01, 0.38 ± 0.06, and 0.11 ± 0.01 at 2, 24, 48, and 72 h of 124I-Her-PEG-dMNPs, compared with the SUVmaxs of 0.87 ± 0.17, 1.47 ± 0.05, 1.72 ± 0.15, and 1.05 ± 0.05 at 2, 24, 48, and 72 h of ¹²⁴I-PEGdMNPs ( Figure 3D ). The SUVmaxs of tumor sites were 3.01 ± 0.59, 1.63 ± 0.07, 1.19 ± 0.06, and 1.04 ± 0.06 of 124I-Her-PEG-dMNPs, compared with the SUVs of 2.86 ± 0.52, 0.90 ± 0.04, 0.45 ± 0.16, and 0.07 ± 0.01 of ¹²⁴I-PEG-dMNPs ( Figure 3E ). The retention effect on the tumor was significantly different, indicating that the probe had good specificity for HER2-positive PDX tumor tissues. These results showed that the majority of ¹²⁴I/⁶⁴Cu-Her-PEG-dMNPs remained in the tumor at 2 h after injection, and a little part was distributed to the liver over time. The probe uptake was low in the liver and gradually decreased after 48 h, which was significantly different from the control group (¹²⁴I-PEG-dMNPs).

Manganese (Mn) as a natural cell component has good biosafety and can be used in cellular and mitochondrial functions to avoid long-term side effects (33). Mn²⁺ saturation can increase the T1-weighted relaxation rate and reduce the dosage of the contrast agent (34). (¹²⁴I, Mn)-Her-PEG-dMNPs can be used for long-term multimodal imaging monitoring over the course of treatment. Therefore, we attempted to use PET/MRI dual enhancement imaging to monitor the high tumor uptake and retention of (¹²⁴I, Mn)-Her-PEG-dMNPs. The gastric cancer PDX tumor-bearing mice were used for imaging monitoring (n = 3). There were significant PET/MRI signals in the tumor site at 2 h after injection of the nanoprobe, and the signals were not concentrated in the liver or other phagocytic organs at 2, 24, and 72 h ( Figure 4A ). The T1-weighted MR signal of the tumor site slightly declined after 72 h, but the PET signal also showed strong retention at the tumor site.

Statistical analysis of the SUVmax and MR signals in the tumor site showed that the tumor/non-tumor (T/NT) values of the two signals decreased slowly ( Figure 4B ). However, the SUVmax signal and the MR signal in the liver were almost not significantly increased, which was basically consistent with the PET/MR images, indicating the strong retention effect of probes in the tumor site. This study truly realized the practical value of the theranostic functions of (¹²⁴I, Mn)-Her-PEG-dMNPs.