Reagents and instruments
Peptides were purchased from ChinaPeptides Co. Ltd (Shanghai, China). All other chemicals were purchased from Sigma-Aldrich Chemical (USA), unless otherwise indicated. Chemicals were used directly without further purification. Rabbit anti-avβ3 and anti-NRP-1 monoclonal antibodies were purchased from Abcam (Shanghai, China). MCF-7 cell lines were purchased from the cell bank of the Chinese Academy of Sciences in Shanghai and utilized for cell studies. [68Ga] GaCl3 was produced with a 68Ge/ [68Ga] generator (China Isotope & Radiation Corporation, China). RGD-ATWLPPR heterodimer was synthesized using our published method [20]. Analytical HPLC had a flow rate of 1 mL/min with a C18 column (10 μm, 250 mm × 4.6 mm, Macherey-Nagel, Nucleosil 100-10). Micro-positron emission tomography/computed tomography (PET/CT) was performed using an Inveon microPET/CT scanner (Siemens).
DOTA-RGD-ATWLPPR synthesis and identification
The Chinese Polypeptide company (Hangzhou) synthesized and purified RGD-ATWLPPR peptide. We also accomplished the synthesis of DOTA-RGD-ATWLPPR peptide using 1,4,7,10-tetraazacyclododecane-N, N',N'',N'''-tetraacetic acid (DOTA) as the chelator via glutamate. The purity of each peptide was above 95%. Subsequently, Peptide DOTA-RGD-ATWLPPR identification was performed using mass spectrometry (MS), and we chose the MS procedure for selecting small molecular samples, and the parameters were properly adjusted to obtain the mass spectrum and store the map.
Radiolabeling and stability
DOTA-RGD-ATWLPPR, DOTA-RGD, or DOTA-ATWLPPR (25 μg) was added to 1 mL buffer (0.25 M sodium acetate, pH 3–4). Then, 4 mL [68Ga] GaCl3 in 0.05 M HCl was added to the solution. The mixture was heated to 100°C and reacted for 5–10 min. After cooling to 25°C, 68Ga-DOTA-RGD-ATWLPPR, 68Ga-DOTA-RGD, and 68Ga-DOTA-ATWLPPR were obtained. The reaction mixture (1 μL) was purified and analyzed by HPLC using the method described above. The gradient program was started from 95% solvent A [0.1% trifluoroacetic acid (TFA) in water] and 5% B [0.1% TFA in acetonitrile (MeCN)] for the first 5 min and then ramped to 35% solvent A and 65% solvent B at 35 min. The in vitro stability of 68Ga-DOTA-RGD-ATWLPPR, 68Ga-DOTA-RGD, and 68Ga-DOTA-ATWLPPR was evaluated by incubating 3.7 MBq probe with fresh mouse serum at 37 °C for 30 and 60 min.
Cell culture and animal model
MCF-7 cell lines were cultured in DMEM supplemented with 10% fetal bovine serum containing 5 % CO2. Animal experiments were approved by the Xiamen University Institutional Animal Care and Use Committee. Female BALB/c nude mice (6–8 weeks old, 18–20 g) were obtained from the Xiamen University Laboratory Animal Center (Xiamen, China). Each mouse was injected subcutaneously with 5×106 cancer cells. Two to three weeks after inoculation, the mice were used for biodistribution and imaging studies.
Tumor-specific integrin αvβ3 and NRP-1 expression
For these experiments, 2 × 105 cells were seeded on coverslips in each well of a 6-well plate. MCF-7 cells were washed 3 times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min. The cells were blocked with PBS containing 1% goat serum for 1 h. Subsequently, the cells were incubated with 5 ug/ml mouse anti-avβ3 monoclonal antibody (Abcam, Cat. No.ab190147) overnight at 4 °C and were washed 5 times with PBS for a total of 3 h, incubated with Alexa Fluor™ 594-labeled tyramide for 1 h at 25 °C, or rabbit anti-neuropilin-1 monoclonal antibody (Abcam, Cat. No.ab81321) overnight at 4 °C and were then washed 5 times with PBS and incubated with Alexa Fluor™ 488-labeled tyramide for 1 h at room temperature. The samples were observed under laser scanning confocal microscope. MCF-7 tumor tissues were collected, fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. The tumor sections (5 μm) were washed three times with PBS. The sections were rinsed with EDTA buffer and blocked with 5% bovine serum albumin. The sections were incubated with primary antibodies (anti-NRP-1, anti-ανβ3; 1:100; Abcam) at 4 °C overnight. After incubation with biotinylated secondary antibody and avidin-biotin-peroxidase reagents for 10 min, 3'3-diaminobenzidine was added for color development. observed using light microscopy.
In vitro cell uptake and blocking studies
MCF-7 cells were cultivated to 90% confluence and counted. The cells were seeded into 12-well plates at a density of 5 × 105 cells per well and incubated overnight at 37 °C to allow adherence. Then, uptake of 68Ga-DOTA-RGD-ATWLPPR, 68Ga-DOTA-RGD, and 68Ga-DOTA-ATWLPPR was evaluated. The cells were incubated with 0.37 MBq probe at 37 °C for 30 min and 1 h. Tumor cells were washed three times with chilled PBS and harvested by trypsinization with 0.25% trypsin/0.02% EDTA (Invitrogen). The cell suspensions were collected and measured using a γ-counter (WIZARD 2480; Perkin-Elmer, USA). For blocking studies, one group of cells was incubated with 68Ga-DOTA-RGD-ATWLPPR, 68Ga-DOTA-RGD, and 68Ga-DOTA-ATWLPPR (100 μL, 3.7 KBq) for 1 h at 37 °C. The other groups were pretreated with excess nonradioactive RGD, ATWLPPR, and RGD-ATWLPPR (1 mg) 15 min before incubation with 68Ga-DOTA-RGD-ATWLPPR, 68Ga-DOTA-RGD, and 68Ga-DOTA-ATWLPPR (100 μL, 7.4 KBq) for 1 h at 37 °C.
MicroPET/CT imaging studies
MicroPET/CT scans were performed using an Inveon microPET/CT scanner (Siemens). Under isoflurane anesthesia, MCF-7 tumor xenograft mice were injected via the tail vein with normal saline, 68Ga iron, or approximately 3.7–7.4 MBq 68Ga-DOTA-RGD-ATWLPPR. Anesthesia was maintained with 2.0% isoflurane in 100% oxygen. Mice were placed on an examination table in the prone position and underwent PET/CT imaging at 30 min and 60 min. Sixty-minute dynamic PET scans were performed after tail vein injection of 68Ga-DOTA-RGD-ATWLPPR (n = 3). To compare 68Ga-DOTA-RGD-ATWLPPR and 68Ga-DOTA-RGD, 68Ga-DOTA- ATWLPPR, these different traces were injected into the tail vein of nude mice. To assess 68Ga-DOTA-RGD-ATWLPPR specificity, blocking experiments were performed in MCF-7 tumor models. The blocking agents RGD, ATWLPPR, and RGD-ATWLPPR (10 mg/kg, approximately 500 μg/mouse) were co-injected with 3.7 MBq 68Ga-DOTA-RGD-ATWLPPR (100 μci). Further, 15 min static microPET/CT scans were acquired 30 min post-injection (n=3/group). After the scan, regions of interests (ROIs) were drawn on the muscle and tumor in the CT images and copied to the PET images using Inveon Research Workplace 4.1 software. The tumor/muscle (T/M) ratio was calculated by dividing the signal intensity of the tumor by that of the muscle.
Biodistribution activity
MCF-7 tumor xenograft mice received a tail vein injection of 0.74 MBq (20 μCi) 68Ga-DOTA-RGD-ATWLPPR to evaluate biodistribution. Mice were euthanized by cervical dislocation at 5-, 15-, 30-, and 60-min post-injection (n=4/group). Blood, tumor, and main organs were harvested, weighed, and counted in a gamma counter (WIZARD2480, PerkinElmer, USA). Then, MCF-7 tumor xenograft mice received a tail vein injection of 0.74 MBq (20 μCi) 68Ga-DOTA-RGD-ATWLPPR, 68Ga-DOTA-RGD, 68Ga-DOTA- ATWLPPR to evaluate the biodistribution at 30 min. The result was reported as the % ID/g.
Statistical analysis
All data are presented as the mean ± SD from at least three independent experiments. statistical analyses were performed using RStudio (R 3.6.1). Means were compared using one-way ANOVA. P < 0.05 was considered statistically significant.