pHLIP (Var7) and the control sequence kVar7 (7) were synthesized by Shanghai Science Peptide Biological Technology Co., Ltd. (Shanghai, China) using solid-phase peptide synthesis. To facilitate ^99mTc labeling, four amino acids, Gly-(D)-Ala-Gly-Gly, were attached to the N-terminus of pHLIP (Var7) and kVar7 to form a strong chelating group containing an N4 structure. To prevent any spatial obstruction, gamma-aminobutyric acid (GABA) was introduced as a spacer between the N4 structure and pHLIP (Var7)/kVar7. The modified pHLIP (Var7) and kVar7 sequences were as follows:pHLIP (Var7), GaGG-GABA-ACEEQNPWARYLEWLFPTETLLLEL-NH₂; kVar7, GaGG-GABA-ACEEQNPWARYLKWLFPTKTLLLKL-NH₂.

pHLIP (Var7) and kVar7 were purified via high-performance liquid chromatography (HPLC) using a Shimadzu-LC2010 instrument (Shimadzu Corporation, Japan) equipped with a Kromasil 100-5C18 chromatographic column (AkzoNobel, Sweden) (5 µm, 4.6 × 150 mm). The peptides were eluted with a gradient from 25 to 85% solvent A (0.1% trifluoroacetic in 100% acetonitrile) and 75 to 15% solvent B (0.1% trifluoroacetic in 100% water) at 1.0 ml/minute, with monitoring at 220 nm. The identities of pHLIP (Var7) and kVar7 were confirmed by mass spectrometric analysis using an Agilent 6125B mass spectrometer (Agilent Technologies, Inc., USA).

Next, 7 µmol/L PHLIP (Var7) and 2 mmol/L 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; diameter ≤ 50 nm) were dissolved in 10 mmol/L phosphate buffer (PB) at pH = 8 and equilibrated at room temperature for at least 10 h. The pH value of the solution was adjusted to 4.0 with 0.1 mol/L HCl.

CD spectra of pHLIP (Var7) were recorded at pH = 8.0 and pH = 4.0 in a 1-mm-path-length quartz cuvette using a spectropolarimeter. The experimental settings were as follows: bandwidth, 1 nm; wavelength range, 190–260 nm; wavelength step size, 1 nm; time-per-point, 0.5 s; temperature, 25°C.

A total of 1 mg peptide powder was dissolved in 1 mL 0.02 M phosphate-buffered saline (PBS). Ten microliters of the above-prepared solution was added to 90 μL PBS. Then, 20 μL of 1 mg/mL stannous chloride (in 0.01 M HCl), 20 μL sodium phosphate solution (8.2 mg/mL), and 74 MBq fresh ^99mTc-pertechnetate were added. The mixture was then placed in a 90°C water bath for 15 min. The product was purified in a Sephadex G25 column (1.0 × 25 cm) and eluted with PBS (0.1 M, pH = 7.4). The radiochemical yield and specific activity of ^99mTc-pHLIP (Var7) and ^99mTc-kVar7 were calculated.

The radiochemical purity of ^99mTc-pHLIP (Var7) and ^99mTc-kVar7 was determined by thin-layer chromatography (TLC). A thin polyamide layer was used as the stationary phase, and acetonitrile was used as the developing solvent. A sufficient amount of the labeled product was aspirated through a capillary pipette and spotted at the origin of the thin polyamide layer. The thin polyamide layer was air-dried and vertically placed in a test tube with developing solvent. The saturated thin polyamide layer was removed, dried, and placed on the TLC scanner for testing.

^99mTc-pHLIP (Var7) and ^99mTc-kVar7 were placed in PBS at room temperature (25°C) and in fresh human serum at 37°C. Samples were taken to test the radiochemical purity at 1, 4, 6, and 24 h after labeling.

MDA-MB-231 human breast cancer cells (Chinese Academy of Sciences) were cultured in an incubator at 37°C with 5% CO₂ in high-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% antibodies (penicillin–streptomycin mixture).

Animal experiments were approved by the ethics committee of Qingdao University, and the management of animals followed ethical standards. We built an MDA-MB-231 tumor model using four- to- five-week-old female BALB/c nude mice (Shanghai SLAC Laboratory Animal Co., Ltd, China). To do so, 0.1 mL of a cell suspension (50 µL PBS + 50 µL Matrigel) containing 1 × 10⁶ MDA-MB-231 cells was subcutaneously inoculated into the right axilla of each mouse.

a. When MDA-MB-231 cells grew to more than 90% confluence, they were digested. The cell concentration was adjusted to approximately 1.0 × 10⁵ cells/mL, and 1 mL of the cell solution was added to each well of a 12-well plate and cultured overnight in a 37°C cell incubator.

b. The medium was discarded, and the cells were washed with PBS twice. Then, 2 mL of fresh medium and 37 kBq ^99mTc-pHLIP (Var7) or ^99mTc-kVar7 (20-30 μL) were added to each well. We adjusted the pH values of the wells to four levels (pH 7.4, 7.0, 6.5, and 6.0).

c. After culturing for 30, 60, 90, 120, and 180 min, the supernatant was aspirated into a supernatant tube, and each well was washed two times with PBS at the corresponding pH (7.4, 7.0, 6.5, or 6.0). The cleaning solution was also added into the supernatant tube. The cells were digested fully with 0.25% trypsin, and the cell suspension was aspirated into a cell tube and washed twice with PBS. The cleaning solution was also added to the cell tube. A γ counter was applied to measure the radioactivity count of the cell tube (B) and supernatant tube (F). The cell-binding fraction of ^99mTc-pHLIP (Var7) or ^99mTc-kVar7 = B/(B+F) × 100%.

When tumor diameters reached 0.8–1.0 cm, 24 tumor-bearing nude mice were selected for an in vivo biodistribution experiment. Each nude mouse was injected with approximately 1480 kBq/200 μL ^99mTc-pHLIP (Var7) or ^99mTc-kVar7 through the tail vein. The mice were sacrificed humanely 1 h, 2 h, 4 h, and 6 h after injection, and their tissues and organs, such as the brain, heart, lung, liver, kidney, stomach, small intestine, blood, muscle, bone, and tumor, were weighed and measured for their radioactivity count. The results are expressed as the percentage of injected dose per gram of tissue (%ID/g).

When tumor diameters reached 0.8–1.0 cm, six tumor-bearing nude mice were selected for imaging. Each nude mouse was injected with approximately 11.1 MBq/200 μL ^99mTc-pHLIP (Var7) or ^99mTc-kVar7 through the tail vein. The tumor-bearing mice were anesthetized with 2% isoflurane at 1 h, 2 h, 4 h, and 6 h after injection and were fixed on the imaging bed of a small-animal SPECT/CT imaging system in the prone position. A pinhole collimator was applied. The main parameters were a matrix size of 256 × 256, magnification power of 1.45, and acquisition time at each time point of 10 min.

SPSS 24.0.0.0 (IBM) statistical software was applied for data processing. Quantitative data are expressed as the mean (M) ± standard deviation (SD). Variables were compared via one-way analysis of variance. P < 0.05 was deemed significant.

pHLIP (Var7) and kVar7 were successfully synthesized via solid-phase peptide synthesis. HPLC analysis of pHLIP (Var7) showed the formation of several compounds consisting of a major product (98.12%, retention time of 12.85 min) accompanied by two smaller peaks at retention times of 12.75 and 13.24 min. HPLC analysis of kVar7 showed the formation of several compounds consisting of a major product (98.03%, retention time of 11.82 min) accompanied by two smaller peaks at retention times of 11.47 and 12.33 min.

MS analysis of pHLIP (Var7) showed two mass peaks with m/z values of 1131.70 [(M+3H)3H+] and 1697.13 [(M+2H)2H+]. MS analysis of kVar7 showed two mass peaks with m/z values of 1130.80 [(M+3H)3H+] and 1695.70 [(M+2H)2H+]. The measured molecular weights of pHLIP (Var7) and kVar7 (3392.10 and 3389.40, respectively) were consistent with the theoretical molecular weights (3391.80 and 3388.98, respectively).

The secondary structure of pHLIP (Var7) was determined by CD at pH 8.0 and 4.0. Figure 1 shows that pHLIP (Var7) exhibited a typical pH-dependent transition from a nonstructural conformation to an α-helical structure when the pH decreased from 8.0 (blue line) to 4.0 (red line) in the presence of POPC.

^99mTc-pHLIP (Var7) had a radiochemical yield of 99.49 ± 0.17% and purity of 99.63 ± 0.44%. ^99mTc-kVar7 had a radiochemical yield of 99.46 ± 0.14% and purity of 99.63 ± 0.35%. The specific activities of ^99mTc-pHLIP (Var7) and ^99mTc-kVar7 were both 7.36 ± 0.01 MBq/μg.

The radiochemical purity of both ^99mTc-pHLIP (Var7) and ^99mTc-kVar7 was > 99% in PBS at room temperature at 24 h and > 96% in fresh human serum at 37°C at 24 h ( Figure 2 ).

At pH = 6.0, 6.5, and 7.0, the binding fractions of ^99mTc-pHLIP (Var7) to MDA-MB-231 cells steadily increased and reached the highest levels at 180 min, which were 3.33 ± 0.12%, 3.02 ± 0.04%, and 2.87 ± 0.03%, respectively. At pH = 7.4, the binding fractions of ^99mTc-pHLIP (Var7) to MDA-MB-231 cells remained low at each time point (30, 60, 90, 120, and 180 min). The binding fractions of ^99mTc-kVar7 to MDA-MB-231 cells also remained low at each time point (30, 60, 90, 120, and 180 min) at all tested pH values (6.0, 6.5, 7.0, and 7.4; Figure 3 ).

The cell-binding fractions of ^99mTc-pHLIP (Var7) at pH = 6.0, 6.5, and 7.0 at various time points (30, 60, 90, 120, 180 min) were significantly higher than the cell-binding fractions at pH = 7.4 (P < 0.01) and than the cell-binding fractions of ^99mTc-kVar7 at pH = 6.0, 6.5, 7.0, and 7.4 (P < 0.01).

The distribution of ^99mTc-pHLIP (Var7) in tumors at 1, 2, 4, and 6 h was 3.63 ± 0.92, 6.11 ± 0.66, 5.33 ± 0.40, and 3.72 ± 0.94% ID/g, respectively, all of which were higher than the distribution levels of ^99mTc-kVar7 in tumors (P < 0.01). Of the measured off-target organs, ^99mTc-pHLIP (Var7) and ^99mTc-kVar7 showed high distribution levels in the liver and blood ( Figure 4A ; n = 3).

The tumor/nontumor (T/NT) ratio increased over time in the ^99mTc-pHLIP (Var7) group, while no significant increasing trend was seen in the T/NT ratio in the ^99mTc-kVar7 group. The T/NT ratio in the ^99mTc-pHLIP (Var7) group was higher at each time point than in the ^99mTc-kVar7 group (P < 0.05; Figure 4B ; n = 3).

SPECT/CT imaging was performed at 1, 2, 4, and 6 h after the injection of ^99mTc-pHLIP (Var7) or ^99mTc-kVar7 ( Figure 5 ; n = 3). Tumors were clearly imaged at each time point after injection of ^99mTc-pHLIP (Var7) but not after injection of ^99mTc-kVar7. The liver was clearly imaged in both groups at each time point.