Micro-PET study of 68Ga-HZ20 in the rhACE2-subcutaneous model
In KM model mice, different doses (0.1, 0.2, 0.4 and 0.8 nmol) of commercial rhACE2 protein were injected into the right shoulder. The molecular weight of the rhACE2 protein was approximately 100 kDa according to mass spectrometry (Fig. S3). PET imaging showed that68Ga-HZ20 had high specificity and sensitivity for rhACE2 in vivo (Fig. 2A, Fig. S4). This probe can locate regions with rhACE2 aggregation and reveal the distribution of rhACE2. Compared to the contralateral side injected with saline control, micro-PET/CT imaging clearly showed the specific uptake of the probe at the rhACE2 injection site, and the uptake rate increased with the rhACE2 injection dose. Similarly, at the same time point after probe injection, SUVmax also increased with the amount of rhACE2 (0.1, 0.2, 0.4 and 0.8 nmol). Furthermore, in the 0.4 nmol and 0.8 nmol rhACE2 injection groups, probe uptake increased over time, reaching a maximum at 120 min after injection (1.70±0.08, 3.13±0.07) and then slowly decreasing (Fig. 2C). The ratio of target site to muscle for response probe targeting and specificity also increased with increasing rhACE2 injection to 2.50±0.41, 8.24±0.47, 15.57±0.49, and 25.45±1.48, respectively, at 240 min (Fig. 2D). Once PET image acquisition was complete, the rhACE2 (0.2 nmol, 0.4 nmol) injection site and the main organs were sampled for ex vivo imaging (Fig. 2E), and the results showed significant dose-dependent uptake of the probe at the rhACE2 injection site. High uptake was detected in the kidneys, but there was almost no uptake in other normal organs. The correlation between SUVmax and rhACE2 dose was analyzed 120 min after injection, and the R2 was 0.9914 (Fig. 2F). We selected the 0.4 nmol rhACE2 group for a 14.63 nmol HZ20 peptide blockade study, and the uptake of probe at the rhACE2 injection site in the blockade group was significantly reduced (Fig. 2B, Fig. S5). At all time points, there was a considerable difference between the SUVmax of the HZ20 block group and that of the non-block group, which was 1.70±0.08 compared to 0.40±0.01 (p<0.0001) at 120 min (Fig. 2G). After micro-PET imaging, rhACE2 residence was verified by western blotting, and the results showed that the rhACE2 protein was successfully localized at the subcutaneous injection site and remained stable in vivo within 240 min (Fig. 2H).