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).