*3.9. Biodistribution Studies*

A total of sixty-three animals were injected in three groups: one group of healthy mice (*n* = 21), and two groups of mice bearing HEP-G2 tumors (*n* = 42). In vivo studies with Balb-C mice (7-week-old; weight of 18–20 g) were performed in accordance with the corresponding ethical regulations for handling laboratory animals ("Official Mexican Standard" NOM-062-ZOO-1999). Mice were injected with the <sup>225</sup>Ac-rHDL system (50 µL, 0.9 MBq) in the tail vein and sacrificed at 0.5, 1, 3, 24, 48, 96, and 192 h (*n* = 3). The spleen, liver, kidneys, lungs, intestine, and heart were removed to measure their activity in a (NaI(Tl)) radioactivity counter, to determine the percentage of injected dose (% ID) per organ, with regard to the total injected activity.

Nude mice bearing HEP-G2 tumors (0.22 ± 0.03 g) were injected intratumorally with the <sup>225</sup>Ac-rHDL system (50 µL, 1.0 MBq) and sacrificed at 0.5, 1, 3, 24, 48, 96, and 192 h (*n* = 3). As a control, the <sup>225</sup>Ac-DOTA-benzene-p-SCN system (50 µL, 1.0 MBq) (*n* = 3) was administered and mice were sacrificed at the same time. In all mice, the tumor, liver, spleen, and kidney were removed for activity measurement in a (NaI(Tl)) radioactivity counter, to determine the percentage of injected dose (% ID) per organ, with regard to the total injected activity.

The values obtained from % ID of each organ or tumor were adjusted to exponential functions to obtain the biokinetic models (*qh*(*t*)). The *Ah*(*t*) functions were obtained by decay-correcting the biokinetic models; that is, by adding to the biological constant (*λB*) the radioactive constant (*λR*), as follows (Equation (2)):

$$A(t) \ = B e^{-(\lambda\_R + \lambda\_B)t} + \mathcal{C}e^{-(\lambda\_R + \lambda\_B)t} + D e^{-(\lambda\_R + \lambda\_B)t} \tag{2}$$

To obtain the biokinetic model, each of the radionuclides generated in the <sup>225</sup>Ac decay chain was considered as described above. The total number of nuclear transformations (N) in each murine organ were obtained by the mathematical integration (from t = 0 to t = ∞) of the biokinetic models. The DF values were obtained via the OLINDA 2.0 software, considering a normalized tumor mass of 1.0 g and all the progeny of <sup>225</sup>Ac.
