of 225Ac and its progeny within the nanosystem. 2.3.2. Cell Viability Assay and Dose to the Nucleus

2.3.2. Cell Viability Assay and Dose to the Nucleus Cell viability of PC-3 and HEP-G2 cells (SR-BI positive), and fibroblasts (negative control), was evaluated after treatment with a) 225Ac-DOTA-benzene-p-SCN (control) and Cell viability of PC-3 and HEP-G2 cells (SR-BI positive), and fibroblasts (negative control), was evaluated after treatment with a) <sup>225</sup>Ac-DOTA-benzene-p-SCN (control) and b) <sup>225</sup>Ac-rHDL at 37 ◦C for 1 h.

b) 225Ac-rHDL at 37 °C for 1 h. The cell viability of PC-3 and HEP-G2 treated with 225Ac-DOTA-benzene-p-SCN was not significantly different between them nor with regard to that of fibroblasts (*p* > 0.05, two-way ANOVA) (Figure 4a). Furthermore, a low 225Ac-DOTA-benzene-p-SCN internalization was observed in all cell lines (Figure 4b). These results indicate that the 225Ac-DOTA-benzene-p-SCN system, despite being a hydrophobic compound, does not have The cell viability of PC-3 and HEP-G2 treated with <sup>225</sup>Ac-DOTA-benzene-p-SCN was not significantly different between them nor with regard to that of fibroblasts (*p* > 0.05, two-way ANOVA) (Figure 4a). Furthermore, a low <sup>225</sup>Ac-DOTA-benzene-p-SCN internalization was observed in all cell lines (Figure 4b). These results indicate that the <sup>225</sup>Ac-DOTA-benzene-p-SCN system, despite being a hydrophobic compound, does not have an interaction mechanism with the surface of the cell membrane that would allow <sup>225</sup>Ac to be internalized into the cell cytoplasm. In contrast, the PC-3 and HEP-G2 cells that received treatment with the <sup>225</sup>Ac-rHDL nanosystem showed cell viability of 54.67<sup>±</sup> 3.16% and 53.12 ± 2.93% at 3 h, respectively, with a slight increase at 24 h (62.08 ± 2.44% for

PC-3 and 64.32 ± 3.38% HEP-G2) (Figure 4c). A possible explanation could be that cell repair mechanisms are stimulated upon receiving an initial dose of radiation, promoting cell proliferation in both cell lines [13,14]. Nevertheless, cell viability decreased to 3.22 <sup>±</sup> 0.72% for the PC-3 cell line at 192 h after <sup>225</sup>Ac-rHDL treatment and to 1.79 <sup>±</sup> 0.23% for the HEP-G2 cell line, as a result of the significant internalization of radiation (Figure 4d). In the case of fibroblasts, a cell viability of 81.2 ± 7.21%, and the lowest internalization index regarding the two cell lines (PC-3 and HEP-G2), was observed after <sup>225</sup>Ac-rHDL treatment (Figure 4d). As is known, fibroblasts poorly express the SR-BI receptor, and when they interact with rHDL, a different mechanism with slight and unspecific internalization can also occur [15–17]. 3 and 64.32 ± 3.38% HEP-G2) (Figure 4c). A possible explanation could be that cell repair mechanisms are stimulated upon receiving an initial dose of radiation, promoting cell proliferation in both cell lines [13,14]. Nevertheless, cell viability decreased to 3.22 ± 0.72% for the PC-3 cell line at 192 h after 225Ac-rHDL treatment and to 1.79 ± 0.23% for the HEP-G2 cell line, as a result of the significant internalization of radiation (Figure 4d). In the case of fibroblasts, a cell viability of 81.2 ± 7.21%, and the lowest internalization index regarding the two cell lines (PC-3 and HEP-G2), was observed after 225Ac-rHDL treatment (Figure 4d). As is known, fibroblasts poorly express the SR-BI receptor, and when they interact with rHDL, a different mechanism with slight and unspecific internalization can also occur [15–17].

an interaction mechanism with the surface of the cell membrane that would allow 225Ac to be internalized into the cell cytoplasm. In contrast, the PC-3 and HEP-G2 cells that received treatment with the 225Ac-rHDL nanosystem showed cell viability of 54.67± 3.16% and 53.12 ± 2.93% at 3 h, respectively, with a slight increase at 24 h (62.08 ± 2.44% for PC-

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**Figure 4.** Cell viability assay: (**a**) PC-3, HEP-G2, and fibroblast cell lines treated with the 225Ac-DOTA-benzene-p-SCN system (control); note that viability above 80% was maintained after 192 h for all cell lines. (**b**) Internalization of 225Ac-DOTA-benzene-p-SCN in PC-3, HEP-G2 cells, and fibroblasts; note that the low cellular internalization of the 225Ac-DOTA-benzene-p-SCN complex (without significant difference among cells, *p* > 0.05, two-way ANOVA) correlates with a relatively low effect on cell viability at 192 h. (**c**) PC-3, HEP-G2, and fibroblast cell lines treated with the 225AcrHDL system; note a viability of 62–64% after 24 h and less than 3.5% at 192 h for the PC-3 and HEP-G2 cell lines, which overexpress the SR-BI protein. (**d**) Internalization of 225Ac-rHDL in PC-3, HEP-G2 cells, and fibroblasts; note a significant cellular internalization of the 225Ac rHDL complex in PC-3 and HEP-G2 cells, which correlates with a significant effect on cell viability. **Figure 4.** Cell viability assay: (**a**) PC-3, HEP-G2, and fibroblast cell lines treated with the <sup>225</sup>Ac-DOTAbenzene-p-SCN system (control); note that viability above 80% was maintained after 192 h for all cell lines. (**b**) Internalization of <sup>225</sup>Ac-DOTA-benzene-p-SCN in PC-3, HEP-G2 cells, and fibroblasts; note that the low cellular internalization of the <sup>225</sup>Ac-DOTA-benzene-p-SCN complex (without significant difference among cells, *p* > 0.05, two-way ANOVA) correlates with a relatively low effect on cell viability at 192 h. (**c**) PC-3, HEP-G2, and fibroblast cell lines treated with the <sup>225</sup>Ac-rHDL system; note a viability of 62–64% after 24 h and less than 3.5% at 192 h for the PC-3 and HEP-G2 cell lines, which overexpress the SR-BI protein. (**d**) Internalization of <sup>225</sup>Ac-rHDL in PC-3, HEP-G2 cells, and fibroblasts; note a significant cellular internalization of the <sup>225</sup>Ac rHDL complex in PC-3 and HEP-G2 cells, which correlates with a significant effect on cell viability.

The calculation of the absorbed dose, from the cytoplasm to the nucleus of the different cell lines at different times after treatment with the <sup>225</sup>Ac-rHDL system, was also evaluated (Table 2). The value used as the dose factor (DF) for the dose calculations was (*DF<sup>α</sup>* <sup>+</sup> *DFe*,*ph* )*Ac*−225(*n*←*Cy*) <sup>=</sup>8.96 <sup>×</sup> <sup>10</sup>−<sup>2</sup> Gy/Bq·s, where the four alpha, electron (e), and photon (ph) emissions of the daughters produced by each <sup>225</sup>Ac nuclear transformation were considered (MIRDcellV2.1 software) [18].

**Table 2.** Mean radiation-absorbed dose (*Gy*) from the cytoplasm (*Cy*) to the cell nucleus (*n*), considering the internalized activity (Bq/cell) with respect to the total initial activity administered as treatment of <sup>225</sup>Ac-rHDL (4.0 kBq/well) (1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well) for each cell line.


The absorbed radiation dose to the cell nucleus at 192 h after <sup>225</sup>Ac-rHDL treatment was 1025.5 Gy (256.4 Gy per kBq administered in the well) for HEP-G2 and 682.5 Gy (170.6 Gy per kBq administered in the well) for PC-3, which is 43 and 29 times higher than in the case of fibroblasts, respectively. Therefore, it was demonstrated that the <sup>225</sup>Ac-rHDL nanosystem is capable of delivering doses of radiation to cause a significant cytotoxic effect, as a result of the ability to internalize <sup>225</sup>Ac into the cell and the multiple alpha-emitting daughter radionuclides generated inside the cell; that is, the SR-BI receptor, expressed on HEP-G2 and PC-3 cells, interacts with the endogenous rHDL lipoproteins, which induces the deposit of the rHDL content (225Ac) directly into the cytoplasm of the cells. An advantage of the alpha radiotherapy system, in addition to its high specificity attributed to rHDL, is that the high number of short-path ionizations, which damage the DNA structure, also inhibit cell repair mechanisms [19,20]. On the other hand, a 20% decrease in cell viability was also observed in fibroblasts with a dose of 8.1 Gy at 48 h after treatment, which can be attributed to the radiosensitivity exhibited by fibroblasts (Figure 4c) [19].
