*2.4. Biodistribution Assay*

The biodistribution profile in healthy mice, after intravenous administration of the <sup>225</sup>Ac-rHDL nanosystem, showed an accumulation of activity, mainly in the liver, with a value of 40.4 ± 2.12% ID, but with a relatively rapid clearance, reaching 6.01 ± 1.07% ID at 192 h (Table 3). Radioactivity in the liver is rapidly removed and excreted due to the liver metabolic dynamics of HDL [21]. Given its nanometric size, <sup>225</sup>Ac-rHDL elimination occurs both through the hepatobiliary (2.88 ± 0.12% ID at 0.5 h in intestine) and renal (4.85 ± 0.89% ID at 0.5 h in kidney) pathways. Because it is inaccurate to establish the percentage of radioactivity accumulated in the total blood of mice, the % ID in blood was not included in Table 3. However, the activity in blood was 3.91 ± 0.92% ID/g and 1.97 ± 0.81% ID/g at 0.5 h and 24 h, respectively. It should be considered that the elimination time of rHDL in blood plasma is relatively prolonged due to its lipoprotein nature (the halflife in plasma fluctuates between 6 h and 24 h after HDL administration) [21]. Therefore, a prolonged blood circulation of <sup>225</sup>Ac-rHDL is expected, which could be an advantage in therapeutic applications given that the nanosystem can produce a significant tumor accumulation of radioactivity before radiopharmaceutical elimination [22]. As expected, rHDL was captured by the liver tissue due to its high expression of SR-BI. These results confirm the specificity and passive targeting of SR-BI receptors, as well as their in vivo affinity for rHDL lipoproteins through a natural process of selective uptake and elimination. This behavior is of particular importance when demonstrating that it is possible to minimize long retention of <sup>225</sup>Ac-rHDL in healthy organs, because, due to the nature of the alphaemitting daughter radionuclides, high damage to healthy tissues could be produced. rHDL lipoproteins through a natural process of selective uptake and elimination. This behavior is of particular importance when demonstrating that it is possible to minimize

long retention of 225Ac-rHDL in healthy organs, because, due to the nature of the alpha-

**Table 3.** Biodistribution of the <sup>225</sup>Ac-rHDL nanosystem in healthy mice (Balb-c) after intravenous injection. The percentage of the injected dose per organ (% ID), at various times, is shown (mean ± SD, *n* = 3). emitting daughter radionuclides, high damage to healthy tissues could be produced. **Table 3.** Biodistribution of the 225Ac-rHDL nanosystem in healthy mice (Balb-c) after intravenous injection. The percentage of the injected dose per organ (% ID), at various times, is shown (mean ±


*Molecules* **2022**, *27*, x FOR PEER REVIEW 8 of 15

Biodistribution in mice with induced HEP-G2 tumors was performed in order to assess tumor uptake activity as a function of time. The biodistribution profile of <sup>225</sup>Ac-rHDL, after intratumoral administration, shows that the radioactivity of the system is retained within the tumor masses (Figure 5a), with an uptake of 90.16 ± 2.52% at 0.5 h, with regard to the initially-administered activity. These results confirm the potential of <sup>225</sup>Ac-rHDL to produce a localized cytotoxic effect. Low <sup>225</sup>Ac-rHDL accumulation was observed in liver (0.65% ID at 24 h), kidney (0.51% ID at 24 h), and spleen (0.34% ID at 24 h), which indicates that <sup>225</sup>Ac does not leak in vivo from the rHDL nanocapsule, because unchelated actinium ( <sup>225</sup>Ac3+) accumulates significantly in the liver [23]. Biodistribution in mice with induced HEP-G2 tumors was performed in order to assess tumor uptake activity as a function of time. The biodistribution profile of 225Ac-rHDL, after intratumoral administration, shows that the radioactivity of the system is retained within the tumor masses (Figure 5a), with an uptake of 90.16 ± 2.52% at 0.5 h, with regard to the initially-administered activity. These results confirm the potential of 225Ac-rHDL to produce a localized cytotoxic effect. Low 225Ac-rHDL accumulation was observed in liver (0.65% ID at 24 h), kidney (0.51% ID at 24 h), and spleen (0.34% ID at 24 h), which indicates that 225Ac does not leak in vivo from the rHDL nanocapsule, because unchelated actinium (225Ac3+) accumulates significantly in the liver [23].

**Figure 5.** Comparison of the biokinetic profile between the (**a**) 225Ac-rHDL nanosystem and (**b**) 225Ac-DOTA-benzene-p-SCN in nude mice bearing HEP-G2 tumors after intratumoral administra-**Figure 5.** Comparison of the biokinetic profile between the (**a**) <sup>225</sup>Ac-rHDL nanosystem and (**b**) <sup>225</sup>Ac-DOTA-benzene-p-SCN in nude mice bearing HEP-G2 tumors after intratumoral administration.

When 225Ac-DOTA-benzene-p-SCN was injected intratumorally in nude mice bearing HEP-G2 tumors, a very rapid clearance of radioactivity from the tumor was observed (Figure 5b); and almost 10-fold higher uptake in liver and spleen was seen with regard to the 225Ac-rHDL biodistribution pattern (Figure 5b). These findings suggest that although the DOTA chelating agent is highly stable for positive trivalent radiometals [12,24,25], 225Ac-DOTA-benzene-p-SCN does not remain in the tumor, because the recoil energy of the 225Ac daughters (1000 times greater than the binding energy of any chemical compound) is possibly causing the breaking of chemical bonds, with the consequent retention When <sup>225</sup>Ac-DOTA-benzene-p-SCN was injected intratumorally in nude mice bearing HEP-G2 tumors, a very rapid clearance of radioactivity from the tumor was observed (Figure 5b); and almost 10-fold higher uptake in liver and spleen was seen with regard to the <sup>225</sup>Ac-rHDL biodistribution pattern (Figure 5b). These findings suggest that although the DOTA chelating agent is highly stable for positive trivalent radiometals [12,24,25], <sup>225</sup>Ac-DOTA-benzene-p-SCN does not remain in the tumor, because the recoil energy of the <sup>225</sup>Ac daughters (1000 times greater than the binding energy of any chemical compound) is possibly causing the breaking of chemical bonds, with the consequent retention of <sup>225</sup>Ac3+ and its progeny in the liver as ionic forms [23].

of 225Ac3+ and its progeny in the liver as ionic forms [23]. Based on the biodistribution results in healthy and tumor-bearing mice, it is possible to propose <sup>225</sup>Ac-rHDL as a convenient natural nanocarrier for the use of <sup>225</sup>Ac in targeted radiotherapy, in a safe and efficient manner.

tion.

Table 4 shows the biokinetic models of the <sup>225</sup>Ac-rHDL nanosystem in tumor and radiation source organs. The results show both the biological or pharmacokinetic model (*qh*(*t*)) and the radiopharmacokinetic model (*Ah*(*t*)), the latter associated with the number of nuclear transformations that occurred in each tissue (N) for the radiation-absorbed dose calculation.


**Table 4.** Biokinetic models and average radiation-absorbed doses in different tissues (liver, kidney, spleen, and HEP-G2 tumor) of mice intratumorally injected with <sup>225</sup>Ac-rHDL (1 MBq).

The highest dose occurred in the tumor (649 Gy), while doses were low to the other organs (Table 4), indicating that the energy deposited by <sup>225</sup>Ac produces ablative radiation doses to the target malignant lesions, avoiding cytotoxic effects on healthy tissues.

As is known, some of the key properties of <sup>225</sup>Ac as a radionuclide for targeted alpha radiotherapy of micrometastases are: (1) range in tissue of a few cell diameters, (2) high linear energy transfer, which leads to direct damage of the DNA structure (LET = 100 KeV/µm), (3) half-life of 10 days, which allows sufficient time for the administration of the dose and its binding and retention in tumor masses, (4) emission of four alpha particles per nuclear transformation [26–29]. Another important aspect to consider is that <sup>225</sup>Ac requires a much lower activity to produce cytotoxic effects at the cellular level with regard to beta emitters, because its energy is deposited in an extremely localized manner. Furthermore, <sup>225</sup>Ac-rHDL have the appropriate physicochemical properties and size to reach the tumor tissue and locally deposit their content (225Ac) within the cytoplasm of tumor cells, being a convenient vehicle for targeted alpha-particle radiotherapy.

Although the mechanism by which the interaction of the SR-BI receptor with HDL occurs has not yet been fully elucidated, it is possible that there is a non-aqueous "channel" in the SR-BI receptor that can accommodate cholesterol esters in such a way that it can couple with rHDL and capture its content through an internal tunnel [30] (Figure 6).

The biokinetic profile of the <sup>225</sup>Ac-rHDL system is comparable to those reported for 99mTc-HYNIC-DA-rHDL, with regard to tumor uptake and clearance because the delivery mechanism is the same [5]. In addition, the accumulation of activity in organs that express SR-BI (mainly liver, spleen, and kidneys) is comparable.

**Figure 6.** Cellular interaction mechanism of rHDL containing 225Ac. Once endogenous rHDL interacts with the SR-BI receptor found on the surface of the cell membrane, its content is released directly into the cell's cytoplasm. Therefore, 225Ac-rHDL is an effective nanosystem for depositing within the malignant cells, an in vivo generator for alpha particle radiotherapy, which delivers lethal radiation doses to cells due to the four alpha particles emitted by 225Ac and its progeny in each nuclear transformation. The ionizations produced by 225Ac and its daughters produce direct damage to the DNA structure, preventing tumor proliferation. **Figure 6.** Cellular interaction mechanism of rHDL containing <sup>225</sup>Ac. Once endogenous rHDL interacts with the SR-BI receptor found on the surface of the cell membrane, its content is released directly into the cell's cytoplasm. Therefore, <sup>225</sup>Ac-rHDL is an effective nanosystem for depositing within the malignant cells, an in vivo generator for alpha particle radiotherapy, which delivers lethal radiation doses to cells due to the four alpha particles emitted by <sup>225</sup>Ac and its progeny in each nuclear transformation. The ionizations produced by <sup>225</sup>Ac and its daughters produce direct damage to the DNA structure, preventing tumor proliferation.

#### The biokinetic profile of the 225Ac-rHDL system is comparable to those reported for 99mTc-HYNIC-DA-rHDL, with regard to tumor uptake and clearance because the delivery **3. Materials and Methods**

mechanism is the same [5]. In addition, the accumulation of activity in organs that express SR-BI (mainly liver, spleen, and kidneys) is comparable. **3. Materials and Methods** Free cholesterol (FC), egg yolk phosphatidylcholine (EYPC), cholesterol oleate (CE), and sodium cholate reagents were supplied by Landsteiner Scientific, and apolipoprotein Apo AI and human plasma by Alfa Aesar (Thermo Fisher, Tewksbury, MA, USA). Buffer Tris-EDTA was prepared in the laboratory. Dialysis tubbing (14000 Daltons) was obtained from MEMBRA CEL® (Thermo Fisher, Tewksbury, MA, USA). Macrocycle S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane acid, DOTA-benzene-p-SCN (p-SCN-Bn-Free cholesterol (FC), egg yolk phosphatidylcholine (EYPC), cholesterol oleate (CE), and sodium cholate reagents were supplied by Landsteiner Scientific, and apolipoprotein Apo AI and human plasma by Alfa Aesar (Thermo Fisher, Tewksbury, MA, USA). Buffer Tris-EDTA was prepared in the laboratory. Dialysis tubbing (14000 Daltons) was obtained from MEMBRA CEL® (Thermo Fisher, Tewksbury, MA, USA). Macrocycle S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane acid, DOTA-benzene-p-SCN (p-SCN-Bn-DOTA) was obtained from Macrocyclics (Dallas, TX, USA). Actinium-225 ( <sup>225</sup>Ac), as <sup>225</sup>AcCl3, was supplied from ITM, Germany. The bladder fibroblast cell human cell line, PC-3, and HEP-G2 cell lines (SR-BI positive) were obtained from American Type Culture Collection (ATCC®, Manassas, VA, USA).

#### DOTA) was obtained from Macrocyclics (Dallas, TX, USA). Actinium-225 (225Ac), as *3.1. Preparation of Lipid Micelles and rHDL*

225AcCl3, was supplied from ITM, Germany. The bladder fibroblast cell human cell line, PC-3, and HEP-G2 cell lines (SR-BI positive) were obtained from American Type Culture Collection (ATCC®, Manassas, VA, USA). *3.1. Preparation of Lipid Micelles and rHDL* The synthesis of the lipid micelles was performed via the microfluidic method with hydrodynamic flow focusing (MFH), using a Dolomite Microfluidics System (Dolomite) equipped with a 150-µm 5-way glass 3D chip, with dimensions of 22.5 mm long, 15 mm wide, and 4 mm high. A 4-way H interface with two 4-way linear connectors and two connector seals were used. Flow patterns in the mixing zone of the microfluidic device The synthesis of the lipid micelles was performed via the microfluidic method with hydrodynamic flow focusing (MFH), using a Dolomite Microfluidics System (Dolomite) equipped with a 150-µm 5-way glass 3D chip, with dimensions of 22.5 mm long, 15 mm wide, and 4 mm high. A 4-way H interface with two 4-way linear connectors and two connector seals were used. Flow patterns in the mixing zone of the microfluidic device were visualized using a high-speed digital microscope (Meros, Dolomite). Fluids were administered by two pressure pumps. The first pump for lipid, ethanol, and chloroform delivery. The second pump for the administration of PBS, with the corresponding flow sensors. The chip used presents a hydrodynamic flow approach that allows the formation of a stable laminar flow confined by two lateral flows.

were visualized using a high-speed digital microscope (Meros, Dolomite). Fluids were administered by two pressure pumps. The first pump for lipid, ethanol, and chloroform delivery. The second pump for the administration of PBS, with the corresponding flow sensors. The chip used presents a hydrodynamic flow approach that allows the formation of a stable laminar flow confined by two lateral flows. rHDL was prepared in two steps. In the first, the formation of lipid micelles was performed via the microfluidic method, for which an organic solution consisting of 2 mL of methanol: chloroform (1:0.02 *v*/*v*), containing a mixture of the following lipids, was used: 300 µL of egg yolk phosphatidylcholine (10 mg/mL in methanol), 7 µL of free cholesterol (10 mg/mL in methanol), and 7.5 µL of cholesterol ester (4 mg/mL in chloroform). PBS (pH 7.4) was used as aqueous phase. Both solutions were filtered through a 0.22-µm PVDF

membrane before being introduced into the microfluidic device. The organic phase was placed in the central channel and the aqueous solution in the coaxial channels, adjusting the organic phase to a flow of 50 µL/min and the aqueous phase to 500 µL/min, with a total flow ratio (TRF) of 550 µL/min and a flow rate ratio (FRR) of 47. Once the micelles were obtained, the size was measured by dynamic light scattering (DLS) (Nanotrac Wave, Model MN401, Microtract, Montgomeryville, PA, USA). Dialysis for purification (5 ◦C; 24 h) was performed using a 14000-kDa membrane (MEBRA CEL®, Thermo Fisher, Tewksbury, MA, USA) with PBS pH 7.4 to remove any residual solvent. Then, 4 mg/mL of Apo AI and 140 µL of sodium cholate (20 mg/mL) were added. Dialysis was performed again with stirring at 5 ◦C for 24 h to remove excess surfactant. The obtained solution was filtered using a 0.45-µm Millipore filter and stored at 4 ◦C.
