*2.1. rHDL Assembly and Chemical Characterization*

rHDL nanoparticles, generated from lipid micelles assembled in a microfluidic system, followed by the incorporation of Apo AI (Figure 1), were obtained with monomodal and monodisperse size distributions, with a hydrodynamic diameter of 13.14 ± 0.20 nm and a polydispersity index of 0.176, as determined by dynamic light scattering (DLS) (Table 1). In addition, transmission electron microscopy (TEM) micrographs demonstrated the presence of nanoparticles with a mean diameter of 11.10 ± 0.17 nm, spherical shape, and a homogeneous and uniform distribution (Table 1 and Figure 2). The protein content analysis of rHDL indicated an Apo AI concentration of 0.131 mg/mL.

**Figure 1.** (**a**) Synthesis of lipid micelles. Preparation of micelles via a microfluidic system with hydrodynamic flow focusing, using a 5-way glass 3D chip that allows the diffusion of lipids in water, as well as water in alcohol, until its concentration decreases below the limit of lipid solubility, triggering the formation of lipid micelles. (**b**) rHDL synthesis. In the second step, the incorporation of apolipoprotein Apo AI was performed for the formation of rigid rHDL. **Figure 1.** (**a**) Synthesis of lipid micelles. Preparation of micelles via a microfluidic system with hydrodynamic flow focusing, using a 5-way glass 3D chip that allows the diffusion of lipids in water, as well as water in alcohol, until its concentration decreases below the limit of lipid solubility, triggering the formation of lipid micelles. (**b**) rHDL synthesis. In the second step, the incorporation of apolipoprotein Apo AI was performed for the formation of rigid rHDL.

**Table 1.** Physical characteristics of micelles and rHDL nanoparticles. **Table 1.** Physical characteristics of micelles and rHDL nanoparticles.


<sup>1</sup> Not applicable. <sup>1</sup> Not applicable.

The FT-IR spectra of the micelles and rHDL are shown in Figure 2. The band at 3400 cm−1, observed in the spectrum of lipid micelles and characteristic of the (OH)ʋ groups of the cholesterol molecule, was shifted to 3287 cm−1 after Apo AI addition, which is associated with the stretching (OH)ʋ vibration of water molecules remaining in the interface between the lipoprotein and cholesterol, as well as indicative of the Apo AI incorporation to the lipid micelles due to interactions of the lysine primary amine residues with the lipid groups [8,9]. The band of highest intensity, observed at 2955 cm−1, corresponds to an asymmetric stretch (CH)ʋ, characteristic of the methyl groups present in the phospholipid chains, while the band at 2854 cm−1 corresponds to the symmetric stretch (CH)ʋ of the methylene groups that constitute the rHDL. However, in the spectrum obtained from the lipid micelles, these signals may be superimposed by the broad band of the vibration (OH)ʋ. The region from 1500 cm−1 to 1800 cm−1 is characteristic of (C = O)ʋ vibrations of the ester bonds present in the phospholipid chains. At 1650 cm−1, there is an important vibration in the rHDL spectrum, which corresponds to Amide I of the α-helix of Apo AI. At 1538 cm−1, a band attributed to amide II corresponding to (N-H)ʋ bending and (C-N)ʋ stretching was observed [8,9]. The FT-IR spectra of the micelles and rHDL are shown in Figure 2. The band at 3400 cm−<sup>1</sup> , observed in the spectrum of lipid micelles and characteristic of the (OH)® groups of the cholesterol molecule, was shifted to 3287 cm−<sup>1</sup> after Apo AI addition, which is associated with the stretching (OH)® vibration of water molecules remaining in the interface between the lipoprotein and cholesterol, as well as indicative of the Apo AI incorporation to the lipid micelles due to interactions of the lysine primary amine residues with the lipid groups [8,9]. The band of highest intensity, observed at 2955 cm−<sup>1</sup> , corresponds to an asymmetric stretch (CH)®, characteristic of the methyl groups present in the phospholipid chains, while the band at 2854 cm−<sup>1</sup> corresponds to the symmetric stretch (CH)® of the methylene groups that constitute the rHDL. However, in the spectrum obtained from the lipid micelles, these signals may be superimposed by the broad band of the vibration (OH)®. The region from 1500 cm−<sup>1</sup> to 1800 cm−<sup>1</sup> is characteristic of (C=O)® vibrations of the ester bonds present in the phospholipid chains. At 1650 cm−<sup>1</sup> , there is an important vibration in the rHDL spectrum, which corresponds to Amide I of the α-helix of Apo AI. At 1538 cm−<sup>1</sup> , a band attributed to amide II corresponding to (N-H)® bending and (C-N)® stretching was observed [8,9].

In the case of lipid micelles, a wide band of lower intensity at 1674 cm−1 was attributed to the (C = C)ʋ vibration characteristic of the second ring of cholesterol. The broad band at 1400 cm−1, in the rHDL spectrum, corresponds to the asymmetric stretching of the (COO-)ʋ groups of the aspartate and glutamate residues, which are believed to be involved in the coupling of lipoproteins [10]. In the case of lipid micelles, a wide band of lower intensity at 1674 cm−<sup>1</sup> was attributed to the (C=C)® vibration characteristic of the second ring of cholesterol. The broad band at 1400 cm−<sup>1</sup> , in the rHDL spectrum, corresponds to the asymmetric stretching of the (COO-)® groups of the aspartate and glutamate residues, which are believed to be involved in the coupling of lipoproteins [10].

**Figure 2.** (**a**) Size distribution of lipid micelles by DLS, (**b**) TEM micrographs of lipid micelles, (**c**) size distribution of rHDL by DLS, (**d**) TEM micrographs of rHDL, (**e**) UV-Vis spectra of lipid micelles (red) and rHDL (blue), and (**f**) FT-IR spectra of lipid micelles (red) and rHDL (blue). **Figure 2.** (**a**) Size distribution of lipid micelles by DLS, (**b**) TEM micrographs of lipid micelles, (**c**) size distribution of rHDL by DLS, (**d**) TEM micrographs of rHDL, (**e**) UV-Vis spectra of lipid micelles (red) and rHDL (blue), and (**f**) FT-IR spectra of lipid micelles (red) and rHDL (blue).

In summary, the spectrum of lipid micelles contains characteristic bands attributable to the cholesterol and free cholesterol esters as components of the mixture for the formation of micelles. In the rHDL spectrum, the Apo AI association with acidic lipid membranes, through interactions between lysine residues and negatively-charged lipid groups, can be appreciated. Interactions with the anionic phospholipids increase the Apo AI α-helix content, which is also an important factor in recognizing the specific molecular In summary, the spectrum of lipid micelles contains characteristic bands attributable to the cholesterol and free cholesterol esters as components of the mixture for the formation of micelles. In the rHDL spectrum, the Apo AI association with acidic lipid membranes, through interactions between lysine residues and negatively-charged lipid groups, can be appreciated. Interactions with the anionic phospholipids increase the Apo AI α-helix content, which is also an important factor in recognizing the specific molecular target [10].

target [10]. The UV-Vis spectrum of the micelles showed a wide absorption band at 207 nm and a shoulder at 244 nm (Figure 2), in agreement with those reported for cholesterol compounds [8]. The band at 244 nm is associated with the π ⟶ π \* transitions of the α and β unsaturated ketones present in the cholesterol structure. After incorporating Apo AI into the micelles, an absorption band at 284 nm was observed, which indicates the coupling of The UV-Vis spectrum of the micelles showed a wide absorption band at 207 nm and a shoulder at 244 nm (Figure 2), in agreement with those reported for cholesterol compounds [8]. The band at 244 nm is associated with the π → π \* transitions of the α and β unsaturated ketones present in the cholesterol structure. After incorporating Apo AI into the micelles, an absorption band at 284 nm was observed, which indicates the coupling of proteins to lipid micelles. A slight band at 231 nm was also observed in the spectrum of rHDL, which is associated with the lipid–protein interaction.

#### *2.2. The 225Ac-rHDL 2.2. The 225Ac-rHDL*

The incorporation of <sup>225</sup>Ac was conducted efficiently with the rHDL vesicles by passive internalization through the encapsulation of <sup>225</sup>Ac previously complexed to a lipophilic molecule (225Ac-DOTA-benzene-p-SCN) with a CLog P of 3.42 (Figure 3). As a result, the <sup>225</sup>Ac-rHDL system was obtained with a labeling efficiency of 85 <sup>±</sup> 3% and a radiochemical purity greater than 99%, as determined by ultrafiltration. The incorporation of 225Ac was conducted efficiently with the rHDL vesicles by passive internalization through the encapsulation of 225Ac previously complexed to a lipophilic molecule (225Ac-DOTA-benzene-p-SCN) with a CLog P of 3.42 (Figure 3). As a result, the 225Ac-rHDL system was obtained with a labeling efficiency of 85 ± 3% and a radiochemical purity greater than 99%, as determined by ultrafiltration.

proteins to lipid micelles. A slight band at 231 nm was also observed in the spectrum of

*Molecules* **2022**, *27*, x FOR PEER REVIEW 5 of 15

rHDL, which is associated with the lipid–protein interaction.

**Figure 3.** Schematic steps of the incorporation of 225Ac into rHDL nanocapsules (225Ac-rHDL). **Figure 3.** Schematic steps of the incorporation of <sup>225</sup>Ac into rHDL nanocapsules (225Ac-rHDL).
