Acetylated Low-Density Lipoprotein-Encapsulated Cholesteryl 1,3-Diiopanoate Glyceryl Ether for the Detection of Atherosclerosis in Rabbits

MATERIALS AND METHODS

C2I was synthesized as illustrated in Figure 1. Compounds a and b were prepared according to a published method (3). To synthesize cholesteryl chloride (compound c), cholesterol (5 g, 0.0129 mol) was added to thionyl chloride (5.5 g, 0.046 mol, 3.5 mL). The mixture was kept at 20°C overnight and then poured into 0.4N NaOH (about 25 mL). The product was extracted 3 times with ether (1:1), and the extracts were combined and dried by use of anhydrous MgSO₄. Ether was evaporated to afford a semipure whitish solid, which was further purified by silica gel column chromatography with hexane as the eluent. A white cholesteryl chloride solid (3.9 g) was obtained at a yield of 76%. C2I (compound d) was synthesized by mixing cholesteryl chloride (55 mg, 0.14 mmol) in 2 mL of benzene, compound b (150 mg, 0.125 mmol) in 2 mL of dimethyl formamide, and KOH (17 mg, 0.29 mmol) in 2 mL of ethanol:water (50:1, v/v). The mixture was refluxed overnight, concentrated in a vacuum at 50°C, and cooled to room temperature. The white crystals obtained were purified by column separation (CHCl₃, silica gel). The yield was about 12.5% (about 25 mg). Infrared details were as follows (per cm): 3,500 and 3,380 (-NH₂), 2,950 and 2,840 (-CH₃), 1,480 (C═C), 1,740 (-C═O), 1,380 [(CH₃)₂-CH-], and 1,600 and 920 (Ar). ¹H nuclear magnetic resonance details were as follows (parts per million): 8.1 (s, 2H, Ar-H), 5.4 (s, 1H, C═C-H), 4.8 (s, 4H, NH₂), 4.1 (m, 4H, CH₂), 3.7 (m, 2H, -O-CH), 3.4–3.2 (m, 4H, CH₂), 2.8 (m, 2H, CHCO₂), 2.2–1.6 (m, 22H, CH₂, CH), 1.1–1.3 (m, 10H, CH₂), 0.9 (d, 15H, CH₃), and 0.7 (s, 6H, CH₃).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Schematic diagram of route of synthesis of C2I.

The partition coefficient of C2I in 1-octanol:phosphate-buffered saline (PBS) was measured according to a published method (4). C2I was labeled with ¹²⁵I via an iodine-exchange reaction (2). ¹²⁵I-C2I was incorporated into AcLDL to form an ¹²⁵I-C2I-AcLDL complex by use of a microemulsion transfer technique. The loading efficiency was determined as described previously (2).

Animal models were established as described previously but with modifications (2). Eight male New Zealand White rabbits (2.5–4.7 kg) were randomly divided into 2 groups. One group was maintained on normal rabbit chow (n = 4, control group) for 12 wk, and the other group was maintained on rabbit chow supplemented with 1% cholesterol (n = 4, cholesterol diet group) for 12 wk to induce early atherosclerotic lesions. The ¹²⁵I-C2I-AcLDL complex was injected via the marginal ear vein into both groups of rabbits at a dose of 555 kBq/kg. Rabbits were killed 24 h after injection, and tissues, including the liver, spleen, lungs, kidneys, adrenal glands, blood, and aorta, were sampled for the determination of radioactivity by use of a γ-counter (2). Sudan IV staining and autoradiography of aortic samples were performed as described previously (2). The in vivo stability of C2I was determined by measuring its degradation in the liver (5).

RESULTS

The compound C2I was synthesized (Fig. 1), and its structure was confirmed by infrared and nuclear magnetic resonance data. The partition coefficient of C2I in octanol and PBS defined as log (C_octanol/C_PBS) was 2.529 ± 0.072. The efficiency of loading of C2I into AcLDL, defined as the number of C2I molecules per AcLDL molecule, was determined to be 162.41 ± 23.38. The tissue distribution at 24 h showed that the accumulation of ¹²⁵I-C2I in the blood and aortic samples from the cholesterol-fed group was significantly higher than that for the control group (2.7 and 2.1 times, respectively) (Table 1). In the adrenal glands, however, less ¹²⁵I-C2I was found in the cholesterol-fed group than in the control group (P < 0.05). No statistically significant difference in ¹²⁵I-C2I accumulation was found in other tissues for the 2 groups. The in vivo stability test showed that 91.2% of the radioactivity in the liver was associated with C2I, indicating that C2I was chemically stable and that the level of radioactivity reflected the content of the intact C2I compound.

The results of Sudan IV staining and autoradiography of aortic samples from the cholesterol-fed group showed a clear superimposition of atherosclerotic lesions and radioactivity deposition (Fig. 2A), suggesting that ¹²⁵I-C2I selectively accumulates at lesion sites in the aorta. The results of both Sudan IV staining and autoradiography were negative for aortic samples from the control group (Fig. 2B).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Results of Sudan IV staining (red staining, left panels) and autoradiography (black regions, right panels) of aortic samples taken at 24 h. (A) Samples from cholesterol diet group. (B) Sample from control group.

DISCUSSION

In a previous study, we examined the potential use of ¹²⁵I-CI-AcLDL for the detection of early atherosclerotic lesions in rabbits (2). In this study, we synthesized a new compound, C2I, for the same purpose. Like CI, C2I also has high in vivo stability because of the chemical nature of the ether linkage between cholesterol and 1,3-diiopanoate glycerol. Compared with CI, which has 3 iodine atoms per molecule, each C2I molecule contains 6 iodine atoms, a factor that could result in twice the specific radioactivity of CI. The higher specific radioactivity of C2I is more desirable for scintigraphic or CT studies.

The tissue distribution study showed that there was no difference in C2I deposition for the cholesterol-fed and control groups, except for blood, aortic, and adrenal gland samples. The accumulation of C2I in blood and aortic samples was 2- to 3-fold higher in cholesterol-fed rabbits than in control rabbits, a finding consistent with the results obtained with CI (2). Reduced accumulation in the adrenal glands of cholesterol-fed animals was also observed (Table 1). When the ratio of the lesion area to the whole area of aortic samples was considered, the actual specific radioactivity of aortic plaque was found to be at least 10 times higher (Fig. 2A).

Sudan IV staining was used to visualize lipid-laden foam cells, a hallmark of early atherosclerosis (2). In this experiment, lesions in cholesterol-fed animals were readily identified by this method (Fig. 2A). The accumulation of ¹²⁵I-C2I in aortic samples, as visualized by autoradiography (Fig. 2A), was superimposed on lesion sites, indicating that C2I accumulates and is retained specifically in regions of plaque.

CONCLUSION

In this study, a new compound, C2I, was synthesized and radiolabeled with ¹²⁵I. Results from animal studies demonstrated selective uptake and retention of radiolabeled C2I in rabbit atherosclerotic lesions when iodinated C2I was carried via AcLDL. It is anticipated that when labeled with ¹²³I or ¹³¹I, C2I incorporated into AcLDL will permit scintigraphic imaging of lesion sites in the aorta. More studies on these topics are under way.