Simultaneous Noninvasive Detection and Therapy of Atherosclerosis Using HDL Coated Gold Nanorods

Abstract

Cardiovascular disease (CVD) is a major cause of death and disability worldwide. A real need exists in the development of new, improved therapeutic methods for treating CVD, while major advances in nanotechnology have opened new avenues in this field. In this paper, we report the use of gold nanoparticles (GNPs) coated with high-density lipoprotein (HDL) (GNP-HDL) for the simultaneous detection and therapy of unstable plaques. Based on the well-known HDL cardiovascular protection, by promoting the reverse cholesterol transport (RCT), injured rat carotids, as a model for unstable plaques, were injected with the GNP-HDL. Noninvasive detection of the plaques 24 h post the GNP injection was enabled using the diffusion reflection (DR) method, indicating that the GNP-HDL particles had accumulated in the injured site. Pathology and noninvasive CT measurements proved the recovery of the injured artery treated with the GNP-HDL. The DR of the GNP-HDL presented a simple and highly sensitive method at a low cost, resulting in simultaneous specific unstable plaque diagnosis and recovery.

Keywords: atherosclerosis; diffusion reflection; gold nanorods; high-density lipoprotein; noninvasive imaging; unstable plaques.

Figure 1

Characterization of GNSs and GNS-HDL. (a) Schematic diagram of the GNS synthesis process and the coating of GNS with m-PEG (85%) and COOH–PEG (15%), followed by covalent conjugation with HDL. (b) FT-IR spectra of GNSs (thin line) and GNS-HDL (bold line). The peaks around 950 cm⁻¹ are the major proof of the formation of the C–N bond. (c) Optical properties of GNSs: ultraviolet-visible spectroscopy of bare GNSs (solid line) and HDL coated GNSs (dotted line), both presenting an absorption peak at 530 nm. Inset: TEM image of the 20 nm gold nanospheres.

Figure 2

In vitro measurements of macrophage cell culture following incubation with GNSs and GNS-HDL. (a) GNSs uptake by macrophages captured by inverted microscopy. Nanoparticles appear as dark dots within cells due to light absorption by the particles. All experiments were performed with a magnification of 200×. (b) Reflectance intensity spectra of macrophages 48 h after their incubation with 0.1 mg/mL of GNSs were extracted from hyperspectral microscopy measurements. The reflectance spectra of macrophages with GNSs (dashed line) and with GNS + HDL (dotted and dashed-dotted lines) present an intensity peak at 530 nm, very similar to the absorption peak of the GNSs measured by the spectrophotometer (Figure 1c). The reflectance spectrum of the macrophages without GNSs (solid line) did not present a peak of around 530 nm. (c) Reflectance intensity at 530 nm was measured for n = 3 plates of macrophages with GNSs and macrophages with GNS + HDL. On average, higher intensity was measured for macrophages that were incubated with GNS + HDL, suggesting their superior uptake by the macrophages. (d) LP-PLA2 levels in macrophage cell culture media, using ELISA assay, after incubation with GNRs coated with HDL or with GNRs alone (n = 10). (e) MTT viable test of cell culture macrophages. Results show that both HDL and GNS-HDL were not toxic to the cells. For all experiments, n = 10.

Figure 3

Characterization of GNRs. (a) Schematic diagram of the GNRs synthesis process and the coating of GNRs with m-PEG (85%) and COOH–PEG (15%), followed by covalent conjugation with HDL. (b) Optical properties of the GNRs: ultraviolet-visible spectroscopy of bare GNRs (solid line) and HDL coated GNRs (dotted line). (c) Transmission electron microscopy image of the GNRs. Average dimensions were 35 × 15 ± 2.2 nm (n = 10).

Figure 4

Noninvasive diffusion reflection measurements of injured rats’ carotids. (a) DR measurements of the rats’ carotids with a 650 nm laser diode illumination and four photodiodes (PDs) placed between 1 and 5 mm from the light source with a 1 mm separation. Representative DR curves of the healthy and injured carotids: (b) GNRs injection: DR profiles of injured arteries before (solid line) and 24 h after (dotted line) the GNRs injection. (c) GNR-HDL injection: Injured artery before (solid line) and 24 h post (dashed-dotted line) the GNRs injection. The Δslopes are due to the increase in the DR slope due to the GNRs injection, indicating that both GNRs and GNR-HDL injections increased the absorption of the carotids.

Figure 5

Ex vivo high-resolution CT scan and histology of rats’ injured arteries two weeks post GNRs injection. Panels (a–c): Hematoxylin and eosin (H and E) staining of normal carotid (a) and balloon-injured carotid artery 4 weeks post-injury and 2 weeks after GNR-HDL injection or GNRs injection (b and c, respectively). Panels (d,e): CD-68 immunostaining for macrophage accumulation in the injured carotid 4 weeks post-injury and 2 weeks after GNR-HDL injection or GNRs injection (d and e, respectively). No positive immunostaining was observed in the carotids treated with GNR-HDL, but a clear CD68 positive staining was observed in carotids treated with GNRs only. Magnifications are 40× in panel (a) and 100× in panels (b–e). (f) CT scan of the injured artery following GNRs injection. Gold is still apparent, indicating the presence of the macrophages within this artery. (g) CT scan of the injured artery following the GNR-HDL injection. The artery presented no GNRs accumulation, suggesting macrophage detachment from the injured site.