Biodistribution of Nanostructured Lipid Carriers in Mice Atherosclerotic Model

Abstract

Atherosclerosis is a major cardiovascular disease worldwide, that could benefit from innovative nanomedicine imaging tools and treatments. In this perspective, we here studied, by fluorescence imaging in ApoE^-/- mice, the biodistribution of non-functionalized and RXP470.1-targeted nanostructured lipid carriers (NLC) loaded with DiD dye. RXP470.1 specifically binds to MMP12, a metalloprotease that is over-expressed by macrophages residing in atherosclerotic plaques. Physico-chemical characterizations showed that RXP-NLC (about 105 RXP470.1 moieties/particle) displayed similar features as non-functionalized NLC in terms of particle diameter (about 60-65 nm), surface charge (about -5 - -10 mV), and colloidal stability. In vitro inhibition assays demonstrated that RXP-NLC conserved a selectivity and affinity profile, which favored MMP-12. In vivo data indicated that NLC and RXP-NLC presented prolonged blood circulation and accumulation in atherosclerotic lesions in a few hours. Twenty-four hours after injection, particle uptake in atherosclerotic plaques of the brachiocephalic artery was similar for both nanoparticles, as assessed by ex vivo imaging. This suggests that the RXP470.1 coating did not significantly induce an active targeting of the nanoparticles within the plaques. Overall, NLCs appeared to be very promising nanovectors to efficiently and specifically deliver imaging agents or drugs in atherosclerotic lesions, opening avenues for new nanomedicine strategies for cardiovascular diseases.

Keywords: ApoE-/- model; active targeting.; atherosclerosis; lipid nanoparticles; macrophage elastase (MMP-12); nanomedicine.

Figure 1

Synthesis of RXP470-modified nanostructured lipid carriers (NLC) and control NLC. (a) Addition of dithiothreitol (DTT), incubation 2 h at room temperature, then dialysis overnight. (b) Addition of RXP470.1-maleimide, 4 h incubation at room temperature, then overnight at 4 °C. (c,d) Addition of 1-(2-hydroxyethyl)-1H-pyrrole-2,5-dione, 30 min incubation at room temperature, then dialysis for 24 h.

Figure 2

Dynamic light scattering (DLS) and SEM characterizations of nanoparticles. (a) Size distribution by intensity of NLC-S-S-Pyr, NLC, and RXP-NLC. (b) Zeta potential distribution of NLC-S-S-Pyr, NLC, and RXP-NLC. (c,d) SEM images of NLC-S-S-Pyr after negative staining.

Figure 3

NLC and RXP-NLC stability when diluted at 5 mg/mL at 37 °C in 1X PBS buffer or in human serum. (a) Z-average particle diameter (nm) measured by DLS and normalized to value measured just after media and particle mixing. (b) DiD fluorescence (670 nm) normalized to value measured just after media and particle mixing.

Figure 4

Blood clearance profiles and biodistribution for naked NLC and RXP-NLC in C57BL/6 ApoE^-/- mice (n = 3 for NLC, n = 3 for RXP-NLC 5 mg injected per mouse). (a) Comparison of blood clearance rates between the two NLCs (naked NLC: Black circles, RXP-NLC: Empty triangles). Time expressed in minutes is reported in logarithm scale. (b) Biodistribution and fluorescence intensity patterns observed for the two NLCs in liver, spleen, kidneys, lungs, femur bone, and brain harvested 24 h after injection. Fluorescence is expressed as arbitrary units (AU) and reported in logarithm scale.

Figure 5

Tissue and histology analysis after NLC or RXP-NLC injection in ApoE^-/- mice. (a) Representative ex vivo fluorescence image of whole aorta harvested 24 h after intravenous injection of 5 mg particles/mouse. (b) Quantitative analysis of fluorescence signal within brachiocephalic artery (ROI = 1 mm²) and comparison between naked nanolipid carriers (NLC, n = 3) and RXP470.1 functionalized ones (RXP-NLC, n = 3). AU: Arbitrary units. (c) Aorta cross-section histology (fluorescence microscopy) of tissues harvested 48 h after intravenous injection. Blue: Nucleus (Hoeschst), Green: Anti-CD68 Ab (macrophage marker) and tissue auto-fluorescence, Red: DiD-loaded NLC. Scale bar: 100 µm.