Modulating Lipoprotein Transcellular Transport and Atherosclerotic Plaque Formation in ApoE-/- Mice via Nanoformulated Lipid-Methotrexate Conjugates

Abstract

Macrophage inflammation and maturation into foam cells, following the engulfment of oxidized low-density lipoproteins (oxLDL), are major hallmarks in the onset and progression of atherosclerosis. Yet, chronic treatments with anti-inflammatory agents, such as methotrexate (MTX), failed to modulate disease progression, possibly for the limited drug bioavailability and plaque deposition. Here, MTX-lipid conjugates, based on 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), were integrated in the structure of spherical polymeric nanoparticles (MTX-SPNs) or intercalated in the lipid bilayer of liposomes (MTX-LIP). Although, both nanoparticles were colloidally stable with an average diameter of ∼200 nm, MTX-LIP exhibited a higher encapsulation efficiency (>70%) and slower release rate (∼50% at 10 h) compared to MTX-SPN. In primary bone marrow derived macrophages (BMDMs), MTX-LIP modulated the transcellular transport of oxLDL more efficiently than free MTX mostly by inducing a 2-fold overexpression of ABCA1 (regulating oxLDL efflux), while the effect on CD36 and SRA-1 (regulating oxLDL influx) was minimal. Furthermore, in BMDMs, MTX-LIP showed a stronger anti-inflammatory activity than free MTX, reducing the expression of IL-1β by 3-fold, IL-6 by 2-fold, and also moderately of TNF-α. In 28 days high-fat-diet-fed apoE^-/- mice, MTX-LIP reduced the mean plaque area by 2-fold and the hematic amounts of RANTES by half as compared to free MTX. These results would suggest that the nanoenhanced delivery to vascular plaques of the anti-inflammatory DSPE-MTX conjugate could effectively modulate the disease progression by halting monocytes' maturation and recruitment already at the onset of atherosclerosis.

Keywords: atherosclerosis; foam cells; inflammation; low-density lipoprotein transport; nanomedicine.

Figure 1

Physicochemical and pharmacological characterization of MTX-loaded nanoparticles. (A, B) Schematic representation of MTX-SPN and MTX-LIP, respectively. (C, D) Scanning electron microscopy images of SPN and LIP, respectively (scale bar: 500 nm; up-right inset scale bar: 100 nm). (E, F) Hydrodynamic diameter and colloidal stability of MTX-SPN and MTX-LIP via dynamic light-scattering analysis. (G) Release studies for MTX from MTX-SPN and MTX-LIP. The table summarizes the absolute drug mass, encapsulation efficiency (%EE), and loading (%LE) data for MTX into MTX-SPN and MTX-LIP.

Figure 2

Macrophage maturation to foam cells and nanoparticle uptake. (A) Correlative light and electron microscopy (CLEM) characterization for macrophages exposed to oxidized low-density lipoprotein (oxLDL)—transmission electron microscopy image (left); confocal fluorescent microscopy image (center) showing the cell nucleus (blue, DAPI), the cell lysosomes (green, LysoTrackerGreen), and oxLDL molecules (red, Dil); light and electron microscopy images overlap (right) (scale bar: 2 μm). (B) Representative confocal images of BMDM and foam cells exposed to SPN (left) and LIP (right) at different time points (2, 8, and 24 h). (C) Flow cytometry analysis BMDM and foam cells exposed to SPN and LIP at different time points (2, 8, and 24 h). *** p < 0.001.

Figure 3

Role of methotrexate in macrophage maturation to foam cells. (A) Representative fluorescence images of different treatments conducted on BMDM forced to become foam cell following exposure to oxLDL (50 μg/mL). Red: Dil-oxLDL; blue: DAPI; green: F-Actin. From top to bottom, untreated foam cells, 24 h free MTX-treated foam cells, 24 h empty LIP-treated foam cells, 24 h MTX-LIP-treated foam cells; 24 h empty SPN-treated foam cells 24 h MTX-SPN-treated foam cells, and BMDM not exposed to oxLDL (scale bar: 50 μm). (B) Quantification of the oxLDL accumulation into cells expressed as the ratio between the size of the red area (Dil-oxLDL) and cell number. (Data are expressed as average ± SEM of n = 10 biological replicates. *** p < 0.0001.) (C, D) Quantification of the total cholesterol in macrophages treated with oxLDL (50 μg/mL) and exposed to MTX, MTX-LIP, or MTX-SPN for 8 and 24 h. (Data are reported as average ± SD of n = 4 biological replicates (* p < 0.01, *** p < 0.0001).

Figure 4

Expression of cholesterol transport and inflammatory genes in macrophages and cell viability. (A) Expression level of foam cells markers (ABCA1, CD36, and SRA-1) in macrophages treated with free MTX, MTX-LIP, and MTX-SPN for 8 h. (B) Expression level of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in macrophages treated with free MTX, MTX-LIP, and MTX-SPN for 24 h. (Data are expressed as average ± SD (n = 5); *** p < 0.0001; ** p < 0.001; * p < 0.01). (C) BMDM viability upon incubation with MTX-LIP (left). The table (right) summarizes the IC50 values on BMDM at 24, 48, and 72 h post exposure to different therapeutic groups, namely, free MTX, DSPE–MTX, empty LIP, MTX-LIP, empty SPN, and MTX-SPN.

Figure 5

Preclinical characterization of MTX-liposomes. (A) Representative photomicrographs (left) of oil red O (ORO)-stained aortic sinuses (scale bar: 500 μm) and quantification of the mean lesion area (right) for empty LIP and MTX-LIP treatments. (B) Representative images of plaque collagen content (left) by picrosirius red staining (scale bar: 500 μm) and quantification of the collagen area (right) for empty LIP and MTX-LIP treatments. For plots in (A) and (B), individual data points represent average value per mouse; horizontal bars denote mean. **p < 0.01. (C) Analyses of cholesterol, IL-1α, and RANTES serum content for empty LIP and MTX-LIP treatments. Individual data points represent average value per mouse, and horizontal bars denote mean. Results are presented as mean ± SEM and analyzed by a Student unpaired t test. * p < 0.05, ** p < 0.01. (D) Cy5-LIP (red signal) biodistribution in liver, spleen, kidneys, and aortic sinus. Green: α-smooth muscle actin (α-SMA). Blue: cell nuclei. In the aortic sinus image: M indicates media and P indicates plaque (scale bar: 20 μm).