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.

Synthesis of Two Methotrexate Prodrugs for Optimizing Drug Loading into Liposomes

“…The coadministration of the two prodrugs and the administration of combo liposomes showed an IC 50 very similar to DSPE-MTX: IC 50 values for the combo were found to be equal to 0.6 ± 0.1 µM and 0.9 ± 0.1 µM (free prodrugs and combo-LIP, respectively). These results were in agreement with results obtained in other works produced by our group and others [ 31 , 45 , 49 ]. In summary, a slight difference in IC 50 among MTX, DSPE-MTX, PEG-MTX and the liposomal formulation derived was found.…”

“…For DSPE-MTX-LIP, the encapsulation efficiency was equal to 79.9 ± 5.6% (799 ± 56 µg), while for PEG-MTX-LIP: 82 ± 7.5% (820 ± 75 µg) and the Combo-LIP: 80.2 ± 1.8% (802 ± 18 µg) as reported in the Table 2 . The direct loading of MTX unmodified molecule into liposomes was extremely difficult due to the extremely poor solubility of the compound both in water and organic solvents as also reported elsewhere [ 31 , 33 ]. A series of MTX modification-based strategies have been pursued by other groups in recent years.…”

“…The DSPE-MTX and PEG-MTX were added during the lipid film formation phase. It might be speculated that the DSPE-MTX could intercalate with the DPPC and DSPE-PEG chains, considering that the lipid part of DSPE-MTX is identical to DSPE-PEG and similar to DPPC [ 31 ]. Regarding the localization of PEG-MTX, there are two options: it could be intercalated into the lipid membranes, with a similar configuration reported for DSPE-MTX, or in the inside of the phospholipid bilayer.…”

“…Liposomes (LIP) were prepared by thin-layer evaporation (TLE) [ 31 ]. Briefly, DPPC, cholesterol, DSPE-PEG (6:3:1) (total amount: 40 mg) and DSPE-MTX/PEG-MTX or both prodrugs (1 mg of prodrug) were dissolved in chloroform in a round-bottomed flask.…”

Curcumin-Loaded Nanoparticles Impair the Pro-Tumor Activity of Acid-Stressed MSC in an In Vitro Model of Osteosarcoma

“…Curcumin-loaded spherical polymeric nanoparticles (SPN-curc) were synthetized using a sonication-emulsion method, as previously described [53,58]. Briefly, an oil phase containing carboxyl-terminated poly(lactic-co-glycolic acid) (PLGA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and curcumin (curc) was added drop by drop to an aqueous phase containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000 (DSPE-PEG-COOH) in 4% ethanol, under sonication (100% amplitude for 1.5 min).…”