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

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.


DSPE-MTX prodrug synthesis.
Considering the poor solubility of the Methotrexate (MTX), a prodrug with amphiphilic properties was generated by binding MTX to 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-amino . In order to generate the prodrug, MTX was pre-activated in loco with a mixture of DCC and NHS, then conjugated with DSPE-NH 2 . Compound purification was achieved by precipitation in cold diethyl ether.

MTX-SPN optimization. Optimization of SPN.
To find the optimal formulation, MTX-SPNs were produced using different amounts of PLGA and DPPC and using PLGA with different molecular weights (25-35KDa or 38-53KDa) as reported in Table S1, Figure S3. To find the optimal formulation, MTX-SPNs were produced using different amounts of PLGA and DPPC and using PLGA with different molecular weights (25-35KDa or 38-53KDa). All the formulations presented average sizes ranging from 200 to 225 nm and showed a negative ζ-potential ranging from -41 to -54 mV. With the increase of PLGA amount a relative increase on the nanoparticles size was retrieved. The negative charge is related to DSPE-PEG-COOH carboxylic groups exposed on SPNs surface. As shown in Figure S3A and B the amount of PLGA also affected DSPE-MTX encapsulation efficiency (EE%) which increases proportionally to PLGA amount, passing from about 0.2% to 0.5 % for the lowest molecular weight PLGA (25-35KDa) and from 0.4% to 1.5% for the highest (38-53kDa). EE% was also affected by the presence of DPPC, by removing DPPC the amount of internalized prodrug increased. This finding is probably related to a competition between DSPE-MTX and DPPC on the stabilization of the PLGA during the addition to the aqueous phase on the preparation of the nanoparticles. Basing on these considerations, SPNs 12 formulation was selected (Table S1, Figure S3). Starting from now on, we will refer to SPNs12 as MTX-SPNs.
5 Table S1: Optimization of SPNs. The SPNs were made in triplicate (n=3) for each single experiment.

Intracellular localization of oxLDL.
To observe oxLDL intracellular distribution at confocal microscopy oxLDL were stained using DiI; for this experiment living RAW 264.7 were treated with 488lysotracker to highlight lysosomes and with a concentration of Dil-oxLDL equivalent to 50 µg/ml of oxLDL. As it is possible to appreciate the green signal from lysosomes and the red signal from oxLDL co-localize almost completely, proving oxLDL accumulation mainly occurs into lysosomes ( Figure   S6A). Control cells were treated only with DiI (instead of DiI-oxLDL) following the same conditions, as it is possible to observe in Figure S6B. No co-localization at lysosomes was observed. Red signal coming from DiI was confined to plasma membrane. It is also important to underline that in cells treated with DiI-oxLDL no fluorescent signal was retrieved at plasma membrane thus proving the absence of any leakage of DiI from fluorescent oxLDL.

Stable association of Dil and oxLDL molecules. To furtherly demonstrate the stability of oxLDL and
DiI a time-lapse microscopy experiment was performed (Movie S1 and S2). Following the same condition living BMDM were incubated overnight with Dil-oxLDL or with Dil. 14 hours after treatment cells were observed at the microscope and movies (Movie S1) were recorded. In Dil-oxLDL treated cells it is possible to note particulate movement inside the cell referring to Dil-oxLDL trafficking; in free Dil treated cells, Movie S2, instead the fluorescent signal is revealed from firm spots most likely located in plasma membrane.  Quantitative assessment of radioactivity distribution in selected tissues 24 h after injection (n = 5).