Development of new Nanoplatforms for the treatment and prevention of Atherosclerosis

Over the last decade, many studies suggest that inflammation has an important role in atherosclerosis progression and development. In the last years, old cheaper drugs, Methotrexate (MTX) and Colchicine (COL) were considered to treat this pathology due to their anti-inflammatory effects. MTX, a conventional chemotherapeutic medicine currently representing the first-line option in inflammatory diseases like rheumatoid arthritis (RA) and psoriatic arthritis, was proposed in the clinical trial Cardiovascular Inflammation Reduction Trial (CIRT). Even though many studies have demonstrated this drug's beneficial activity on animals/patients with atherosclerosis, the clinical trial failed. Col is known for its therapeutic use in gout and Familial Mediterranean Fever (FMF). In these patients, it was noticed a reduction of myocardial infarctions after the use of Colchicine. Its effect is associated with a stronger effect in the inhibition of inflammasome activation and IL-1β secretion. These observations are at the base of different clinical trials like Low-Dose Colchicine (LoDoCo), A second Low Dose Colchicine (LoDoCo2), Low Dose Colchicine after Myocardial Infarction (LoDoCo-MI). In general, their clinical use is limited by their several side effects and, furthermore, in MTX’s case is even more difficult due to its poor bioavailability and solubility in biological fluids. Taking this into account, nanomedicine might become a valid ally in furnishing potent tools to ease combined treatment and increase drug accumulation at the target site. Thus, this work aims to propone nanoparticles, polymeric, and lipid to improve the stability and solubility of encapsulated cargos, promote transport across membranes, and prolong circulation times to increase safety and efficacy. Here, it was proposed two different strategies for modifying the methotrexate by conjugating the DSPE and PEG. The prodrugs obtained (DSPE-MTX and PEG-MTX) were used to synthesize liposomes, including one or both of them. Physiochemical features were tested. The DSPE-MTX was selected (due to the instability at pH 6.8 of the system) and delivered by liposomes (MTX-LIP) and spherical polymeric nanoparticles (MTX-SPNs). Both nanoparticle formulations presented similar features. For the lipidic nanoparticles, the size is 174±2 nm (PdI: 0.15 ± 0.0007), and Zeta Pot -48 ± 0.02 mV; for the polymeric nanocarriers, the size is 208±2 nm (PdI: 0.15 ± 0.02), and Zeta Pot 45.8 ± 0.02 mV. MTX encapsulation efficiency (EE%) 70±5% for Lip-MTX and 1.5±0.2% for SPNs-MTX. Foam cells were obtained by treating rat bone marrow-derived monocytes (BMDM) with oxLDL. The treatment with the two nanoformulations was able to reverse foam cells maturation into macrophages. Both MTX-LIP and MTX-SPNs decreased cholesterol amounts after 24 hours in BMDM. The efficacy of the treatment was also proved by gene expression analysis. RT-PCR showed that the CD36 and SRA-1 gene expression (regulating oxLDL influx) was minimal. The reverse cholesterol transporter (ABCA1) in foam cells treated with the nanoformulations was inducing a 2-fold compare the CTRL. MTX-LIP and MTX-SPNs also reduced inflammatory gene expression (IL-6, IL-1β, and TNFα). MTX-LIP was used in vivo in a murine model of atherosclerosis. ApoE-/- mice, fed with a high-fat diet for 28 days, were treated for 4 weeks (once every three days) with MTX-LIP. Plaque burden was measured, revealing a reduction in plaque area for the treated mice. These results influenced a new line of thinking; it could be possible to boost the liposomes' anti-inflammatory effect combining in the same particles the MTX and the Colchicine (Col). Particles have a size around 100 nm with stability upon 4 days. The liposomes' encapsulation efficiency was ~ 28 % for MTX and ~ 33 % for COL with a ratio of 1:16. The anti-inflammatory effect was tested on the primary cell line (BMDM). Specifically, liposomes with MTX and Col were able to reduce the inflammation caused by the LPS. At the same time, they were able to modulate the foam cell marker (ABCA1), reflecting a reduction of the expression of IL-1β by 3-fold, IL-6 by 2-fold, and moderately of TNF-α. These results would suggest that combining the chemotherapeutic drugs in the liposomes could strongly reduce the inflammation modulating the disease progression. These data suggest that MTX and COL loaded nanoparticles could be the new strategy for the treatment of atherosclerosis.