66.    Novel respirable nanomicelles for encapsulation of anti-tuberculous 

Rifampicin (RIF) is one of the most powerful and effective first line drugs used in the treatment of tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb). It is a borderline class II drug, according to the Biopharmaceutical Classification System, due to its poor aqueous solubility (2.04mg/mL, at 25°C). Its biolimitations as its pH-dependent aqueous solubility and its poor chemical stability under acid and basic pH conditions hampers the development of novel RIF liquid dosage forms. Furthermore, the RIF inhalatory administration route has been recently investigated as a potential adjuvant therapy to the conventional oral TB treatment. In this framework, the development of novel RIF formulations with improved drug aqueous solubility and stability for inhalatory administration, by means of nanotechnology, represents a technological challenge. The present study investigates, for the first time, RIF encapsulation within nanosized polymeric micelles (PMs), employing the commercially available poly(vinyl caprolactam)- poly(vinyl acetate)-poly(ethylene glycol) graft copolymer (Soluplus®). This biomaterial has an excellent colloidal stability mainly due to its low critical micellar
concentration (2.24x10-3%w/v, 25°C), as determined by dynamic light scattering. Several RIF-loaded Soluplus® (Solu) micelles were prepared by a solvent-diffusion technique, using acetone, increasing RIF aqueous solubility up to 29.0mg/mL (Solu 10%w/v). RIF-loaded PMs (3%w/v) showed the best micellar solubilization capacity, with a 6.65-fold increment of RIF apparent aqueous solubility. Also, this system exhibited a hydrodynamic diameter of 114.6nm at 25°C, along with an optimal in vitro RIF stability (100.5±9.7%, 28 days, 25°C). Regarding the previous data, RIFloaded (10mg/mL) micellar system (3%w/v) was chosen as the best candidate for further experiments. Further, the in vitro cumulative release profiles (pH 7.4, 37°C, 72h) of the micellar systems were 61.8±2.6%. Additionally, these nanocarriers represented a suitable system for inhalation and drug delivery to the deep lung, according to its in vitro aerodynamic behavior. Further, the RIF minimal inhibitory concentration was not modified after its encapsulation within PMs, as determined by the resazurin colorimetric assay on Mtb ΔRD1 mc26230. The in vitro RIF cellular uptake of drug-loaded PMs in RAW 264.7 (murine macrophages) demonstrated
higher drug intracellular/cell levels (μg RIF/mg protein) for RIF-Solu (0.20±0.02) than RIF control solution (0.13±0.03) over 2h. Finally, in vitro preliminary studies showed the microbicidal effect of the RIF-loaded PMs vs a RIF solution. Human THP-1 derived macrophages cells infected with Mtb showed a decrease in bacterial load after treatment with drug-loaded PMs at a concentration of 20μg/ml. Overall, our new Solu-based polymeric nanocarrier denotes a first-rate and simple nanotechnological platform for its potential application in TB respirable therapy.