Respirable nano/micro systems for prevention and treatment of pulmonary infectious diseases

BACKGROUND - Pulmonary drug delivery is a promising substitute for traditional drug delivery routes, which proved efficient in treating local and systemic diseases, including tuberculosis. The lungs may also represent the target site for administering active ingredients intended to prevent infectious diseases, for example, stimulating the immune response. Among immunostimulants imiquimod, now approved for topical administration and employed extensively in off-label therapeutic trials, is appealing. However, it has been successfully formulated to date only as semi-solid lipophilic formulations for topical application, is poorly soluble in most solvents, and solubility literature data are discordant. AIM - This work aims to investigate imiquimod solubility in solvents suitable to develop innovative formulations and to clarify literature discrepancies to collect reliable data. Another aim is to design an imiquimod powder suitable for inhalation and helpful in preventing infectious diseases due to its ability to stimulate innate immunity in the lungs, but also potentially valuable for fighting them, as an adjuvant in allergen immunotherapy or intended to be combined with an antigen for the formulation of vaccines for inhalation or nasal administration. Finally, this work aims to exploit the benefits of inhalation administration by designing a spray-dried powder of microparticles embedding innovative pyrazinoic acid nanoconjugates targeting alveolar macrophages with the final aim of improving the actual, disadvantageous treatment of tuberculosis. RESULTS - Imiquimod solubility in water, ethanol, methanol, acetonitrile, and dimethyl sulfoxide was accurately determined at different temperatures. Experimental conditions like temperature and stirring time were found to significantly affect the time required to achieve complete dissolution. The Scatchard-Hildebrand equation does not apply to imiquimod solutions studied because of association phenomena due to intermolecular hydrogen bonds and/or π-stacking, as supported by the hyperchromic effect very pronounced in highly polar solvents. Micronization and spray drying were found to be suitable methods for producing highly crystalline and respirable imiquimod inhalation powders, with the possibility to modify the administered dose by making adhesive mixtures with coarse lactose, which proved to maintain a favorable aerodynamic behavior. In vitro studies showed powders’ non-toxicity and ability to induce a controlled production of IL-6 and IL-8. Conjugates between pyrazinoic acid and poly (malic acid) proved to self-assemble, after simple nanoprecipitation in water, into nanometric, negatively surface-charged, spherical, and stable over time nanoconjugates, which gave a sustained release of active ingredients. Nanoconjugates proved to be degraded if exposed to aqueous enzyme solutions, which would reasonably happen also inside alveolar macrophages. A potentially respirable spray-dried microparticle powder was produced starting from a solution of these nanoconjugates, leucine, and sodium hyaluronate, and proved to be able to yield original nanoconjugates upon redispersion in aqueous media, maintaining drug concentration over time and avoiding burst release that usually affects nanosystems, and reducing doses and frequency of administration, reducing side effects, and improving tuberculosis therapy. CONCLUSION - This thesis highlights the peculiar imiquimod behavior in solution for the first time, revealing the association between imiquimod molecules in very polar solvents and providing reliable solubility data. Nanoparticle-based imiquimod crystalline and respirable powders were produced, proposing for the first time an inhalation administration of this active ingredient with the primary aim of designing an inhalation platform that stimulates the immune response to prevent infectious diseases. Finally, a significant step towards improving tuberculosis therapy has been made by developing an inhalation powder of microparticles embedding innovative nanoconjugates of pyrazinoic acid that target alveolar macrophages, providing a sustained release of active ingredients.