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Antimicrobial resistance is a current public health concern, limiting the available therapeutic options used for the treatment of common bacterial infections. The development of new drug entities via biotechnological processes is however expensive and time-consuming. Therefore, old antimicrobial agents have been recovered for clinical use. An example of these drugs is polymyxin, which is known for its serious adverse side effects, such as nephrotoxicity, neurotoxicity and promotion of skin pigmentation. To overcome these limitations, the use of biodegradable nanoparticles has been proposed to allow site-specific targeting, increasing the drug's bioavailability and decreasing its side effects. The aim of this work was the development of an optimized pharmaceutical formulation composed of solid lipid nanoparticles (SLN) loading polymyxin B sulphate (PLX) for the treatment of bacterial infections. The PLX-loaded SLN were produced by a double emulsion method (w/o/w), obtaining particles with a mean size of approximately 200 nm, polydispersity of 0.3 and zeta potential of − 30 mV. The encapsulation efficiency reached values above 90% for all developed formulations. SLN remained stable for a period of 6 months of storage at room temperature. The occlusive properties of the SLN was shown to be dependent on the type of lipid, while the antimicrobial properties of PLX-loaded SLN were effective against resistant strains of Pseudomonas aeruginosa. Results from the differential scanning calorimetry (DSC), wide angle X-ray diffraction (WAXD) and small angle X-ray scattering (SAXS) analyses confirmed the crystallinity of the inner SLN matrices, suggesting the capacity of these particles to modify the release profile of the loaded drug.PLX-loaded SLN produced by a double emulsion method (w/o/w), obtaining unilamelar versus multilamelar particles with a crystalline matrix confirmed by X-ray diffraction.