Multifunctional Hybrid Lipid-PLGA Nanoparticles For Dual-Drug delivery: Surface Functionalization with M12 peptide for Muscle Targeting

Sarcopenia, defined as the progressive decline in skeletal muscle mass, strength, and functional capacity, arises from a multifactorial pathophysiology involving structural degeneration, inadequate protein-energy intake, endocrine dysregulation, chronic low-grade inflammation and oxidative stress. Despite its substantial clinical burden, including increased disability, falls, osteosarcopenia, and mortality, no targeted pharmacological interventions are currently available, largely due to the complexity of the underlying mechanisms. Within this framework, trimetazidine (TMZ) and palmitoylethanolamide (PEA) emerge as promising candidates based on their complementary biological activities. TMZ enhances cellular energy efficiency and preserves ATP homeostasis, effects also documented in skeletal muscle. PEA, an endogenous lipid mediator, exerts anti-inflammatory actions relevant to the persistent inflammatory milieu characteristic of sarcopenia. Their combined administration may therefore enable simultaneous modulation of metabolic and inflammatory pathways implicated in disease progression. However, effective co-delivery to muscle tissue remains challenging because of the distinct physicochemical properties of TMZ and PEA, which hinder their incorporation and controller release from a single nanocarrier system. On this basis, the present thesis focused on the development of a functionalized nanosystem of hybrid lipid-polymeric nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) for the co-delivery of PEA and TMZ. Initially, single-loaded and co-loaded non-PEGylated formulations were prepared and subsequently compared with the corresponding PEGylated co-loaded nanoparticles. PEGylation was a fundamental step in increasing its stability and half-life once administered. Surface functionalization of the nanoparticles with the M12 peptide, with the aim of developing a nanosystem designed for active targeting of skeletal muscle, represented the central aspect of this work. The functionalization strategy was developed using L-Phe as a probe molecule and subsequently applied to the M12 peptide. The successful conjugation of the peptide was supported by 1H-NMR analysis, allowing the resulting PLGA-PEG-M12 polymer to be employed in the formulation of functionalized nanoparticle. The obtained nanoparticles, both functionalized and non-functionalized, were characterized by evaluating their size by PCS, their morphology by TEM/STEM analysis and their capacity to incorporate both compounds by HPLC analysis. Furthermore, the in vitro release profiles of PEA and TMZ-B and the behaviour of the nanoparticles during incubation in FBS were also analysed. Overall, this work enabled the development of lipid-PLGA hybrid co-loaded with PEA and TMZ-B and functionalized with the M12 peptide, while preserving suitable physicochemical features and loading efficiency. These results provide a foundation for future studies, which will assess in vivo biodistribution and the ability of M12 functionalization to enhance skeletal muscle targeting.