Hybrid PLGA Nanoparticles co-delivering Trimetazidine and Palmitoylethanolamide: Design and Preliminary Study on Functionalisation for Muscle Cell Active Targeting

Sarcopenia is a pathological condition characterised by a progressive and generalised decline in skeletal muscle mass, strength, and function, representing an increasing public health concern in the context of population ageing. Its pathogenesis is multifactorial, involving interplay between inflammaging, mitochondrial dysfunction, oxidative stress, hormonal decline, physical inactivity, malnutrition, and obesity. These factors lead to structural and functional alterations of muscle tissue, impairing mobility and autonomy in older adults and leading to an increased risk of disability, frailty, and premature mortality. Current clinical management of sarcopenia relies primarily on non-pharmacological strategies, including physical exercise, nutritional supplementation, and lifestyle adjustments. However, such interventions may prove insufficient in the advanced stages. Since no pharmacological therapy is yet approved for the specific treatment of sarcopenia, several investigational agents are growing interest; among them, Trimetazidine (TMZ) and Palmitoylethanolamide (PEA) have emerged as particularly promising. TMZ, a piperazine derivative traditionally used as an antianginal agent, inhibits fatty acid β-oxidation via selective 3-ketoacyl-CoA thiolase block. This metabolic shift increases glucose oxidation, thereby improving energy efficiency and reducing oxidative stress in cardiac and skeletal muscle. PEA, an endogenous lipid amide with potent anti-inflammatory properties, acts as endocannabinoids analogue. Since chronic systemic inflammation is a recognised key driver of sarcopenic muscle degeneration, PEA’s ability to modulate inflammatory pathways (through the activation of the PPAR-α receptor) represents a promising complementary therapeutic candidate. The potentially complementary mechanisms of action of TMZ and PEA -simultaneously targeting muscle metabolic dysfunction and inflammation- gives hope their co-administration could provide synergistic benefits, even though their opposite physicochemical properties (TMZ being hydrophilic and PEA lipophilic) complicate the co-encapsulation into a single nano-formulation. In this thesis, hybrid polymeric nanoparticles (NPs) co-loaded with TMZ and PEA were developed using a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA). The formulation process was optimised by employing various excipients and production techniques to achieve desirable particle size, stability, and drug loading. Physicochemical characterisation of the NPs was performed by dynamic light scattering and microscopy, determining particle size, polydispersity index, zeta potential, and morphology. Spectrophotometry and HPLC were employed to evaluate encapsulation efficiency and drug loading, while differential scanning calorimetry (DSC) provided insights into drug-polymer interactions. Furthermore, as the project aims for parental administration, NPs were PEGylated to enhance circulation time and endosomal escape, while preliminary studies on the NPs’ functionalisation with a myocyte-targeting peptide (M12: Arg-Arg-Gln-Pro-Pro-Arg-Ser-Ile-Ser-Ser-His-Pro) to obtain an active targeting have been initiated. In vitro release profiles of both active ingredients were assessed by dialysis in phosphate buffer, followed by HPLC analysis. Moreover, in collaboration, in vivo biodistribution, muscle cell targeting and anti-sarcopenic efficacy studies were initiated in senescent murine models. In conclusion, this work investigates an innovative nanomedicine-based therapeutic approach for sarcopenia, which includes M12 peptide functionalisation for muscle cells targeting, aimed at improving clinical outcomes in older patients and at mitigating the growing socioeconomic impact of this age-related condition.