Sustainable avocado seed starch nanoparticles for limonene encapsulation: a novel immunomodulatory delivery system

Abstract

D-limonene is a bioactive monoterpene with recognized antioxidant, anti-inflammatory, and antimicrobial properties, whose therapeutic application is severely constrained by its high volatility, sensitivity to oxidation, and poor aqueous solubility. Nanoencapsulation within biopolymeric matrices represents a promising strategy to overcome these limitations. Therefore, this study aimed to develop and characterize chemically modified avocado seed (Persea americana Miller) starch nanoparticles, produced via a simple one-step nanoprecipitation method, for D-limonene encapsulation, evaluating their physicochemical properties, long-term stability, and immunomodulatory potential. Acetylation of the starch was confirmed by FTIR, ¹³C NMR, and degree of substitution (DS) determination, which collectively demonstrated successful introduction of acetyl groups into the polymer backbone and the resulting increase in hydrophobicity that underlies the encapsulation capacity of the nanocarrier. Thermogravimetric analysis further characterized the physicochemical properties of the modified starch and nanoparticles. The resulting nanoparticles displayed mean diameters lower than 350 nm, predominantly homogeneous size distributions (PdI<0.3), zeta potentials ranging from −10.46 to −13.77 mV and encapsulation efficiency higher than 80% in two formulations. Long-term stability assessment demonstrated preservation of colloidal integrity after 10 months under refrigerated storage, with retention of approximately 69% of initial encapsulation efficiency. TEM analysis revealed small, globular particles with smooth surfaces and narrow size distribution, while FT-IR spectroscopy provided surface chemistry characterization consistent with limonene confinement within the nanoparticle core. In vitro release studies demonstrated a sustained, gradual release profile, with 59.1% of the encapsulated DL released after 24 h and release kinetics best described by a first-order model, with Fickian diffusion as the dominant transport mechanism. Immunological analysis demonstrated a potent and context-dependent immunomodulatory potential of ASNP-DL1. In vitro assays showed the suppression of pro-inflammatory cytokines (TNF-α and IL-6) in macrophages and dendritic cells, concurrently maintaining anti-inflammatory IL-10 at basal levels, indicating a coordinated shift toward an anti-inflammatory phenotype. These findings underscore the potential of this sustainable nanocarrier as a versatile platform that effectively addresses the physicochemical limitations of D-limonene, positioning this sustainable, agriculturally-derived nanocarrier as a promising candidate for immunomodulatory therapies with broad pharmaceutical and biotechnological relevance