Purification and Anti-Inflammatory Activity of Walnut Exosome-like Nanoparticles

This study reports the first successful isolation and characterization of exosome-like nanoparticles from walnut kernels (WELNs). The isolated WELNs exhibited a typical cup-shaped morphology with an average diameter of 139.7 ± 67.5 nm, a concentration of 7.4 × 10¹¹ particles/mL, and a zeta potential of -17.47 ± 4.06 mV. Proteomic and small RNA sequencing analyses confirmed the presence of diverse proteins and microRNAs within WELNs. In vitro assays demonstrated their potent antioxidant capacity, with radical scavenging rates of 67.54% against ABTS⁺ and 48.59% against DPPH⁺ at 102 μg/mL and IC₅₀ values of 89.7 μg/mL and >102 μg/mL for scavenging of ABTS⁺ and DPPH⁺ radicals, respectively. Cytotoxicity assays indicated no adverse effects on RAW264.7 macrophage viability at concentrations up to 60 μg/mL. In LPS-stimulated RAW264.7 macrophages, WELN treatment (20-60 μg/mL) dose-dependently mitigated oxidative stress by reducing intracellular ROS levels (down to 81.22% of the control at 60 μg/mL) and malondialdehyde (MDA) content while restoring the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). Furthermore, WELNs significantly suppressed the production of nitric oxide (NO) and pro-inflammatory cytokines TNF-α, IL-6, and IL-1β (reduced to approximately 30.8%, 22.7%, and 23.6% of LPS-induced levels, respectively, at 60 μg/mL). Mechanistic investigation revealed that the anti-inflammatory effect was mediated through the inhibition of the MAPK signaling pathway, as evidenced by decreased phosphorylation of p38, ERK, and JNK. In conclusion, WELNs exhibit dual anti-inflammatory and antioxidant properties. This study provides the first evidence of bioactivity for walnut-derived exosome-like nanoparticles, advancing the mechanistic understanding of walnuts' health benefits and highlighting their potential as a natural component for functional food applications.