Isolation and characterization of plant-derived exosome-like nanoparticles from Carica papaya L. fruit and their potential as anti-inflammatory agent

Inflammation is an immune system response that identifies and eliminates foreign material. However, excessive and persistent inflammation could disrupt the healing process. Plant-derived exosome-like nanoparticles (PDENs) are a promising candidate for therapeutic application because they are safe, biodegradable and biocompatible. In this study, papaya PDENs were isolated by a PEG6000-based method and characterized by dynamic light scattering (DLS), transmission Electron Microscopy (TEM), bicinchoninic acid (BCA) assay method, GC-MS analysis, total phenolic content (TPC) analysis, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. For the in vitro test, we conducted internalization analysis, toxicity assessment, determination of nitrite concentration, and assessed the expression of inflammatory cytokine genes using qRT-PCR in RAW 264.7 cells. For the in vivo test, inflammation was induced by caudal fin amputation followed by analysis of macrophage and neutrophil migration in zebrafish ( Danio rerio ) larvae. The result showed that papaya PDENs can be well isolated using the optimized differential centrifugation method with the addition of 30 ppm pectolyase, 15% PEG, and 0.2 M NaCl, which exhibited cup-shaped and spherical morphological structure with an average diameter of 168.8±9.62 nm. The papaya PDENs storage is stable in aquabidest and 25 mM trehalose solution at -20˚C until the fourth week. TPC estimation of all papaya PDENs ages did not show a significant change, while the DPPH test exhibited a significant change in the second week. The major compounds contained in Papaya PDENs is 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP). Papaya PDENs can be internalized and is non-cytotoxic to RAW 264.7 cells. Moreover, LPS-induced RAW 264.7 cells treated with papaya PDENs showed a decrease in NO production and downregulation mRNA expression of pro-inflammatory cytokine genes (IL-1B and IL-6) and an upregulation in mRNA expression of anti-inflammatory cytokine gene (IL-10). In addition, in vivo tests conducted on zebrafish treated with PDENs papaya showed inhibition of macrophage and neutrophil cell migration. These findings suggest that PDENs papaya possesses anti-inflammatory properties.