Characterization of Matrix-Adapted PEG Isolation of Exosome-Like Nanovesicles from Pandanus conoideus

Plant-derived exosome-like nanovesicles (PDENs) have recently gained prominence as biocompatible, renewable nanocarriers, but lipid-dense tropical plants remain an underutilized vesicle reservoir and present substantial hurdles for efficient isolation. Here, we establish Pandanus conoideus (red fruit), a Papuan endemic with unusually high carotenoid, tocopherol, and unsaturated fatty acid content, as a new source of PDENs and develop a matrix-adapted polyethylene glycol (PEG 6000) workflow for their scalable isolation from fresh fruit (RFF), dried simplicia (RFS), and long-stored red fruit oil (RFO). Differential centrifugation coupled with matrix-specific PEG precipitation and syringe filtration yielded RFO-derived PDENs (RFO-PDENs) with exosome-like hydrodynamic diameters (predominantly 30-150 nm by nanoparticle tracking analysis), low polydispersity, and spherical, bilayered morphology by transmission electron microscopy, whereas RFF and RFS favored larger, more polydisperse vesicle/aggregate populations. Optimization uncovered narrow matrix-dependent PEG "windows", with 8-10% PEG producing the most homogeneous, highly concentrated RFO-PDENs fraction (up to 1.2 × 10¹⁰ particles/mL) using only benchtop equipment, thus enabling straightforward scale-up. Untargeted LC-HRMS metabolomics of bulk RF-PDEN preparations revealed a complex metabolite landscape dominated by fatty acids and oxylipins, fatty acid amides, sphingolipids/ceramides, amino acids, and cyclic peptides, together with signature P. conoideus antioxidants (α-tocopherol, α/β-carotene) and dermatologically relevant metabolites such as azelaic acid. However, the presence of highly abundant signals with plausible nonendogenous or process-related origins (e.g., 4-undecylbenzenesulfonic acid) indicates that these data represent a composite PDENs preparation rather than a definitive catalog of enclosed vesicular cargo, and potential coprecipitation of matrix- or process-derived components cannot be excluded. Complementary label-free proteomics identified a low-complexity proteome enriched in plastid/mitochondrial ribosomal proteins, Cytochrome b ₆f complex subunit VI, and a dense cohort of lectin and lectin receptor-like kinases that is consistent with translation/redox-associated components and a lectin-enriched, signaling-candidate vesicle surface, while remaining preliminary and qualitative in nature. Collectively, these findings show that matrix-optimized PEG precipitation enables isolation of a high-yield RF-PDEN reservoir from challenging, lipid-rich red fruit oil with preserved membrane architecture and a composition consistent with bioactive lipid and antioxidant enrichment, while underscoring that proposed antioxidant, anti-inflammatory, barrier-repair, or depigmenting applications of RFO-PDENs remain hypothesis-generating and will require future functional validation.