RGD-functionalized Wolffia globosa-derived Exosome-like Nanoparticles for Targeted Doxorubicin Delivery in Breast Cancer Therapy.

INTRODUCTION Plant-derived exosome-like nanoparticles (ELNs) have attracted considerable attention as biocompatible drug delivery vehicles. Wolffia globosa (Kai-pum), a native Thai aquatic plant, represents a novel and underexplored source of ELNs. This study describes the isolation, characterization, and functional evaluation of W. globosa-derived exosome-like nanoparticles (WGENs) that are surface-modified with the integrin-targeting Arg-Gly-Asp (RGD) peptide and loaded with doxorubicin (Dox) for targeted breast cancer therapy.

Methods

WGENs were isolated by differential ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). RGD functionalization was achieved via DSPE-PEG-RGD membrane insertion, followed by doxorubicin loading through electroporation. Cellular uptake was assessed by fluorescence microscopy and live-cell imaging in 4T1 and CL-S1 cell lines. Cytotoxicity was evaluated by LDH assay.

Results

NTA revealed WGEN mode sizes of 94.1 and 106.9 nm. TEM confirmed cup-shaped vesicular morphology. RGD-Exo-Dox treatment resulted in 4-fold greater intracellular doxorubicin accumulation in 4T1 cells than in CL-S1 cells (4.3% vs. 1.1% red fluorescence at 16 h). No statistically significant difference in cytotoxicity between RGD-Exo-Dox and free doxorubicin was detected in 4T1 cells (all p > 0.05); the observed numerical differences did not constitute positive evidence of differential cytotoxicity.

Discussion

Preferential intracellular doxorubicin accumulation was observed in αvβ3-overexpressing 4T1 cells compared with CL-S1 cells, which is consistent with RGD-mediated integrin targeting. This interpretation is subject to important limitations, including the absence of a scrambled-peptide control nanoparticle and unconfirmed surface RGD density; the differential uptake may therefore reflect nonspecific factors. Comparative formulation data are presented as preliminary, descriptive data pending formal statistical validation.

Conclusion

RGD-functionalized WGENs constitute a promising plant-derived nanocarrier platform for proof-of-concept doxorubicin delivery, with enhanced uptake in αvβ3-overexpressing 4T1 cells. These preliminary findings warrant further investigation with appropriate mechanistic controls and in vivo validation.