Plant-derived extracellular nanovesicles: a promising biomedical approach for effective targeting of triple negative breast cancer cells

Introduction: Triple negative breast cancer (TNBC), a highly aggressive subtype accounting for 15–20% of all breast cancer cases, faces limited treatment options often accompanied by severe side effects. In recent years, natural extracellular nanovesicles derived from plants have emerged as promising candidates for cancer therapy, given their safety profile marked by non-immunogenicity and absence of inflammatory responses. Nevertheless, the potential anti-cancer effects of Citrus limon L.-derived extracellular nanovesicles (CLENs) for breast cancer treatment is still unexplored. Methods: In this study, we investigated the anti-cancer effects of CLENs on two TNBC cell lines (4T1 and HCC-1806 cells) under growth conditions in 2D and 3D culture environments. The cellular uptake efficiency of CLENs and their internalization mechanism were evaluated in both cells using confocal microscopy. Thereafter, we assessed the effect of different concentrations of CLENs on cell viability over time using a dual approach of Calcein-AM PI live-dead assay and CellTiter-Glo bioluminescence assay. We also examined the influence of CLENs on the migratory and evasion abilities of TNBC cells through wound healing and 3D Matrigel drop evasion assays. Furthermore, Western blot analysis was employed to investigate the effects of CLENs on the phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal- regulated kinase (ERK) expression. Results: We found that CLENs were internalized by the cells via endocytosis, leading to decreased cell viability, in a dose- and time-dependent manner. Additionally, the migration and evasion abilities of TNBC cells were significantly inhibited under exposed to 40 and 80 μg/mL CLENs. Furthermore, down-regulated expression levels of phosphorylated phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK), suggesting that the inhibition of cancer cell proliferation, migration, and evasion is driven by the inhibition of the PI3K/AKT and MAPK/ERK signaling pathways. Discussion: Overall, our results demonstrate the anti-tumor efficiency of CLENs against TNBC cells, highlighting their potential as promising natural anti-cancer agents for clinical applications in cancer treatment.


Figure S3 .
Figure S3.Wound healing assay performed in HCC-1806 cells scratched to cause a wound.Cells were incubated without or with 40 and 80 μg/mL CLENs.Representative images are shown the cell migration into wound area 48h (A) and 72h (B) post-wounding.Graphical representation of data from three independent experiments performed in triplicate.Bars, Mean +/-SEM.* p<0.05; ** p<0.01;One-way ANOVA followed by Tukey's multiple comparisons test.

Figure S4 .
Figure S4.Original Western blot image of PI3K, samples of control (extracted proteins from untreated 4T1 cells), CLENs treatment for 72h cell lysate from three independent experiments.Samples were added in the following order: control, 40 μg/mL CLENs, 80 μg/mL CLENs.

Figure S6 .
Figure S6.Original Western blot image of AKT, samples of control (extracted proteins from untreated 4T1 cells), CLENs treatment for 72h cell lysate from three independent experiments.Samples were added in the following order: control, 40 μg/mL CLENs, 80 μg/mL CLENs.

Figure S8 .
Figure S8.Original Western blot image of ERK, samples of control (extracted proteins from untreated 4T1 cells), CLENs treatment for 72h cell lysate from three independent experiments.Samples were added in the following order: control, 40 μg/mL CLENs, 80 μg/mL CLENs.

Figure S10 .
Figure S10.Original Western blot image of β-actin, samples of control (extracted proteins from untreated 4T1 cells), CLENs treatment for 72h cell lysate from three independent experiments.Samples were added in the following order: control, 40 μg/mL CLENs, 80 μg/mL CLENs.

Figure S11 .
Figure S11.Original Western blot image of PI3K, samples of control (extracted proteins from untreated HCC-1806 cells), CLENs treatment for 72h cell lysate from three independent experiments.Samples were added in the following order: control, 40 μg/mL CLENs, 80 μg/mL CLENs.

Figure S13 .
Figure S13.Original Western blot image of AKT, samples of control (extracted proteins from untreated HCC-1806 cells), CLENs treatment for 72h cell lysate from three independent experiments.Samples were added in the following order: control, 40 μg/mL CLENs, 80 μg/mL

Figure S15 .
Figure S15.Original Western blot image of ERK, samples of control (extracted proteins from untreated HCC-1806 cells), CLENs treatment for 72h cell lysate from three independent experiments.Samples were added in the following order: control, 40 μg/mL CLENs, 80 μg/mL CLENs.