Tier 4 — mechanistic

Targeting Cellular Senescence with Liposome-Encapsulated Fisetin: Evidence of Senomorphic Effect

Henschke A, Grześkowiak B, Ivashchenko O, Sánchez-Cerviño MC, Coy E, Moya S

International Journal of Molecular Sciences 2025 26(15):7489

Bibliography

DOI: 10.3390/ijms26157489
PubMed: PMID 40806616
PubMed Central: PMC12347707
Funding: Funded by the National Science Centre of Poland under the OPUS programme (grant 2019/33/B/ST5/01495). No industry funding declared.
Competing interests: The authors declare no conflicts of interest. All authors are academic researchers at public institutions in Poland, Argentina, and Spain, with no disclosed commercial relationships with fisetin or liposome manufacturers.

Study snapshot

Design	Doxorubicin-induced senescence model in two human lung cell lines (WI-38 normal lung fibroblasts; A549 lung adenocarcinoma). Cells were exposed to fisetin — either in free form or encapsulated in DOPC/DSPE/cholesterol liposomes prepared by thin-film hydration — across concentrations from 1.25 to 160 μM. Outcomes measured by live/dead viability assay, SA-β-galactosidase staining, EdU proliferation, cell morphology (CLSM), imaging flow cytometry, cellular uptake of Nile red-labelled liposomes, and ELISA quantification of IL-6 and IL-8 secretion.
Model	Doxorubicin-induced senescence in two human lung cell lines cultured in vitro: WI-38 (normal diploid lung fibroblasts) and A549 (lung adenocarcinoma). Senescence induced by 72 h DOX exposure (1 μM for WI-38, 0.2 μM for A549), followed by 72 h drug-free recovery before intervention.
Sample	In vitro, biological triplicate (n = 3) for all assays. Imaging flow cytometry acquired 1000 events per sample. No animal or human subjects.
Intervention	Free fisetin versus liposome-encapsulated fisetin, at matched concentrations of 1.25, 2.5, 5, 10, 20, 40, 80 and 160 μM. Liposomes were approximately 95–116 nm hydrodynamic diameter, encapsulation efficiency 13.68%.
Duration	Approximately 9 days per experimental arm: 72 h senescence induction + 72 h recovery + 48 h fisetin/liposome treatment + 24 h medium conditioning before ELISA readout. Liposome stability studies ran 30 days at 4 °C. Cellular uptake incubation was 4 h.
Endpoints	Cell viability (LIVE/DEAD Fixable Far Red membrane-integrity stain, imaging flow cytometry); SA-β-galactosidase activity (colorimetric staining, quantified as percentage-positive cells); Cell proliferation (EdU incorporation, fluorescence imaging); Cell morphology (CLSM with phalloidin-488 actin stain and Hoechst 33342 nuclear counterstain); Cell size distribution (imaging flow cytometry, area in μm²); Senescence-associated heterochromatin foci (SAHF) presence (Hoechst nuclear pattern, WI-38 only); IL-6 secretion (ELISA); IL-8 secretion (ELISA); Liposome hydrodynamic diameter, polydispersity index, and zeta potential (DLS, day 1 and day 30); Liposome morphology (Cryo-SEM); Fisetin encapsulation efficiency (fluorimetry against calibration curve); Cellular uptake of Nile red-labelled liposomes (CLSM); Metabolic activity (WST-1 assay, supplementary)

What the study showed, in plain terms

Fisetin is one of the most-studied senotherapeutics — compounds that target the "zombie" cells (senescent cells) that accumulate with age and after cancer treatment. Most of the literature classifies fisetin as a senolytic, meaning it kills senescent cells outright. Does that classification hold up when fisetin is tested in a controlled cell-culture setting on lung cells damaged by doxorubicin, a widely used chemotherapy drug?

Henschke and colleagues at Adam Mickiewicz University tested this. They induced senescence in two human lung cell lines — WI-38 (normal lung fibroblasts) and A549 (lung adenocarcinoma) — using doxorubicin, then treated the resulting senescent cells with fisetin at concentrations from 1.25 to 160 μM, either as free drug or encapsulated in liposomes.

The result was surprising. Fisetin did not selectively kill the senescent cells in either line. What it did do was quieten them: it significantly reduced their secretion of two pro-inflammatory signals, interleukin-6 and interleukin-8, which together form part of the inflammatory profile that senescent cells release into surrounding tissue. Compounds that suppress this profile without killing the cell are called senomorphics. The liposomal formulation delivered maximal suppression at the lowest tested dose — a potency advantage over free fisetin.

The bottom line. This is a mechanistic in-vitro study on two lung cell lines, not a human trial. It does not tell us what happens to senescent cells in the body. But it does complicate the common description of fisetin as a "senolytic" — at least in this model, on these cell types, fisetin worked by dampening the inflammatory output of senescent cells rather than eliminating them. For readers thinking about fisetin as a longevity supplement, the practical implication is that the senolytic-versus-senomorphic distinction may depend heavily on cell type and context, and that firm claims in either direction outrun the evidence.

Key findings

Fisetin did not exhibit senolytic activity in either cell line at any tested concentration — viability of senescent cells tracked non-senescent cells across the whole dose range. Fisetin did exhibit senomorphic activity: it significantly reduced IL-6 and IL-8 secretion in senescent WI-38 and A549 cells. The liposomal formulation reached maximal SASP suppression at the lowest tested concentration (1.25 μM) in A549 cells and matched or exceeded free fisetin across the concentration range. Liposome encapsulation efficiency was 13.68% (approximately 245 μg/mL fisetin, corresponding to 850 μM). Liposomes measured 95–116 nm by DLS and remained stable in size over 30 days, though zeta potential drifted from −20.3 to −8.3 mV. Cellular uptake was limited at 4 h, with most liposomes remaining at the cell surface.

What this study can and cannot tell us

In vitro only — no animal or human data. Only two cell lines tested, both from lung tissue, so cell-type generalisability is unproven. The senolytic-versus-senomorphic distinction reported here contradicts the wider fisetin senotherapeutic literature and may reflect the specific senescence-induction method used (doxorubicin) rather than a universal property. Encapsulation efficiency of 13.68% is low, and the authors themselves flag deviations from the source protocol (Mignet 2012). Cellular uptake was limited at 4 h and was not quantified, so the reported potency advantage of liposomal fisetin cannot be attributed unambiguously to enhanced delivery. WST-1 results showed high apparent cytotoxicity that other viability assays did not, an inconsistency the authors attribute to metabolic modulation rather than true cytotoxicity — but this is not directly demonstrated.