A folate-functionalized chitosan nanoparticle loaded with an ethanolic stembark extract of Ailanthus excelsa Roxb. suppressed key malignant behaviors in triple-negative breast cancer cells and in mouse xenografts by restoring the tumor-suppressor microRNA miR-34a and repressing NOTCH1 signaling, according to a study published on 3 September 2026 in Clinical Phytoscience. The work, led by Cletus Anes Ukwubile with colleagues, combined nanoparticle formulation, cell assays, RNA sequencing, animal experiments and public TCGA data analysis to probe a signaling axis that the authors link to cancer stemness, epithelial-mesenchymal transition and metastasis in this aggressive breast cancer subtype.
Triple-negative breast cancer lacks the three receptors that guide most targeted breast cancer therapy and accounts for roughly 15 to 20 percent of breast cancer cases, the authors note. NOTCH1, a signaling pathway component that helps maintain cancer stem cell populations and promotes epithelial-mesenchymal transition, is frequently activated in these tumors and correlates with poor prognosis and therapeutic resistance. miR-34a, a small noncoding RNA regulated by the p53 pathway, normally restrains genes involved in cell cycle progression, apoptosis and stemness, but its loss in many cancers, including triple-negative disease, permits cancer stem cell enrichment and invasiveness. Because miRNA-based therapies suffer from poor stability in circulation, rapid nuclease degradation and inefficient cellular uptake, the researchers sought a delivery vehicle that could protect and target their plant-derived payload.
The team collected fresh stembark of Ailanthus excelsa, authenticated it taxonomically, and extracted it with 70 percent ethanol in a Soxhlet apparatus for 48 hours. High-performance liquid chromatography and liquid chromatography-mass spectrometry profiling revealed multiple well-resolved phytochemical peaks, with quassinoids and flavonoid derivatives among the major constituents quantified against reference standards. The extract was then encapsulated in chitosan nanoparticles prepared by ionic gelation, in which positively charged chitosan interacts electrostatically with negatively charged tripolyphosphate. A subset of particles was functionalized with folic acid through EDC/NHS carbodiimide coupling to exploit folate receptors that triple-negative breast cancer cells overexpress, enabling receptor-mediated uptake.
Physicochemical characterization showed predominantly spherical particles under transmission electron microscopy, with hydrodynamic diameters averaging 150 to 160 nanometers for the untargeted formulation and 180 to 190 nanometers after folic acid modification, and polydispersity indices below 0.2 in both cases. Zeta potentials were approximately negative 28 millivolts for the untargeted particles and negative 32 millivolts for the folate-functionalized version, values the authors attribute to carboxyl moieties on the surface ligand and to electrostatic repulsion that limits aggregation. In vitro release in phosphate-buffered saline at 37 degrees Celsius followed a biphasic pattern: an initial burst over roughly 8 to 12 hours, attributed to surface-adsorbed drug, followed by slower diffusion from the polymer matrix up to 48 hours, with the functionalized particles releasing slightly more slowly because of the added surface barrier.
Biological testing centered on MDA-MB-231, a human triple-negative breast cancer cell line. Quantitative reverse-transcription PCR showed a stepwise increase in miR-34a expression from free extract to untargeted nanoparticles to folate-targeted nanoparticles, with the targeted formulation producing the strongest rise relative to controls. A dual-luciferase reporter assay established that miR-34a directly targets NOTCH1: co-transfection of a miR-34a mimic with a reporter carrying the wild-type NOTCH1 3-prime untranslated region significantly reduced normalized luciferase activity, whereas a mutant construct with a disrupted binding site showed no repression, indicating sequence-specific binding. Rescue experiments reinforced the mechanism, as transfecting a miR-34a inhibitor before nanoparticle exposure restored NOTCH1 protein expression and reversed the nanoparticles’ suppression of cell viability, migration, invasion and tumorsphere formation.
Functional assays quantified these effects. In MTT assays, the folate-targeted nanoparticles achieved half-maximal inhibitory concentrations of 72.5 plus or minus 3.6 micrograms per milliliter at 24 hours, 31.8 plus or minus 1.9 at 48 hours and 18.2 plus or minus 1.1 at 72 hours in MDA-MB-231 cells, outperforming both the free extract and untargeted particles at every time point. Against normal MCF-10A breast epithelial cells, the same formulation required 97.9 plus or minus 4.7 micrograms per milliliter at 72 hours, yielding a selectivity index of 5.38 that indicates preferential cytotoxicity toward the cancer cells. Wound-healing and Matrigel-coated Transwell assays showed the greatest reductions in migration and invasion with the targeted nanoparticles, and tumorsphere assays under non-adherent conditions revealed marked decreases in sphere-forming efficiency and average sphere diameter, consistent with impaired self-renewal of cancer stem-like cells.
RNA sequencing of nanoparticle-treated versus control cultures identified more than 2,000 differentially expressed genes at an adjusted p-value below 0.05 and an absolute log2 fold change of at least 2, with 1,138 genes upregulated and 1,024 downregulated. Pathway enrichment highlighted PI3K-Akt, NOTCH1, Wnt and TGF-beta signaling alongside epithelial-mesenchymal transition and stem cell pluripotency terms. Stemness genes including Nanog, SOX2, Oct4 and BMI1 fell sharply, as did mesenchymal and invasive markers such as VIM, SNAI1, ZEB1 and MMP9, while the epithelial marker CDH1 rose, a pattern the authors interpret as reversal of epithelial-mesenchymal transition. NOTCH pathway elements, including NOTCH1 through 4, HES1 and JAG1, were consistently downregulated, with NOTCH1 showing one of the strongest reductions.
In female athymic nude mice bearing subcutaneous MDA-MB-231 xenografts, treatment began once tumors reached approximately 100 cubic millimeters and continued for four weeks by intraperitoneal injection. The targeted nanoparticles produced the largest reductions in tumor volume, excised tumor size and final tumor weight among the nanoparticle groups, approaching the efficacy of doxorubicin while causing markedly less toxicity. In a tail-vein metastasis model, the targeted formulation yielded the fewest lung metastatic nodules, and Kaplan-Meier analysis showed significantly prolonged overall survival compared with controls. Blood chemistry parameters including ALT, AST, ALP, BUN and creatinine remained unchanged in nanoparticle-treated animals, whereas doxorubicin elevated these markers, and histopathological examination of heart, liver, kidney and spleen showed no visible structural damage in nanoparticle-treated mice. Pharmacokinetic and biodistribution studies using fluorescently labeled formulations demonstrated prolonged circulation, increased maximum plasma concentration, delayed time to peak, longer half-life and greater area under the curve for the nanoparticle form, with preferential tumor accumulation and only moderate liver and spleen uptake at 24 hours.
Analysis of The Cancer Genome Atlas breast cancer cohort, stratified by receptor status into normal, non-TNBC and TNBC groups, showed that miR-34a was significantly decreased in TNBC tissues and inversely correlated with NOTCH1, with a strong negative Spearman correlation (r below 0, p below 0.001). Patients with high miR-34a expression had better overall and disease-free survival, while high NOTCH1 expression predicted worse outcomes; univariate Cox modeling identified NOTCH1 as a risk factor with a hazard ratio above 1 and miR-34a as protective with a hazard ratio below 1. When the nanoparticle formulation was combined with doxorubicin in vitro and in vivo, combination index values fell below 1 at all tested effect levels and time points, indicating synergy, and the combination produced greater tumor suppression, lower metastatic burden and less systemic toxicity than either agent alone, with the combination group receiving the same doxorubicin dose as the monotherapy group.
The authors emphasize several caveats. The relatively high effective concentrations reflect the use of a crude plant extract rather than purified compounds, and future work should identify the principal active constituents. Although miR-34a restoration was accompanied by reduced NOTCH1 expression, direct mechanistic validation through NOTCH1 overexpression or rescue was not performed, so the causal contribution of NOTCH1 to the observed effects remains to be established. The 24-hour pharmacokinetic sampling window may not have captured the terminal elimination phase, making half-life estimates preliminary. The study also relies on established cell lines and immunodeficient xenografts, which do not fully reproduce the genetics and immune context of human triple-negative breast cancer, and the authors call for patient-derived models, immunocompetent or humanized animals, longer safety assessments and biomarker-guided patient selection before clinical translation.
Subject of Research: Nanoparticle delivery of Ailanthus excelsa phytochemicals to restore miR-34a and suppress NOTCH1-driven cancer stemness in triple-negative breast cancer
Article Title: Nanocarrier-enabled restoration of miR-34a by Ailanthus excelsa Roxb. phytochemicals directly target NOTCH1 signaling to suppress cancer stemness in triple-negative breast cancer
Article References: Ukwubile, C. A., Ahuchaogu, A. A., Robert, A. E., Yesufu, H. B., Odugu, J. A., Ida, P. A., & Ikpefan, E. O. (2026). Nanocarrier-enabled restoration of miR-34a by Ailanthus excelsa Roxb. phytochemicals directly target NOTCH1 signaling to suppress cancer stemness in triple-negative breast cancer. Clinical Phytoscience, 12(1), Article 25. https://doi.org/10.1186/s40816-026-00440-6
Image Credits: AI Generated
DOI: 10.1186/s40816-026-00440-6
Keywords: triple-negative breast cancer, miR-34a, NOTCH1, chitosan nanoparticles, folic acid targeting, Ailanthus excelsa, cancer stemness, epithelial-mesenchymal transition, MDA-MB-231 xenograft, RNA sequencing, doxorubicin synergy, TCGA analysis
News Source: Nathaniel Bowman. (October 28, 2026). Plant-Derived Nanoparticles Restore Tumor-Suppressor miR-34a to Block NOTCH1 in Triple-Negative Breast Cancer. Scienmag.
