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<p>Hepatocellular carcinoma remains difficult to treat effectively, motivating delivery strategies that improve the performance of bioactive natural products under chemically traceable conditions. Here, a hydroethanolic propolis extract (EEP) was first standardized by high-performance liquid chromatography with diode-array detection (HPLC–DAD) using a marker-based approach, then associated with umbilical mesenchymal stem cell-derived small extracellular vesicle-enriched exosome preparations (EXOs) by sonication-assisted membrane permeabilization followed by size-exclusion re-purification. The resulting formulation was characterized by transmission electron microscopy, nanoparticle tracking analysis, immunoblotting of extracellular vesicle markers, and post-loading HPLC quantification of representative propolis markers. EEP-EXOs retained vesicle-like morphology and canonical extracellular vesicle marker detectability, with a modest hydrodynamic size increase and preserved particle recovery after loading. The apparent encapsulation efficiencies of the two selected representative markers were 25.93 ± 0.34% for caffeic acid phenethyl ester and 15.82 ± 0.28% for caffeic acid, with nearly quantitative recovery of both analytes. In HepG2 hepatocellular carcinoma cells, the EXO-associated EEP formulation elicited a more pronounced cytotoxic response after 48&#xa0;h than the free extract, decreasing the IC₅₀ from 42.94 to 23.20&#xa0;µg/mL on a caffeic acid phenethyl ester (CAPE)-equivalent basis. Under IC₅₀-equivalent exposure conditions, the loaded formulation further increased the proportion of total Annexin V-positive cells, enhanced caspase-3/7 activity, and induced greater 2′,7′-dichlorofluorescin diacetate (DCFH-DA)-based oxidant-sensitive intracellular fluorescence compared with the free extract, whereas empty EXOs exerted only minimal effects. RT-qPCR analysis demonstrated a consistent transcriptional response, marked by upregulation of BAX, CASP3, CDKN1A, NRF2, NQO1, and SOD2, together with downregulation of BCL2. The most pronounced changes in gene expression were consistently observed in cells treated with the EXO-associated formulation. Collectively, these findings provide preliminary in vitro proof-of-concept evidence in the HepG2 model that EXO-associated formulation enhances the cellular bioactivity of chemically standardized propolis polyphenols under matched CAPE-equivalent conditions. However, the present data do not define the mechanisms governing cellular uptake, intracellular trafficking, or cargo release. Further validation in additional hepatocellular carcinoma models, non-malignant hepatic cells, and more physiologically relevant experimental systems is required before firm conclusions can be drawn regarding tumor selectivity, broader applicability to HCC, or translational relevance.</p> Graphical Abstract <p></p>

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Umbilical MSC-Derived Exosome-Associated Standardized Propolis Polyphenols Enhance Apoptosis-Associated and Oxidant-Responsive Signals in HepG2 Cells

  • 期刊论文

  • 发表日期：

    2026年7月1日

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  • Chunmei Wang,
  • Yuanyuan Cui,
  • Arezu Golabi,
  • Hosna Ahmadi,
  • Zhongying Chen,
  • Li Dong,
  • Rongping Ning

摘要

Hepatocellular carcinoma remains difficult to treat effectively, motivating delivery strategies that improve the performance of bioactive natural products under chemically traceable conditions. Here, a hydroethanolic propolis extract (EEP) was first standardized by high-performance liquid chromatography with diode-array detection (HPLC–DAD) using a marker-based approach, then associated with umbilical mesenchymal stem cell-derived small extracellular vesicle-enriched exosome preparations (EXOs) by sonication-assisted membrane permeabilization followed by size-exclusion re-purification. The resulting formulation was characterized by transmission electron microscopy, nanoparticle tracking analysis, immunoblotting of extracellular vesicle markers, and post-loading HPLC quantification of representative propolis markers. EEP-EXOs retained vesicle-like morphology and canonical extracellular vesicle marker detectability, with a modest hydrodynamic size increase and preserved particle recovery after loading. The apparent encapsulation efficiencies of the two selected representative markers were 25.93 ± 0.34% for caffeic acid phenethyl ester and 15.82 ± 0.28% for caffeic acid, with nearly quantitative recovery of both analytes. In HepG2 hepatocellular carcinoma cells, the EXO-associated EEP formulation elicited a more pronounced cytotoxic response after 48 h than the free extract, decreasing the IC₅₀ from 42.94 to 23.20 µg/mL on a caffeic acid phenethyl ester (CAPE)-equivalent basis. Under IC₅₀-equivalent exposure conditions, the loaded formulation further increased the proportion of total Annexin V-positive cells, enhanced caspase-3/7 activity, and induced greater 2′,7′-dichlorofluorescin diacetate (DCFH-DA)-based oxidant-sensitive intracellular fluorescence compared with the free extract, whereas empty EXOs exerted only minimal effects. RT-qPCR analysis demonstrated a consistent transcriptional response, marked by upregulation of BAX, CASP3, CDKN1A, NRF2, NQO1, and SOD2, together with downregulation of BCL2. The most pronounced changes in gene expression were consistently observed in cells treated with the EXO-associated formulation. Collectively, these findings provide preliminary in vitro proof-of-concept evidence in the HepG2 model that EXO-associated formulation enhances the cellular bioactivity of chemically standardized propolis polyphenols under matched CAPE-equivalent conditions. However, the present data do not define the mechanisms governing cellular uptake, intracellular trafficking, or cargo release. Further validation in additional hepatocellular carcinoma models, non-malignant hepatic cells, and more physiologically relevant experimental systems is required before firm conclusions can be drawn regarding tumor selectivity, broader applicability to HCC, or translational relevance.

Graphical Abstract