ROS‑Responsive Piperlongumine‑Eluting GI Stent: A Novel Nanofiber Strategy to Suppress Cholangiocarcinoma

Abstract

Background

We engineered a drug‑eluting gastrointestinal (GI) stent that releases piperlongumine (PL) via reactive oxygen species (ROS)‑sensitive nanofiber mats. The PL‑laden mats were designed to provide a localized, ROS‑triggered anticancer effect against cholangiocarcinoma (CCA) cells.

Methods

We synthesized a diselenide‑linked block copolymer (LEse) by conjugating selenocystamine to methoxy poly(ethylene glycol) (MePEG) and then to poly(lactide) (PLA). By electrospinning mixtures of poly(ε‑caprolactone) (PCL) and LEse in various ratios, we produced PL‑containing nanofiber mats. These mats were coated onto a silicone‑membrane‑covered biliary stent. In vitro drug‑release, ROS responsiveness, and anticancer activity were evaluated, followed by an in vivo HuCC‑T1 xenograft study.

Results

Nanofibers with higher LEse content exhibited ROS‑triggered acceleration of PL release, whereas PCL‑only mats showed minimal responsiveness. PL released from the mats retained potent cytotoxicity against several CCA cell lines, induced ROS generation, and promoted apoptosis. In HuCC‑T1 tumor‑bearing mice, the PL‑eluting stent significantly inhibited tumor growth and increased caspase‑3/9 expression compared to controls.

Conclusion

LEse‑sensitive nanofiber mats successfully convert a conventional GI stent into a ROS‑responsive, locally acting anticancer platform. This strategy offers a promising, targeted approach for treating unresectable cholangiocarcinoma.

Background

Cholangiocarcinoma (CCA) originates from the bile duct epithelium and is among the most aggressive malignancies, with rising incidence worldwide. Because most patients present at advanced stages, surgical resection is rarely feasible, and conventional therapies—radiotherapy, systemic chemotherapy, and metal stenting—offer limited benefit. Stent placement is essential to relieve biliary obstruction, yet metal stents lack curative properties and are prone to tumor overgrowth, prompting research into drug‑eluting stents (DES). Early DES trials using paclitaxel, sorafenib, and vorinostat demonstrated modest improvements in stent patency and tumor inhibition, yet a curative effect remains elusive.

Stimuli‑responsive nanomaterials, particularly ROS‑sensitive systems, have emerged as powerful drug‑delivery platforms for cancer therapy. Diselenide linkages in polymers degrade in the oxidative tumor microenvironment, triggering rapid drug release. Piperlongumine (PL), a natural alkaloid from Piper longum, selectively elevates ROS in cancer cells, inducing DNA damage and apoptosis while sparing normal tissues. However, PL’s rapid systemic clearance and renal toxicity necessitate localized delivery.

We therefore fabricated a redox‑responsive, PL‑loaded nanofiber coating for a GI stent, leveraging the tumor’s ROS milieu to achieve on‑demand drug release and potent anticancer activity.

Materials and Methods

Materials

PL (LKT Labs, MN, USA), PLA (MW ≈ 5 kDa, Wako), MePEG‑NHS (MW ≈ 5 kDa, Sunbio), selenocystamine (Sigma‑Aldrich), PCL (MW ≈ 80 kDa, Sigma‑Aldrich), and other reagents were of analytical grade. The silicone‑membrane‑covered biliary stent (1 cm × 10 cm) was sourced from M.I. Tech (Pyeongtaek, Korea).

Synthesis of LEse Block Copolymer

MePEG‑NHS was reacted with excess selenocystamine in DMSO, followed by dialysis (MWCO 1 kDa) and lyophilization. The resulting MePEG‑selenocystamine was then coupled to PLA via EDAC/NHS chemistry, yielding the LEse block copolymer (yield > 94 %). ¹H‑NMR confirmed characteristic peaks at 1.7 ppm and 2.9 ppm (selenocystamine) and 3.5–3.7 ppm (MePEG). GPC analysis gave Mn ≈ 8.2 kDa and Mw ≈ 9.5 kDa.

Preparation of PL‑Loaded Nanofiber Mats and Stent Coating

PL was dissolved in acetone (10 mg mL⁻¹). LEse (100–400 mg) and PCL (600–900 mg) were added, stirred for 2 h, and electrospun onto the stent (15 kV, 100 µL min⁻¹, 500 rpm). The coated stent was vacuum‑drying for 24 h and stored at 4 °C.

Drug Loading and Release Studies

Drug content was quantified by UV‑Vis at 325 nm after dissolving 5 mg of mats in DMSO. Release was monitored in PBS (0.01 M, pH 7.4) at 37 °C with and without 10 mM H₂O₂. Samples were collected at predetermined intervals and analyzed by UV‑Vis.

Cell Culture and Anticancer Assays

Human CCA lines (SNU‑478, SNU‑245, SNU‑1196, HuCC‑T1) were cultured in RPMI‑1640 with 10 % FBS. Cells were treated with PL, released PL (day 5 or 15), or vehicle for 48 h, and viability assessed via MTT. IC₅₀ values were calculated.

ROS Measurement

DCFH‑DA fluorescence (485 nm/535 nm) quantified intracellular ROS after 6–12 h exposure to PL or released PL.

Western Blotting

Cells were lysed 24 h post‑treatment, and protein extracts (50 µg) were resolved by SDS‑PAGE, transferred to PVDF, and probed for BAX, caspase‑3/7/9, and cleaved PARP. Bands were visualized by chemiluminescence and quantified with ImageJ.

In Vivo Tumor Xenograft Model

BALB/c nude mice received subcutaneous HuCC‑T1 cells (1 × 10⁶ cells). When tumors reached 4–5 mm, PL‑loaded or empty nanofiber disks (10 mg PL kg⁻¹) were implanted beneath the tumor. Tumor volumes were measured every 5 days, calculated as V = (a × b²)/2. All procedures were approved by the PNUIACUC (Approval No. PNU‑2017‑1608).

Immunohistochemistry

Tumor sections were stained for caspase‑3 and caspase‑9 using standard protocols. Staining intensity was scored qualitatively.

Statistical Analysis

Data were analyzed by Student’s t‑test; p < 0.05 was considered significant.

Results

Polymer Characterization

The LEse block copolymer was successfully synthesized, as evidenced by ¹H‑NMR and GPC. Blend ratios of PCL to LEse were varied to optimize fiber morphology and drug release.

Nanofiber Morphology and Drug Loading

FE‑SEM images revealed that pure PCL produced fine, uniform fibers, whereas LEse addition induced granules and irregularities at higher loadings (≥ 50 % LEse). Optimal mats (60 % LEse) maintained acceptable fiber morphology and incorporated PL at theoretical loadings (~ 10 % w/w).

ROS‑Responsive Drug Release

PL release was sustained over 25 days, with a burst phase up to 4 days. Mat compositions with higher LEse accelerated release, especially in the presence of H₂O₂, confirming ROS sensitivity. PCL‑only mats exhibited negligible responsiveness.

In Vitro Anticancer Activity

Released PL from day 5 and day 15 retained cytotoxicity comparable to freshly dissolved PL across all CCA lines, with IC₅₀ values only slightly higher. ROS assays showed that both PL and released PL induced significant intracellular ROS. Western blotting demonstrated upregulation of BAX, caspase‑3/7/9, and cleaved PARP, indicating apoptosis induction.

In Vivo Efficacy

In the HuCC‑T1 xenograft model, PL‑loaded stents reduced tumor volume to one‑third of controls and enhanced caspase‑3/9 expression, whereas empty stents or vehicle had no effect.

Discussion

The tumor microenvironment’s elevated ROS levels both trigger PL‑induced apoptosis and accelerate release from the diselenide‑linked nanofibers, creating a synergistic, localized therapeutic effect. This dual action mitigates systemic exposure and addresses the challenges of conventional DES, which lack tumor‑targeted release.

Conclusion

We have demonstrated that a ROS‑responsive PL‑loaded nanofiber coating on a GI stent effectively delivers anticancer therapy to cholangiocarcinoma tumors in vitro and in vivo. This platform offers a promising strategy for local, controlled treatment of unresectable CCA.