Enhanced Synergy of Lovastatin and Doxorubicin Delivered via Pullulan Nanoparticles Against Triple‑Negative Breast Cancer

Abstract

Triple‑negative breast cancer (TNBC) remains highly resistant to conventional therapy. We engineered an amphiphilic pullulan–lovastatin conjugate (PLV) and loaded it with doxorubicin (DXR) to produce PLV/DXR nanoparticles (NPs). The PLV conjugate was prepared at three pullulan:LV ratios, with the 1:2 ratio yielding the highest degree of substitution (7.87 %) and the smallest particle size (≈180 nm). PLV/DXR NPs exhibited a sustained, pH‑sensitive release profile (97.9 % DXR released after 72 h at pH 5.4 vs. 76.2 % at pH 7.4) and an encapsulation efficiency of 21 %. In vitro, PLV/DXR NPs markedly inhibited proliferation of the TNBC cell line MDA‑M‑B‑231 (IC₅₀ 0.60 µM) while sparing the non‑TNBC line MDA‑M‑B‑453 (IC₅₀ 11.05 µM). FITC‑labelled NPs demonstrated time‑dependent uptake, with MDA‑M‑B‑231 cells internalizing more particles than MDA‑M‑B‑453 cells. Free drug studies revealed a strong synergistic interaction between LV and DXR, reducing the required dose of each agent by up to 40‑fold. These results support PLV/DXR NPs as a promising platform for TNBC therapy.

Introduction

TNBC lacks ER, PR, and HER2, limiting targeted options and driving poor outcomes. Nanoparticles can exploit the enhanced permeability and retention (EPR) effect to deliver therapeutics selectively to solid tumors. Lovastatin (LV), a lipophilic statin, has shown selective cytotoxicity toward TNBC cells by targeting cancer stem cells and inhibiting the efflux transporter P‑glycoprotein, thereby potentiating chemotherapeutics such as DXR. However, the poor water solubility of LV and the pharmacokinetic mismatch of co‑administered drugs hamper synergistic therapy. Encapsulating LV and DXR within a single, biocompatible nanocarrier addresses these limitations.

Materials and Methods

Pullulan (200 kDa) and LV were conjugated via succinic anhydride activation, yielding PLV conjugates with pullulan:LV molar ratios of 1:2, 1:3, and 1:4. FTIR and ¹H NMR confirmed successful esterification; the 1:2 conjugate had a degree of substitution (DS) of 7.87 %. PLV self‑assembled into micelles (≈180 nm) in aqueous buffer; DXR was incorporated by solvent‑exchange, producing PLV/DXR NPs (≈226 nm). Drug loading and encapsulation efficiency were quantified by UV‑vis spectroscopy. In vitro release was measured in PBS (pH 7.4 and 5.4) at 37 °C via dialysis. MDA‑M‑B‑231 and MDA‑M‑B‑453 cells were cultured in DMEM + 10 % FBS. Cytotoxicity was assessed with CellTiter‑Blue after 48 h exposure; synergism was evaluated using isobologram analysis. Cellular uptake of FITC‑labelled NPs was visualised by fluorescence microscopy.

Results

Drug Combination Synergy

Free LV and DXR exhibited dose‑dependent inhibition of MDA‑M‑B‑231 cells. Increasing DXR concentration reduced LV’s IC₅₀ from 10.92 µM to 0.28 µM (39‑fold), and vice versa. Isobologram analysis identified three synergistic ratios (DXR/LV = 0.025/7.09, 0.3033/3.0, 0.625/0.28 µM). No significant LV activity was observed in MDA‑M‑B‑453 cells, underscoring TNBC specificity.

PLV Conjugate Characterisation

FTIR showed new ester carbonyl peaks at 1725 cm⁻¹; ¹H NMR revealed LV methylene signals (0.5–2.7 ppm). Dynamic light scattering (DLS) and TEM confirmed monodisperse, spherical particles; the 1:2 conjugate (PLV (1/2)) was chosen for further work due to its highest DS and smallest size.

PLV/DXR Nanoparticle Profile

Encapsulation efficiency 20.9 %, loading capacity 1.93 %. FITC loading increased particle size to 254 nm and zeta potential to −15 mV. Release studies showed a rapid burst (≈30 % in 8 h) followed by sustained release; pH 5.4 accelerated cumulative release to 97.9 % at 72 h versus 76.2 % at pH 7.4.

Cellular Uptake

Fluorescence microscopy revealed a time‑dependent increase in NP internalisation; MDA‑M‑B‑231 cells accumulated more FITC than MDA‑M‑B‑453 cells (statistically significant at 1 h).

Cytotoxicity of PLV/DXR NPs

PLV/DXR NPs produced lower IC₅₀ values than free DXR in MDA‑M‑B‑231 cells (0.60 µM vs. 0.86 µM), while efficacy in MDA‑M‑B‑453 cells remained low, mirroring free drug behaviour.

Discussion

PLV/DXR NPs combine the anticancer potency of LV with the cytotoxicity of DXR, delivering both agents in a tumor‑targeted, pH‑sensitive manner. The enhanced uptake and selective cytotoxicity in TNBC cells confirm the platform’s therapeutic promise. Although the current study is limited to in vitro assays, the design aligns with the EPR effect and offers a feasible route to co‑delivery of poorly soluble drugs.

Conclusion

We established a dual‑drug nanoparticle system that leverages pullulan’s biocompatibility to solubilise lovastatin and co‑encapsulate doxorubicin. The resulting PLV/DXR NPs display monodispersity, high drug loading, pH‑responsive release, and selective, synergistic inhibition of TNBC cells, providing a strong foundation for future in vivo evaluation.