Targeted Silica Nanoparticles Co‑Deliver miR‑495 and Doxorubicin to Overcome Multidrug Resistance in Lung Cancer

Abstract

Multidrug resistance (MDR) and non‑specific distribution limit the effectiveness of conventional chemotherapy. We engineered a cancer‑cell‑membrane (CCM) coated silica (SLI) nanoparticle capable of co‑loading miR‑495 and doxorubicin (DOX) to address both challenges. The CCM, derived from the MDR lung‑cancer line A549/DOX, confers tumor‑homing properties, while miR‑495 silences the P‑glycoprotein (P‑gp) efflux pump, enhancing intracellular drug retention. In vitro and in vivo studies demonstrated that CCM/SLI/R‑D markedly outperformed free DOX, CCM/SLI/DOX, and CCM/SLI/miR‑495 alone, achieving superior tumor regression and minimal systemic toxicity. This multifunctional platform offers a promising strategy for MDR‑challenged lung‑cancer therapy.

Introduction

Multidrug resistance, largely mediated by ATP‑binding cassette transporters such as P‑glycoprotein (P‑gp), remains a primary obstacle in effective chemotherapy [1,2,3]. P‑gp actively effluxes chemotherapeutics, reducing intracellular concentrations and clinical outcomes [4,5]. Targeting P‑gp, therefore, represents a rational approach to resensitize resistant tumors.

MicroRNAs (miRNAs) regulate gene expression post‑transcriptionally and have emerged as potent modulators of drug sensitivity. miR‑495, in particular, has been shown to downregulate P‑gp in ovarian and gastric cancers [8] and is now investigated in the context of MDR lung cancer.

Nanoparticle‑based drug delivery systems (DDS) have transformed cancer therapeutics by improving bioavailability and reducing off‑target effects [9‑12]. Silica nanoparticles (SLI) offer high drug loading, tunable surface chemistry, and excellent biocompatibility [17,18]. Yet, most DDS rely on ligand conjugation for targeting, which can trigger immunogenicity and cytotoxicity. In contrast, decorating nanoparticles with homologous cancer‑cell membranes (CCM) provides innate tumor‑homing capabilities while maintaining biocompatibility [24,25].

Here we combine CCM‑mediated targeting with miR‑495–induced P‑gp inhibition in a single silica nanoparticle platform (CCM/SLI/R‑D) to achieve synergistic reversal of MDR and enhanced chemotherapeutic efficacy in lung cancer.

Materials and Methods

Materials

All reagents, including MTT, AEAPS, TEOS, DOX, Triton X‑100, and miR‑495, were obtained from Sigma‑Aldrich or Cell Biolabs. Cell lines (A549, A549/DOX, NIH3T3) were cultured in DMEM supplemented with 10% FBS, penicillin, and streptomycin. A549/DOX was generated by incremental DOX exposure as described previously [26].

Nanoparticle Fabrication

Amine‑functionalized silica (SLI) nanoparticles were synthesized via a water‑in‑oil microemulsion. DOX was entrapped during hydrolysis of TEOS and AEAPS. Subsequent loading of miR‑495 at optimal weight ratios yielded SLI/R‑D binary complexes, which were finally cloaked with CCM extracted from A549/DOX cells, producing the final CCM/SLI/R‑D formulation.

Characterization

Dynamic light scattering (DLS) and transmission electron microscopy (TEM) confirmed a uniform ~120 nm diameter and core‑shell morphology. Zeta potential measurements showed a surface charge shift from +26 mV (SLI/R‑D) to −28 mV after CCM coating, indicating successful membrane decoration. Gel retardation assays demonstrated complete miR‑495 binding at a 20:1 SLI:miR‑495 weight ratio. Drug loading capacities were 17.96% for DOX (HPLC) and 1.64% for miR‑495 (UV).

Stability and Release

CCM/SLI/R‑D remained stable in PBS (pH 7.4) and mouse plasma over 48 h, with <33% drug release extracellularly. Acidic conditions (pH 5.5) accelerated release to ~76%, mimicking the tumor microenvironment.

In Vitro Studies

Transfection of A549/DOX with CCM/SLI/miR‑495 reduced P‑gp expression in a dose‑dependent manner (Western blot). Consequently, intracellular DOX accumulation increased by up to 1.85‑fold compared to controls. Cytotoxicity assays (MTT) showed that CCM/SLI/R‑D achieved a 38.5% viability at 50 µM DOX, outperforming free DOX (72.6%) and single‑agent formulations (≈60%). The combination index (CI) of 0.84 indicated strong synergy. Apoptosis markers (cleaved caspase‑3, cytochrome C, low Bcl‑2) confirmed enhanced mitochondrial‑mediated cell death.

Multicellular Tumor Spheroid (MCTS) Assay

In 3D cultures, CCM/SLI/R‑D inhibited spheroid growth to negative volume change over five days, whereas free DOX promoted continued expansion, underscoring the platform’s efficacy against resistant tumor masses.

In Vivo Evaluation

Balb/c nude mice bearing A549/DOX xenografts received intratumoral injections of CCM/SLI/R‑D (5 mg/kg DOX, 0.25 mg/kg miR‑495) seven times over 14 days. Tumor volumes decreased to 303 ± 25 mm³, the lowest among all groups. Body weight remained stable, indicating reduced systemic toxicity compared with free DOX, which caused progressive weight loss.

Targeting Efficiency

Fluorescently labeled CCM/SLI/R‑D displayed superior tumor accumulation in vivo (24 h post‑injection) and markedly higher cellular uptake in A549/DOX cells than uncoated SLI/R‑D. Pre‑incubation with excess CCM competitively inhibited uptake, confirming membrane‑mediated endocytosis.

Conclusion

We have successfully fabricated a CCM‑coated silica nanoparticle that co‑delivers miR‑495 and DOX, achieving tumor‑homing, MDR reversal, and synergistic cytotoxicity in lung cancer models. The platform’s robust stability, pH‑responsive release, and minimal toxicity make it a promising candidate for clinical translation in MDR‑driven tumors.