A QbD optimization of a pH-responsive Eudragit S100–chitosan nanoformulation for the co-delivery of pentoxifylline and simvastatin in colorectal cancer therapy

Abstract

Colorectal cancer (CRC) presents a significant global health challenge, where the efficacy of conventional treatments is often hampered by systemic toxicity, poor bioavailability, and drug resistance. Drug repurposing and nanotechnology offer promising avenues to overcome these limitations. While pH-responsive Eudragit S100–chitosan hybrid systems are established as robust pH-responsive oral delivery carriers, the present study introduces a novel therapeutic strategy by combining these platforms with the co-delivery of the repurposed drugs pentoxifylline (PTX) and simvastatin (SIM) for synergistic CRC therapy. A hybrid nanoparticle system was synthesized using ionic gelation, combining the mucoadhesive properties of chitosan with the pH-dependent release of Eudragit S100.

The formulation was optimized using a Quality by Design (QbD) Box–Behnken design to yield nanoparticles with a particle size of 152 ± 5 nm, a zeta potential of +31.2 ± 1.5 mV, and high entrapment efficiency for both drugs (PTX: 85.4 ± 3.1%; SIM: 78.9 ± 2.8%). In vitro release studies in simulated gastrointestinal media demonstrated minimal drug release in acidic conditions (PTX <8.3%; SIM <5.7%) and sustained release at colonic pH (cumulative release: PTX 82.3 ± 3.7%, SIM 78.6 ± 3.2% at 24 h; plateau levels of 89.7% and 85.4% by 48 h), confirming the pH-responsive release behavior of the formulation, with preferential drug liberation at neutral-to-alkaline pH conditions mimicking the colonic environment. These in vitro findings demonstrate pH-triggered release characteristics consistent with a mechanistic rationale for preferential colonic drug exposure; however, in vivo validation is required to confirm whether site-specific delivery to the colon is achieved under physiological conditions. Molecular docking simulations revealed strong binding affinities of simvastatin against key cancer targets EGFR, CA9, and GSK3β (ΔG: −7.5 to −8.7 kcal mol⁻¹), providing a mechanistic rationale for its repurposing.

The optimized nanoformulation (NP-PTX/SIM) exhibited significant synergistic anti-proliferative cytotoxic effects against HCT-116 cells (IC₅₀ = 10.21 µg mL⁻¹) compared to free drugs through caspase-3 activation and suppression of proliferative (Ki-67) and angiogenic vascular endothelial growth factor (VEGF) markers confirming its apoptotic effects. By integrating the established Eudragit S100–chitosan carrier with the novel co-delivery of pentoxifylline and simvastatin, coupled with QbD optimization and comprehensive therapeutic evaluation, this work presents a distinct and innovative multi-targeted therapeutic strategy for CRC with improved efficacy and reduced off-target effects.

Introduction

Colorectal cancer (CRC) persists as a leading cause of global cancer-related morbidity and mortality, characterized by high rates of recurrence and metastasis despite advances in surgical and chemotherapeutic interventions.1–3 The limitations of conventional chemotherapy including severe off-target toxicity, poor solubility of active agents, and the rapid development of multidrug resistance underline an urgent need for innovative therapeutic strategies.4 In this study, drug repurposing, the investigation of existing drugs for new therapeutic applications, presents a time- and cost-effective strategy to accelerate oncology drug discovery. Simultaneously, nanotechnology-based drug delivery systems have emerged as powerful tools to enhance drug bioavailability, enable targeted delivery, and mitigate systemic side effects.5

Statins, particularly simvastatin (SIM), traditionally prescribed for dyslipidemia, have garnered significant attention in oncology due to their well-documented pleiotropic effects.6 These extend beyond cholesterol lowering to include the inhibition of cancer cell proliferation, induction of apoptosis, suppression of angiogenesis, and modulation of the tumor microenvironment.7,8 The primary anticancer mechanism involves the inhibition of the mevalonate pathway, which disrupts the prenylation and activation of small GTPases like Ras and Rho, proteins critical for oncogenic signaling.6 Clinical and preclinical evidence increasingly supports the role of statins in reducing CRC-specific mortality, highlighting their potential as adjunctive or primary anticancer agents.8

Pentoxifylline (PTX), a methylxanthine derivative and non-selective phosphodiesterase inhibitor, is another repurposing candidate with potent anti-inflammatory, anti-fibrotic, and immunomodulatory properties. It functions primarily by inhibiting tumor necrosis factor-alpha (TNF-α) production and suppressing Nuclear Factor kappa-B (NF-κB) activation, pathways intimately linked to cancer-associated inflammation, cell survival, and therapy resistance.9–11 The rational combination of SIM and PTX is designed to create a multi-mechanistic attack on CRC, simultaneously targeting intracellular proliferative signaling and the pro-tumorigenic inflammatory microenvironment.12

To fully harness the synergistic potential of this drug combination and ensure its preferential delivery to the colonic site of malignancy, an advanced carrier system is essential. Oral pH-responsive drug delivery remains a preferred yet technically challenging strategy for achieving preferential colonic drug exposure. Chitosan, a natural cationic polysaccharide, is an ideal biopolymer for this purpose. Its excellent biocompatibility, biodegradability, and mucoadhesive properties promote prolonged intestinal residence and enhanced epithelial permeability.13,14 Furthermore, chitosan nanoparticles can be produced under mild conditions via ionic gelation, a process that preserves drug stability and bioactivity.13 To confer precise pH-responsive release, chitosan is strategically combined with Eudragit S100, an anionic copolymer soluble at pH > 7.0.15,16 This hybrid system is engineered to remain intact in the stomach and small intestine, thereby protecting the encapsulated payload, and to dissolve upon reaching the neutral-to-alkaline environment of the colon, ensuring pH-triggered drug release that is mechanistically consistent with a rationale for preferential colonic drug exposure. While Eudragit S100–chitosan hybrid systems are well-recognized as robust, established models for pH-responsive oral delivery; for instance, in the context of curcumin delivery for ulcerative colitis;17 the present study introduces a distinct therapeutic innovation. Our contribution lies not in the carrier platform itself, but in the synergistic co-delivery of the repurposed drugs pentoxifylline and simvastatin, the systematic QbD-driven optimization of this specific combination, and the comprehensive in vitro and in silico evaluation of its therapeutic potential against colorectal cancer. This approach directly addresses the pharmacokinetic shortcomings of both drugs, particularly the poor oral bioavailability and extensive first-pass metabolism of SIM.

This study introduces a novel, comprehensive strategy integrating QbD formulation, computational validation, and extensive biological testing. We hypothesize that the co-encapsulation of PTX and SIM within a Eudragit S100–chitosan hybrid nanoparticle system will create a synergistic, pH-responsive therapeutic platform with superior anticancer efficacy against CRC. The work is structured to first optimize the formulation using a Box–Behnken statistical design, followed by thorough physicochemical characterization. Molecular docking simulations are employed to elucidate the binding interactions of the drugs with key CRC-associated protein targets, providing a robust in silico rationale for the combination. Finally, the therapeutic potential is rigorously validated through in vitro drug release studies, cytotoxicity assays against HCT-116 cells, and the assessment of apoptotic and proliferative biomarkers.

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Materials

Low molecular weight chitosan (150 kDa, deacetylation degree ≥95%), pentoxifylline (PTX, 99.5% purity), simvastatin (SIM, 98% purity), glacial acetic acid, dimethyl sulfoxide (DMSO), and sodium tripolyphosphate were procured from Sigma-Aldrich (St. Louis, MO, USA). Eudragit S100 was generously provided by Evonik Operations GmbH (Darmstadt, Germany). Ethanol, acetone, and sodium hydroxide were obtained from Merck KGaA (Darmstadt, Germany). High-glucose Dulbecco’s Modified Eagle Medium (DMEM), RPMI-1640 medium, fetal bovine serum (FBS), HEPES buffer solution, 0.25% trypsin–EDTA, l-glutamine, and gentamycin were purchased from Lonza (Belgium). Trypan blue dye, MTT, and DMSO from Sigma (St. Louis, Mo., USA). The human colorectal carcinoma cell line (HCT-116) was obtained from the American Type Culture Collection (ATCC, Rockville, MD). All other chemicals and solvents were of analytical or HPLC grade.

Samar M. Mahgoub, Seham M. Hamed, Ahmed A. Allam, Doaa R. I. Abdel-Gawad, Ahmed G. Soliman, Khaled Metwally and Rehab Mahmoud, A QbD optimization of a pH-responsive Eudragit S100–chitosan nanoformulation for the co-delivery of pentoxifylline and simvastatin in colorectal cancer therapy, RSC Adv.,2026,16, 27847, Received 8th March 2026, Accepted 28th April 2026, DOI: 10.1039/d6ra02003h, rsc.li/rsc-advances

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