Efficient Colon Cancer Gene Therapy Using Cationic DOTAP–PEG‑PCL Micelles for siRNA Delivery

Abstract

siRNA-based gene therapy offers a promising route for colon cancer treatment, yet safe and efficient delivery remains a bottleneck. We engineered a cationic micelle (DMP) by integrating the biocompatible polymer mPEG‑PCL with the cationic lipid DOTAP, yielding a stable, positively charged carrier. DMP/siRNA complexes targeting Bcl‑xl and Mcl‑1 demonstrated high transfection efficiency (>85 %) in C26 colon cancer cells, minimal cytotoxicity (IC₅₀ = 3.7 µg/mL in 293T cells), and robust gene silencing. In a subcutaneous C26 xenograft model, intratumoral injections of DMP/siBcl‑xl and DMP/siMcl‑1 reduced tumor volumes by 65 % and 55 %, respectively, with no observable organ toxicity. These findings establish DMP micelles as a safe, effective siRNA delivery platform for colon cancer gene therapy.

Introduction

Colorectal cancer remains a leading cause of cancer mortality worldwide, demanding innovative therapeutic strategies. Gene therapy, particularly via siRNA-mediated knockdown of oncogenes, can selectively trigger apoptosis in tumor cells. However, clinical translation hinges on non‑viral vectors that combine high transfection efficiency, low immunogenicity, and biocompatibility. The amphiphilic polymer mPEG‑PCL offers biodegradability and stealth properties, while the cationic lipid DOTAP supplies robust nucleic‑acid binding. Previous work has shown DMP micelles to deliver plasmid DNA safely; here we evaluate their capacity to transport siRNA targeting the anti‑apoptotic proteins Bcl‑xl and Mcl‑1, key drivers of colon cancer survival.

Materials and Methods (Condensed)

• DMP Preparation: mPEG‑PCL (Mn ≈ 4 kDa) and DOTAP (5–20 mg) were co‑dissolved in dichloromethane, evaporated to a thin film, and rehydrated in water. The resulting micelles had a mean diameter of 145 nm and zeta potential of +46 mV.
• siRNA Complexation: siRNA (50 nM) was mixed with DMP at a mass ratio of 30:1 (DMP:siRNA) to form stable complexes, confirmed by agarose gel retardation.
• In Vitro Assays: Transfection efficiency was measured by flow cytometry using FAM‑labelled siRNA; cytotoxicity via MTT; apoptosis by Annexin V/PI staining; gene knockdown assessed by qPCR.
• In Vivo Study: BALB/c mice bearing subcutaneous C26 tumors received intratumoral injections of DMP/siRNA (10 µg per dose) every other day for five cycles. Tumor volume and weight were recorded; histology (H&E) and TUNEL assay evaluated safety and apoptosis.

Results

Micelle Characterization

DMP micelles displayed a narrow size distribution (PDI = 0.32), positive surface charge (+46 mV), and remained stable for >96 h in aqueous solution. Gel retardation revealed complete siRNA encapsulation at DMP/siRNA ratios ≥30:1.

Low Cytotoxicity and High Transfection

IC₅₀ values: 3.7 µg/mL (293T) and 0.50 mg/mL (C26), markedly lower than PEI25K (IC₅₀ < 0.1 µg/mL). Transfection efficiency surpassed 85 % in serum‑free and 71–81 % in 30 % FBS conditions, comparable to PEI.

siRNA-Mediated Gene Silencing

qPCR showed >70 % reduction of Bcl‑xl and Mcl‑1 transcripts at 50–100 nM DMP/siRNA. Clone‑formation assays and MTT assays confirmed significant inhibition of C26 proliferation (cell viability 56–70 %). Apoptosis rates increased to 33–38 % in treated cells.

In Vivo Antitumor Efficacy

Intratumoral DMP/siBcl‑xl and DMP/siMcl‑1 injections reduced tumor volumes by 65 % and 55 %, respectively, and tumor weights dropped to 0.34–0.42 g from 0.85 g in controls. No histopathological abnormalities were observed in heart, liver, spleen, lung, or kidney.

Discussion

The partial shielding of DOTAP’s positive charges by mPEG‑PCL reduces serum protein interference and cytotoxicity while preserving siRNA binding and delivery efficiency. This design addresses key limitations of conventional cationic lipids and demonstrates the feasibility of DMP micelles as a versatile, low‑toxic siRNA carrier for solid tumors.

Conclusion

DMP micelles effectively deliver Bcl‑xl and Mcl‑1 siRNAs, inducing apoptosis and inhibiting tumor growth in vitro and in vivo with minimal toxicity. They represent a promising non‑viral platform for colon cancer gene therapy.