Targeted lung cancer therapy: preparation and optimization of transferrin-decorated nanostructured lipid carriers as novel nanomedicine for co-delivery of anticancer drugs and DNA

Purpose: Lung cancer chemotherapy is limited by multidrug resistance, severe adverse effects, and poor tumor targeting. A nanocarrier that actively targets lung cancer cells and co-delivers a cytotoxic drug plus therapeutic DNA could improve efficacy and reduce off-target toxicity.

Hypothesis: Transferrin (Tf)-decorated nanostructured lipid carriers (NLCs) can co-deliver paclitaxel (PTX) and plasmid DNA, be recognized by transferrin receptor-overexpressing NCI-H460 lung cancer cells, undergo receptor-mediated endocytosis, and produce enhanced gene transfection and antitumor activity compared with non-targeted NLCs or free drug.

Aims: Synthesize Tf-PEG-PE ligands with different PEG molecular weights. - Prepare PTX- and DNA-loaded NLCs and decorate them with Tf. - Characterize particle size, zeta potential, drug/gene loading, release, and cytotoxicity. - Evaluate in vitro and in vivo gene transfection and antitumor efficacy in NCI-H460 lung cancer models.

Delivery system: Platform: Nanostructured lipid carriers (NLCs) prepared by microemulsion technique. - Core lipids: Glyceryl monostearate, oleic acid, soya lecithin. - Cationic surfactant: DOTMA. - Payloads: Paclitaxel (PTX) and enhanced green fluorescent protein plasmid DNA. - Targeting ligand: Transferrin (Tf) conjugated to PEG-PE; PEG molecular weights 2, 5, and 10 kDa tested. - Lead formulation: Tf₅ₖ-PTX-DNA-NLC (PEG 5 kDa). - Physicochemical properties: Tf₅ₖ-PTX-DNA-NLC ~133 nm, zeta potential ~+19 mV; Tf₁₀ₖ-PTX-DNA-NLC ~236 nm, zeta ~+8 mV; DNA loading ~90%; PTX encapsulation ~83–87%. - Route: Intravenous tail-vein injection in mice.

Approach: In vitro: NCI-H460 human non-small cell lung carcinoma cells; transfection and cytotoxicity assays. - In vivo: Male BALB/c mice bearing subcutaneous NCI-H460 tumors; five groups, six mice per group; treatments included Tf-PTX-DNA-NLC, PTX-DNA-NLC, Taxol, saline control, and related controls. - Dosing: PTX 1.25–1.3 mg/mL, IV every other day for 14 days. - Controls: Blank NLC, naked DNA, Lipofectamine-DNA complexes, Taxol, saline.

Key methods: TEM for morphology. - Dynamic light scattering for size and zeta potential. - PicoGreen assay for DNA loading. - HPLC for PTX encapsulation and release. - MTT assay for cytotoxicity. - Flow cytometry for EGFP transfection efficiency. - Tumor volume measurement and tumor inhibition rate (TIR) for in vivo efficacy.

Key results: Tf₅ₖ-PTX-DNA-NLC: ~133 nm, zeta +19 mV; Tf₁₀ₖ-PTX-DNA-NLC: ~236 nm, zeta +8 mV. - DNA loading ~90–92%; PTX encapsulation ~83–87%. - In vitro IC₅₀: Tf₅ₖ-PTX-DNA-NLC 3.35 µM; Tf₁₀ₖ-PTX-DNA-NLC 7.12 µM; PTX-DNA-NLC 7.36 µM; Taxol 14.45 µM. - Tf₅ₖ-PTX-DNA-NLC showed the strongest antitumor effect in vivo; reported tumor inhibition rates were approximately 51% for the best-performing Tf₅ₖ group, 42% for PTX-DNA-NLC, and 23% for Tf₁₀ₖ-PTX-DNA-NLC. - In vivo transfection: Tf₅ₖ-PTX-DNA-NLC and Lipo-DNA were higher than other formulations at 36 and 72 h; at 72 h Tf₅ₖ exceeded Lipo-DNA. Tf₁₀ₖ performed poorly. - PEG 10 kDa was not suitable; PEG 5 kDa was optimal.

Interpretation: Tf₅ₖ-PTX-DNA-NLC combines active transferrin receptor targeting with stable PTX/DNA co-delivery, producing superior gene transfection and antitumor efficacy. The authors propose this as a promising combined drug/gene nanomedicine strategy for lung cancer.

Limitations: Only NCI-H460 cells and a mouse xenograft model were used; no large-animal validation. - No survival study; efficacy followed for only 14 days. - No comprehensive biodistribution, long-term toxicity, or repeated-dose safety data. - In vitro vs. in vivo transfection results were not fully consistent. - Tf receptor specificity was not directly blocked or knocked down. - Optimal PEG length was empirical; 10 kDa PEG impaired transfection and release. - No comparison with clinical standard combination regimens.