Efficient gene delivery by EGF-lipoplexes in vitro and in vivo

Purpose: Nonviral gene delivery vectors are safer than viral vectors but generally suffer from low and nonspecific transfection efficiency. EGFR is overexpressed on many tumor cells, making EGF-mediated targeting an attractive strategy to improve selectivity and gene delivery into cancer cells.

Hypothesis: If EGF is incorporated into cationic DOTAP/cholesterol lipoplexes, then the resulting EGF-lipoplexes will bind EGFR-overexpressing tumor cells, enhance transgene delivery via receptor-mediated endocytosis, and improve transfection in vitro and in vivo without substantial cytotoxicity.

Aims: Prepare DOTAP/cholesterol liposomes modified with EGF at different lipid/DNA (+/-) charge ratios. - Characterize EGF-lipoplexes for size, zeta potential, DNA protection against DNase I, and cytotoxicity. - Evaluate in vitro transfection in EGFR-overexpressing cell lines using reporter and therapeutic genes. - Confirm receptor-mediated uptake using an EGFR-deficient cell line. - Assess in vivo transfection and biodistribution after systemic administration in mice.

Delivery system: Core carrier: DOTAP/cholesterol liposomes at a 1:0.9 molar ratio; small unilamellar vesicles. - Targeting ligand: EGF, mixed with cationic liposomes and plasmid DNA to form EGF-lipoplexes. - Payload: Plasmid DNA encoding luciferase (`pCMVLuc`) or IL-12 (`pCMVIL12`). - Charge ratios tested: 0.5/1, 1/1, 2/1, and 5/1 (+/-). - In vivo formulation: 5/1 (+/-) charge ratio, containing 1 µg EGF per µg DNA and 60 µg plasmid per mouse.

Approach: In vitro models: HepG2 and DHDK12proB cells overexpressing EGFR; SW620 cells used as an EGFR-deficient model. - Transfection conditions: 4 h incubation in the presence of 60% serum, followed by 48 h gene expression. - Controls: Naked plasmid DNA, plain lipoplexes without EGF, and untreated cells. - In vivo model: Female Balb-c mice, 7–8 weeks old; groups of eight; tail-vein injection of PBS, naked pDNA, or EGF-lipoplexes. - In vivo imaging: Luciferase bioluminescence measured over time using an IVIS CCD camera.

Key methods: Dynamic light scattering for particle size. - Zeta-potential measurement for surface charge. - DNase I protection assay with agarose gel electrophoresis. - Luciferase reporter assay for transfection efficiency. - ELISA for IL-12 expression. - Alamar Blue assay for cell viability. - In vivo bioluminescence imaging for gene expression and organ localization.

Key results: EGF-lipoplexes were approximately 300 nm in size, with positive surface charge at charge ratios ≥2/1 (+/-); zeta potentials ranged from about 15 to 40 mV. - EGF-lipoplexes protected plasmid DNA from DNase I degradation at 1/1, 2/1, and 5/1 (+/-) charge ratios. - In HepG2 cells, the optimal 5/1 EGF-lipoplex gave a 3.6-fold increase in luciferase expression versus plain lipoplexes. - In DHDK12proB cells, the same formulation gave a 40-fold increase versus plain lipoplexes. - In EGFR-deficient SW620 cells, EGF-lipoplexes did not enhance transfection: 160 vs 165 ng luciferase/mg protein compared with plain lipoplexes. - IL-12 expression in HepG2 cells was higher with EGF-lipoplexes: 47 vs 32 ng IL-12/ml. - In vivo, EGF-lipoplexes produced higher gene expression than naked DNA or control; bioluminescence was mainly in the lung and maximal at 24 h after injection. - No significant cytotoxicity was observed in the tested formulations.

Interpretation: The authors conclude that EGF-lipoplexes are simple, stable, noncytotoxic nanovectors that protect DNA, target EGFR-overexpressing cells via receptor-mediated endocytosis, and improve transfection both in vitro and in vivo. They suggest these nanovectors may be a promising alternative to viral vectors for gene therapy, particularly for EGFR-overexpressing tumors such as lung cancer.

Limitations: In vivo experiments used healthy mice, not tumor-bearing animals, so tumor targeting was not directly demonstrated. - Biodistribution was assessed mainly by luciferase imaging, with no detailed cellular-level uptake or organ-level quantification. - Gene expression was followed only up to 48 h; longer-term expression and repeat dosing were not evaluated. - The paper does not report large-animal validation or detailed safety/immunogenicity data.