DC-CholDOPE cationic liposomes A comparative study of the influence factors on plasmid pDNA and siRNA gene delivery

Purpose: DC-Chol/DOPE cationic liposomes are efficient non-viral vectors, but the effects of DC-Chol/DOPE molar ratio, PEGylation, and serum on pDNA versus siRNA delivery were unclear. The study also aimed to test whether pDNA and siRNA have different transfection mechanisms in these liposomes.

Hypothesis: Because siRNA acts in the cytoplasm while pDNA must enter the nucleus, factors such as DC-Chol/DOPE molar ratio, PEGylation, and serum will have different effects on siRNA versus pDNA transfection.

Aims: Prepare DC-Chol/DOPE liposomes with varying molar ratios and PEGylation degrees. - Characterize lipoplex size, zeta potential, and pDNA/siRNA binding affinity. - Compare pDNA and siRNA transfection efficiency under different DC-Chol/DOPE ratios, DC-Chol/nucleic acid weight ratios, PEGylation, and serum conditions. - Evaluate siRNA silencing and discuss differences between pDNA and siRNA delivery mechanisms.

Delivery system: Platform: Cationic liposomes composed of DC-Chol and DOPE; optionally PEGylated with mPEG-DSPE. - Payloads: pGPH1/GFP/Neo plasmid; FAM-labeled negative control siRNA; Cy3-labeled siRNA; anti-HER2 siRNA. - Preparation: Dry-film method; hydration in PBS; sonication; extrusion through 200, 100, and 80 nm membranes; 0.22 µm sterilization. - Lipoplex formation: Dilution method; liposomes and nucleic acid mixed and incubated 20 min at room temperature. - Targeting ligand: None. - Key variables: DC-Chol/DOPE molar ratio; DC-Chol/pDNA or DC-Chol/siRNA weight ratio; PEGylation degree; serum vs serum-free.

Approach: Model: In vitro only. - Cell line: Human breast cancer SK-BR3 cells. - Transfection format: 48-well plates; 7.5 × 10⁴ cells/well; lipoplex containing 0.5 µg pDNA or siRNA; 8 h incubation; assays 48 h later. - Serum conditions: Serum-containing and serum-free transfection compared. - Controls: Lipo2000 transfection; negative control siRNA; untreated cells. - Replication: Data generally mean ± SD; n = 3 or 4.

Key methods: Dynamic light scattering and zeta potential for particle size/surface charge. - Gel retardation assay for pDNA/siRNA binding. - Ultrafiltration centrifugation for siRNA encapsulation efficiency. - Flow cytometry for GFP expression and FAM-siRNA uptake. - Fluorescence microscopy for GFP and Cy3-siRNA visualization. - Flow cytometry for HER2 mean fluorescence intensity to measure siRNA silencing.

Key results: Optimal DC-Chol/DOPE molar ratio: 1:2 for pDNA transfection; 1:1 for siRNA transfection. - Weight ratio effects: pDNA transfection efficiency decreased as DC-Chol/pDNA weight ratio increased from 3/1. siRNA transfection efficiency increased with increasing DC-Chol/siRNA weight ratio, plateauing above 5–10. - PEGylation: Decreased both pDNA and siRNA transfection. At ≥3% PEG, pDNA transfection was below 5% in most cases. PEGylated liposomes showed no measurable siRNA silencing. - Serum: Did not significantly inhibit pDNA or siRNA transfection efficiency, except slight impairment for DC-Chol/DOPE = 2/1 siRNA. However, siRNA silencing in serum was <6% versus <25% in serum-free conditions. - Binding/encapsulation: pDNA fully retarded at DC-Chol/pDNA weight ratio ≥2. siRNA fully retarded only at high ratios (~7.5); PEGylated liposomes could not retard siRNA even at ratio 15. siRNA encapsulation >90% at weight ratio ≥5 for non-PEGylated and ≥10 for PEGylated liposomes. - Size/zeta: Liposomes were 130–150 nm. Lipoplex size increased near charge neutralization; PEGylation reduced size and surface charge.

Interpretation: The authors conclude that DC-Chol/DOPE molar ratio, PEGylation, and serum influence pDNA and siRNA delivery differently, indicating distinct transfection mechanisms. DOPE is highlighted as important for pDNA endosomal escape, whereas siRNA transfection depends more on internalization. Understanding these factors should aid rational design of DC-Chol/DOPE liposomes for gene therapy.

Limitations: In vitro only; no in vivo validation. - Single cell line (SK-BR3). - siRNA silencing efficiency was low, especially in serum (<6%). - No cytotoxicity or long-term stability data. - No detailed intracellular trafficking or mechanism study. - PEGylation tested only at 1–5%. - No targeting ligand or disease-specific delivery.