Composite liposome-PEInucleic acid lipopolyplexes for safe and efficient gene delivery and gene knockdown
Pinnapireddy Sr, Duse L, Strehlow B, Schäfer J, Bakowsky UDOI 10.1016/j.colsurfb.2017.06.022
Summary
Polyethylenimine (PEI)-based gene delivery is limited by cytotoxicity, rapid degradation, and low cellular uptake. A safer, more stable non-viral system was needed for both gene delivery and gene knockdown. Physicochemical properties: DDC liposomes ~198.7 nm, ζ = −30.9 mV; IPEI polyplexes ~166.2 nm, ζ = +19.1 mV; bPEI polyplexes ~180.5 nm, ζ = +27.3 mV; DDC-IPEI lipopolyplexes ~211.4 nm, ζ = +3.9 mV. - Transfection:.
Purpose: Polyethylenimine (PEI)-based gene delivery is limited by cytotoxicity, rapid degradation, and low cellular uptake. A safer, more stable non-viral system was needed for both gene delivery and gene knockdown.
Hypothesis: Encapsulating PEI/nucleic acid polyplexes inside anionic DOPE/DPPC/cholesterol liposomes will shield the cationic charge of PEI, reduce cytotoxicity, improve complex stability, and enhance transfection and knockdown efficiency compared with bare polyplexes.
Aims: Formulate and characterize PEI-based liposome-encapsulated delivery vehicles (lipopolyplexes) for pDNA and siRNA. - Compare branched PEI 25 kDa (bPEI) and deacetylated linear PEI 22 kDa (IPEI) polyplexes and their lipopolyplexes. - Evaluate transfection efficiency, gene knockdown, cytotoxicity, storage stability, and haemocompatibility. - Establish structure–activity relationships for the composite liposome–PEI/nucleic acid platform.
Delivery system: Platform: Composite lipopolyplex — PEI/nucleic acid polyplex core encapsulated within an anionic liposome. - Polyplex core: pDNA or siRNA condensed with bPEI 25 kDa or deacetylated linear PEI 22 kDa (IPEI). Standard N/P ratios: 15 for bPEI, 9.5 for IPEI. - Liposomal shell: DOPE/DPPC/cholesterol (DDC) at 70:15:15 mol/mol; anionic, ~199 nm, ζ ≈ −30.9 mV. - Optimized formulation: DDC-IPEI lipopolyplexes at liposome/PEI mass ratio 0.39. - Payloads: pCMV-luc (luciferase reporter), pEGFP-N1 (GFP reporter), siGL3 (anti-luciferase siRNA), siControl. - Targeting ligand: None. - Preparation: Polyplexes formed by electrostatic mixing, ultrafiltered to remove free PEI, then mixed with liposomes for 1 h.
Approach: Model: In vitro only. - Cell lines: Human ovarian adenocarcinoma SK-OV-3 and luciferase-expressing SK-OV-3 luc. - Transfection/knockdown: 10,000 cells/well in 96-well plates; 0.2 µg pDNA or 7.2 pmol siRNA; quadruplicate wells; experiments repeated three times. - GFP imaging: 90,000 cells/well in 12-well plates; 1.75 µg pEGFP-N1. - Cytotoxicity: 0.4 µg pDNA per well; LDH assay; Triton X-100 positive control. - Controls: Naked pDNA, lipoplexes, bare polyplexes, untreated cells, siControl.
Key methods: Dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) for size and ζ-potential. - SEM and TEM with gold-labeled PEI for structural elucidation. - Gel retardation and heparin competition assays for complex integrity/stability. - Ethidium bromide intercalation assay for pDNA binding affinity. - Luciferase assay for transfection efficiency. - Luciferase knockdown assay in SK-OV-3 luc cells. - Confocal laser scanning microscopy for GFP expression. - LDH assay for cytotoxicity. - Activated partial thromboplastin time (aPTT) for haemocompatibility. - Storage stability assay over 30 days at room temperature.
Key results: Physicochemical properties: DDC liposomes ~198.7 nm, ζ = −30.9 mV; IPEI polyplexes ~166.2 nm, ζ = +19.1 mV; bPEI polyplexes ~180.5 nm, ζ = +27.3 mV; DDC-IPEI lipopolyplexes ~211.4 nm, ζ = +3.9 mV. - Transfection: DDC-IPEI lipopolyplexes at liposome/PEI mass ratio 0.39 gave a ~10-fold increase in luciferase expression over IPEI polyplexes. - Knockdown: DDC-IPEI lipopolyplexes achieved up to ~80% luciferase knockdown with siGL3. - Stability: Lipopolyplex size increased by only 9% after 30 days at room temperature; biological activity loss was ~10% at 3 days and ~20% at 7 days; stable in heparin at normal plasma concentrations (15–17 IU/100 mL). - Cytotoxicity: Lipopolyplexes showed low cytotoxicity; bPEI polyplexes were more toxic; liposomal shielding reduced surface charge and improved cell viability. - Haemocompatibility: aPTT: plasma control 32.2 ± 0.1 s; lipopolyplexes 34.2 ± 0.4 s; IPEI polyplexes 37.1 ± 0.3 s; bPEI polyplexes 61.8 ± 0.4 s. - GFP expression: Confocal microscopy confirmed higher GFP expression with DDC-IPEI lipopolyplexes than with polyplexes.
Interpretation: The authors conclude that liposomal shielding of PEI polyplexes reduces surface charge, decreases cytotoxicity, improves stability, and enhances transfection and knockdown. The DDC-IPEI lipopolyplex is proposed as a biocompatible non-viral vector suitable for further in vivo gene therapy development.
Limitations: In vitro study only; no in vivo efficiency, toxicity, or biodistribution data. - Uses commercial PEI and a single ovarian adenocarcinoma cell line. - No targeting ligand or disease-specific delivery. - Storage stability evaluated only up to 30 days. - Mechanism of cellular uptake and intracellular trafficking not fully resolved. - The paper notes future work is needed to determine in vivo efficiency and toxicity.
Related articles
Let's engineer the next delivery breakthrough together
We co-develop nanocarrier and biosensing programs with pharma, biotech and academic groups — from target selection through GMP supply.