European Journal of Pharmaceutical Sciences2010ReviewNon-viral Gene Delivery

Gene delivery by lipoplexes and polyplexes

Conchita Tros De Ilarduya, Yan Sun, Nejat Düzgüneş29 September 2026DOI 10.1016/j.ejps.2010.03.019

Summary

Viral vectors achieve high gene transfer efficiency but can induce immune/inflammatory responses, have limited DNA carrying capacity, and raise recombination and cost concerns. Non-viral vectors such as cationic lipids and polymers are safer alternatives, but their transfection efficiency—especially in vivo—remains relatively low. A mechanistic understanding of how lipoplex and polyplex structure, charge, and formulation relate to stability and. Membrane charge density is a universal parameter for lamellar lipoplexes: transfection efficiency increases exponentially with membrane charge density (σM). At low σM (~0.005 e/Å²), intact.

Keywords

PolymericGene deliveryDNATransfectionPolyethylenimineViral vectorsEndosomal escape

Purpose: Viral vectors achieve high gene transfer efficiency but can induce immune/inflammatory responses, have limited DNA carrying capacity, and raise recombination and cost concerns. Non-viral vectors such as cationic lipids and polymers are safer alternatives, but their transfection efficiency—especially in vivo—remains relatively low. A mechanistic understanding of how lipoplex and polyplex structure, charge, and formulation relate to stability and transfection is needed to design better non-viral vectors.

Hypothesis: As a review, there is no single formal hypothesis. The central thesis is:

> If the supramolecular structure, charge, and formulation of lipoplexes and polyplexes are matched to the biological barriers they encounter, then transfection efficiency can be improved. > - For lipoplexes: lipids that facilitate transformation to non-bilayer phases (e.g., DOPE-containing systems) mediate high transfection in vitro, whereas serum-stable lipids such as cholesterol are more suitable for in vivo delivery. > - For polyplexes: efficiency depends on the polymer’s ability to condense DNA while still allowing DNA dissociation once inside the cell.

Aims: Review the interactions between cationic vectors and DNA that lead to complex formation. - Describe the supramolecular structures of lipoplexes and polyplexes. - Outline the mechanisms of DNA transfer, including cell binding, endosomal escape, dissociation, and nuclear import. - Relate vector structure, charge, and formulation to stability and transfection efficiency, providing a framework for rational design of optimal non-viral vectors.

Delivery system:

Feature: Vector types; Description: Cationic liposomes (lipoplexes) and cationic polymers (polyplexes)

Feature: Cationic lipids; Description: DOTAP, DOTMA, DC-Chol, DODAC, DOSPA, DOGS, DMRIE, DDAB, SAINT-4, BGTC, BGSC, etc.

Feature: Helper/neutral lipids; Description: DOPE, cholesterol, DOPC; DOPE promotes inverted hexagonal HII phase; cholesterol improves serum stability

Feature: Cationic polymers; Description: Poly(ethylenimine) (PEI), poly(L-lysine) (PLL), chitosan, PAMAM dendrimers, pDMAEMA, protamine sulfate

Feature: Payload; Description: Nucleic acids: plasmid DNA, oligodeoxynucleotides, RNA (reviewed generally as DNA/gene material)

Feature: Complex formation; Description: Electrostatic interaction between cationic vector and anionic DNA phosphate backbone

Feature: Key formulation parameters; Description: Charge ratio (+/−), N/P ratio, lipid composition, polymer molecular weight, branching, ionic strength, mixing order

Approach: This is a narrative review synthesizing mechanistic and structural studies from the literature.

No primary experimental model is used.
It covers in vitro cell culture systems (e.g., COS-7, HUH-7, HeLa, A549, CHO-K1, B16F10, OVCAR-3, SK-OV-3, human tracheal epithelial cells) and in vivo contexts (respiratory tract, brain, systemic administration) from cited studies.
Disease context: gene therapy for acquired and genetic diseases.
Group structure/controls are not applicable because this is a review.

Key methods: Techniques discussed from cited primary studies include:

Colloidal characterization: light scattering, zeta potential, particle size.
DNA condensation: ethidium bromide fluorescence quenching.
Lipid phase behavior: differential scanning calorimetry (DSC), 31P NMR, ESR.
Supramolecular structure: small-angle X-ray scattering (SAXS), X-ray diffraction (XRD), electron microscopy, scanning probe microscopy.
Intracellular trafficking: laser scanning confocal microscopy (LSCM).
Structure–activity modeling: quantitative structure–activity relationship (QSAR).
Transfection/functional assays: gene expression, cytotoxicity, serum stability.

Key results: Membrane charge density is a universal parameter for lamellar lipoplexes: transfection efficiency increases exponentially with membrane charge density (σM). At low σM (~0.005 e/Å²), intact lipoplexes remain inside cells and DNA is trapped; at high σM (~0.012 e/Å²), DNA is released inside cells. - DOPE vs. cholesterol: DOPE promotes inverted hexagonal HII phase and high in vitro transfection, but DOPE-containing complexes tend to aggregate in blood. Cholesterol-containing lipoplexes are structurally more stable in serum and are more suitable for in vivo delivery. - PEI polyplex formulation: stable complexes require N/P ≈ 2–3. Increasing N/P from 2 to 20 decreases particle size from >1000 nm to 100–200 nm and reduces polydispersity. - Polyplex aggregation: branched 25 kDa PEI polyplexes grow from ~120 to 370 nm over 3 h; linear 22 kDa PEI aggregates much faster, reaching ~750 nm after 20 min and >6 µm after 3 h. - Polymer dissociation matters: low-molecular-weight PLL polyplexes dissociate more readily and transfect better than high-molecular-weight counterparts. Reducible PLL polymers can increase gene expression up to 187-fold. - Proton sponge effect: PEI-containing endosomes show ~140% relative volume increase; quaternization of protonatable amines decreases transfection ~20-fold. - Chitosan: forms 50–100 nm particles and transfects HeLa cells even in 10% serum.

Interpretation: The authors conclude that no single ideal gene delivery vector exists; each vector must be tailored to the disease, cell type, and route of administration. Rational design based on chemical structure, mixture composition, and supramolecular organization should help overcome barriers to transfection. Lipoplexes that transform into the inverted hexagonal HII phase have higher in vitro activity but poor serum stability, whereas cholesterol-containing lamellar complexes are more stable in blood but less efficient. For polyplexes, the key challenge is balancing DNA condensation/protection with intracellular dissociation. The review aims to provide a framework for designing optimal non-viral vectors from the ground up.

Limitations: Stated or evident from the review: - The review provides no new experimental data; conclusions depend on the cited literature. - Mechanistic understanding remains incomplete, especially for nuclear import and polyplex endosomal escape. - The proton sponge hypothesis does not fully explain PEI/PAMAM efficiency. - Conflicting reports exist on polyplex uptake pathways (clathrin-dependent, caveolae-mediated, lipid-raft-dependent, fluid-phase endocytosis). - Many findings are from in vitro cell culture and may not translate to in vivo. - No universal vector has been identified; transfection efficiency depends on cell type, polymer/lipid structure, and administration route. - Clinical translation, long-term safety, and manufacturing scalability are not deeply addressed. - Serum stability remains a major barrier for in vivo applications.

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Gene delivery by lipoplexes and polyplexes

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