Purpose: Non-viral gene delivery vectors are safer alternatives to viral vectors but often suffer from low transfection efficiency, especially in hard-to-transfect primary cells and 3-D tissue models. There is a need to understand how small changes in polymer structure—specifically terminal functionalization and molecular weight—affect gene delivery in both traditional 2-D monolayer and more physiologically relevant 3-D organotypic cultures of mammary epithelial cells.
Hypothesis: If small modifications are made to the terminal groups and molecular weight of a poly(beta-amino ester) (PBAE) polymer, then transfection efficacy in mammary epithelial cells will be significantly enhanced or abolished. Furthermore, the relative efficacy of polymers in 2-D culture will correlate with that in 3-D organotypic culture, though absolute transfection levels will be lower in 3-D.
Aims: Synthesize and characterize ten modified versions of the base PBAE polymer poly(1,4-butanediol diacrylate-co-5-amino-1-pentanol) (B4S5) with varying terminal groups and molecular weights. - Evaluate transfection efficacy in 2-D EPH4 mouse mammary epithelial monolayer cultures. - Evaluate transfection efficacy in 3-D primary mouse mammary organoid cultures. - Compare performance with the commercial transfection reagent FuGENE® HD. - Determine relationships between polymer structure (terminal group, molecular weight) and transfection efficacy in both culture systems.
Delivery system:
Component: Polymer class; Details: Poly(beta-amino ester) (PBAE)
Component: Base polymer; Details: Poly(1,4-butanediol diacrylate-co-5-amino-1-pentanol) (B4S5)
Component: End-capping groups; Details: E4 (2-methylpentane-1,5-diamine), E7 (1-(3-aminopropyl)-4-methylpiperazine), E8 (1-(3-aminopropyl)pyrollidine), E9 (4-aminophenyl disulfide), E10 (cystamine)
Component: Non-end-capped; Details: Amino-alcohol terminated B4S5
Component: Payload; Details: Plasmid DNA: EGFP-N1 (GFP) and CMV-Luc (luciferase)
Component: Nanoparticle formation; Details: Electrostatic self-assembly in sodium acetate buffer (pH 5)
Component: Size; Details: ~100 nm (majority component by NTA); some formulations 69–150 nm
Component: Targeting ligand; Details: None
Component: Key feature; Details: Biodegradable PBAE; terminal groups chosen to modulate DNA binding, endosomal escape, or triggered release (disulfide)
Approach: In vitro only. No in vivo animal studies. - 2-D model: EPH4 mouse mammary epithelial cells; 96-well and 24-well plates; transfection with polymer/DNA nanoparticles; 4 h incubation; analysis 48 h post-transfection. - 3-D model: Primary mouse mammary organoids embedded in Matrigel; 24-well low-adherent plates; transfection with polymer/DNA nanoparticles; 4 h incubation; analysis 48 h post-transfection. - Controls: FuGENE® HD (commercial reagent), untreated cells. - Doses: 2-D: 0.6–1.8 µg DNA per well, polymer:DNA wt/wt 60 or 100. 3-D: 6 µg DNA per well, 60 wt/wt. - Replicates: n = 4 for flow cytometry; experiments performed in duplicate for 3-D. - Statistics: One-way ANOVA with Dunnett’s multiple comparison test; mean ± SEM.
Key methods: Polymer synthesis and characterization: Gel permeation chromatography (GPC) for molecular weight. - Nanoparticle sizing: Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). - Transfection efficacy (2-D): Luciferase assay (CMV-Luc) for initial screening; flow cytometry and confocal microscopy for GFP expression. - Transfection efficacy (3-D): Flow cytometry for GFP-positive live cells; confocal microscopy for visualization of mosaic expression. - Viability: Not explicitly quantified by MTT, but cell morphology and viability noted; flow cytometry gating excluded dead cells.
Key results: 2-D transfection: Best polymer B4S5E7 transfected 57 ± 6% of cells; B4S5E8 = 44 ± 3%; B4S5E4 = 40 ± 5%. FuGENE® HD = 4 ± 2%. Leading polymers were ~10-fold more effective than FuGENE® HD. - 3-D transfection: Best polymer B4S5E8 transfected 6 ± 1% of cells; B4S5E7 = 4.0 ± 0.6%; B4S5 = 3.0 ± 0.3%. FuGENE® HD = 3 ± 1%. Leading polymers were ~2-fold more effective than FuGENE® HD. - Molecular weight effect: Higher synthesis temperature (90 °C vs 40 °C) increased molecular weight and increased transfection efficacy (e.g., B4S5 90 °C = 3 ± 1% vs 40 °C = 1 ± 0.4% in 3-D). - Terminal group effect: Small changes in end-capping molecules dramatically altered efficacy; tertiary amine end groups (E7, E8) performed best; disulfide-containing groups (E9, E10) showed lower efficacy. - 2-D vs 3-D correlation: Generally positive linear relationship, with 3-D efficacy approximately 10% of 2-D efficacy; some outliers (e.g., B4S5E4 and B4S5E7 had lower 3-D efficacy than predicted).
Interpretation: The authors claim that small polymer structural changes can tune gene delivery efficacy in both 2-D and 3-D mammary epithelial cultures. The degradable PBAE polymers formed ~100 nm nanoparticles and were more effective than FuGENE® HD. These polymeric nanoparticles may be useful as reagents and therapeutics for breast cancer and as tools to study mammary gland development, including mosaic delivery of Cre recombinase.
Limitations: In vitro only: No in vivo validation, biodistribution, or therapeutic efficacy. - Low 3-D efficacy: <10% transfection in organoids, likely due to limited nanoparticle penetration into 3-D tissue. - Single cell type: Only mouse mammary epithelial cells (EPH4 and primary organoids); generalizability to other cell types or human cells not established. - No targeting ligand: Delivery relies on nonspecific electrostatic interactions. - No long-term safety or toxicity data. - No mechanistic studies: Endosomal escape, intracellular trafficking, and DNA release were not directly visualized. - No comparison with viral vectors. - Modest sample size: n = 4 for 2-D; 3-D experiments performed in duplicate. - No disease model or therapeutic gene testing.
