Purpose: Effective and safe gene delivery to primary endothelial cells in the presence of serum proteins is particularly challenging. Endothelial cells are important targets for cardiovascular disease and cancer, but existing nonviral vectors have low efficacy and/or cytotoxicity under serum-containing conditions.
Hypothesis: If poly(β-amino ester) (PBAE) vectors are optimized for serum-containing conditions, then they can achieve high transfection efficacy and low cytotoxicity in human umbilical vein endothelial cells (HUVECs), and their serum-phase biophysical properties (size, stability, ζ potential) will correlate with transfection efficacy better than properties measured in buffer alone.
Aims: Synthesize and screen a library of biodegradable PBAEs for gene delivery to primary human endothelial cells in serum. - Optimize vector parameters: polymer type, polymer-to-DNA weight ratio, DNA loading, and N/P ratio. - Characterize biophysical properties (particle size, ζ potential, stability over time) in buffer and serum-containing media. - Compare lead PBAE vectors against commercial reagents (jetPEI, jetPEI-HUVEC, Lipofectamine 2000, 25k PEI) for efficacy and cytotoxicity in HUVECs.
Delivery system:
Component: Polymer class; Details: Biodegradable poly(β-amino esters) (PBAEs), synthesized by conjugate addition of amine monomers to acrylate esters
Component: Library; Details: 2,350 structurally unique PBAEs; top 486 resynthesized; 11 selected for HUVEC optimization
Component: Lead polymer; Details: C32 (acrylate C, amine 32); other notable vectors: JJ32, E28
Component: Payload; Details: Plasmid DNA encoding luciferase (pCMV-Luc) or enhanced green fluorescent protein (EGFP)
Component: Nanoparticle formation; Details: Electrostatic self-assembly in 25 mM sodium acetate buffer (pH 5); 10 min complexation
Component: Polymer:DNA ratios; Details: w/w varied (10–120); N/P ratios varied; higher w/w and N/P generally required for serum-containing HUVEC transfection
Component: Targeting ligand; Details: None
Component: Key feature; Details: Biodegradable, serum-stable, non-cytotoxic polymeric nanoparticles for primary endothelial cell gene delivery
Approach: In vitro model: Primary human umbilical vein endothelial cells (HUVECs), passage 5, cultured in EGM-2 media. - Transfection format: 96-well plates for luciferase assays; 24-well plates for GFP/FACS. Cells grown to confluence; transfected in media containing 2% or 12% serum. - DNA dose: 0.3 or 0.6 µg/well for luciferase; 3 µg/well for GFP. - Incubation time: 1–4 h, then replaced with fresh media; expression measured 48 h later. - Controls: Naked DNA, 25k PEI, jetPEI, jetPEI-HUVEC, Lipofectamine 2000. - Group structure: n ≥ 2–10 depending on assay; no in vivo studies. - Disease context: Cardiovascular disease and cancer (endothelial cell targeting for angiogenesis/vascular function).
Key methods: Luciferase expression: Bright-Glo assay with luminometer. - GFP transfection: Flow cytometry (FACS) using two-dimensional density plot (GFP vs. autofluorescence) to gate true positives. - Biophysical characterization: ZetaPALS dynamic light scattering for particle size and ζ potential in NaAc buffer, OptiMem, 2% serum, and 12% serum over time (up to 8 days). - Cell viability: MTS assay (CellTiter 96 AQueous One Solution). - Polymer characterization: Gel-permeation chromatography for molecular weight. - Statistics: Unpaired, two-tailed Student’s t-tests with 99% confidence.
Key results: C32 transfection in 12% serum: 47 ± 9% positive HUVECs (n = 10), significantly higher than jetPEI (11 ± 2%, p < 0.001), jetPEI-HUVEC (6 ± 5%, p < 0.001), Lipofectamine 2000 (29 ± 1%, p < 0.01), and 25k PEI (2 ± 1%, p < 0.001). - Serum-dependent particle properties: In buffer, all PBAE/DNA particles were <150 nm. In 12% serum, top vectors (C32, JJ32, E28) formed small, stable particles (~193–236 nm), while poor vectors (e.g., D24) aggregated to ~1 µm. ζ potential shifted from positive in PBS to negative in serum. - Cytotoxicity: C32 was non-cytotoxic to HUVECs across w/w ratios and DNA loadings (up to ~500 µg/mL polymer, 600 ng DNA, 120 w/w), whereas jetPEI was cytotoxic. - Structure–function: Single-carbon differences in acrylate and amine monomers caused dramatic efficacy differences. Amine monomer length correlated with efficacy: 32 (5 carbons) > 28 (4 carbons) > 24 (3 carbons + hydroxyl) > 20 (3 carbons). C32, JJ32, and E28 transfected HUVECs well in serum, while C28 and U28 (good in COS-7 serum-free) performed poorly in HUVEC serum.
Interpretation: The authors claim that poly(β-amino esters) as a class, and C32 specifically, transfect human primary endothelial cells better than several leading commercial reagents, with high serum stability and low cytotoxicity. They conclude that biophysical characterization in physiologically relevant serum-containing media correlates with transfection efficacy and may help predict in vivo performance. PBAE vectors are proposed as promising biodegradable alternatives for cardiovascular gene therapy.
Limitations: In vitro only: No in vivo animal studies or disease models were performed. - Single primary cell type: Only HUVECs were tested; generalizability to other endothelial or primary cells is unclear. - No targeting ligand: Vectors rely on nonspecific electrostatic interactions. - Efficacy below viral vectors: C32 achieved ~47% positive cells, compared with ~70% reported for lentiviral vectors in similar HUVEC settings. - Short-term expression: Transfection measured at 48 h; long-term expression and stability not assessed. - Serum interactions not fully mechanistically resolved: Correlation between serum-phase size and efficacy is observed, but causal mechanisms are not fully proven. - No in vivo biocompatibility or safety data in this study.
