Purpose: Human umbilical vein endothelial cells (HUVECs) are relevant to tissue engineering, cancer therapy, and angiogenesis-related diseases. Non-viral vectors are safer than viruses but often less efficient. Biodegradable poly(ester amine) (PEA) and poly(amido amine) (PAA) nanoparticles were developed and screened for efficient siRNA and DNA delivery to HUVECs, and to determine whether small changes in polymer structure can tune efficacy for different nucleic acid cargos.
Hypothesis: If PEA and PAA polymer structures are systematically varied, then some nanoparticles will effectively deliver siRNA and/or DNA to HUVECs; small changes in polymer chemistry will tune efficacy, and physicochemical properties such as particle size, zeta potential, and particle concentration will correlate with transfection efficiency.
Aims: Synthesize and characterize a library of biodegradable PEA and PAA polymers. - Form nanoparticles with siRNA and plasmid DNA. - Screen nanoparticles for siRNA-mediated GFP knockdown and DNA-mediated DsRed expression in HUVECs. - Compare efficacy with Lipofectamine 2000. - Identify relationships between polymer structure, nanoparticle physicochemical properties, and delivery efficacy. - Determine whether some polymers are effective for siRNA, DNA, or both.
Delivery system:
Component: Polymer classes; Details: Poly(ester amine)s (PEAs) and poly(amido amine)s (PAAs)
Component: Synthesis; Details: Michael addition of backbone diacrylates (B4, B5, BSS) with side-chain amines (S3, S4, S5), then end-capped with small amine molecules (E1, E3, E4, E6, E7, E10)
Component: Degradability; Details: PEAs degrade hydrolytically; PAAs contain disulfide bonds (BSS/E10) and degrade by bioreduction
Component: Payloads; Details: siRNA against eGFP; plasmid DsRed-Max DNA
Component: Nanoparticle formation; Details: Electrostatic self-assembly in sodium acetate buffer (pH 5)
Component: Effective size range; Details: ~50–150 nm for effective delivery
Component: Zeta potential; Details: Positive, ~1–20 mV in PBS
Component: Targeting ligand; Details: None
Component: Key feature; Details: Biodegradable; different polymer structures can be tuned for siRNA vs. DNA delivery; siRNA required higher polymer-to-nucleic acid w/w ratios (60–150) than DNA (20–90)
Approach: In vitro only. No in vivo animal studies. - Cell model: HUVECs; stably eGFP-expressing HUVECs for siRNA knockdown. - siRNA transfection: 60 nM siRNA; polymer w/w ratios 60, 100, 150; 4 h incubation; GFP signal measured over 3 days. - DNA transfection: DsRed-Max plasmid; polymer w/w ratios 30, 60, 90; 48 h; flow cytometry. - Controls: Lipofectamine 2000; untreated cells; scrambled siRNA (scrRNA); no-polymer controls. - Replicates: Transfection experiments n = 4; sizing/zeta experiments n = 3. - Statistics: Student’s t-test; one-way ANOVA with Dunnett’s test; significance P < 0.05, P < 0.01, **P < 0.001.
Key methods: Polymer degradation: Gel permeation chromatography (GPC) in aqueous buffer at 37°C. - Nanoparticle characterization: Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), dynamic light scattering (DLS) for zeta potential. - Nucleic acid binding: Gel retardation assays. - Viability: MTS assay 24 h post-transfection. - siRNA knockdown: Fluorescence plate reader and flow cytometry for GFP signal; fluorescence microscopy. - DNA transfection: Flow cytometry for DsRed expression; fluorescence microscopy.
Key results: siRNA delivery: Up to 60–75% GFP knockdown in HUVECs. Best formulation 454 at 100 w/w achieved 72.3 ± 0.6% knockdown, superior to Lipofectamine 2000 (51.6 ± 5.2%) with comparable or better viability. - DNA delivery: Up to 50–60% transfection; best formulation 456 at 30 w/w achieved 59.7 ± 2.0% DsRed-positive HUVECs. - Polymer grouping: PEAs and PAAs separated into groups effective for siRNA delivery, DNA delivery, or both. - Physicochemical correlations: Effective particles typically had mean/mode diameters of 50–150 nm. Particle concentration correlated positively with siRNA knockdown (r² = 0.4678) and DNA transfection (r² = 0.4344). Zeta potential correlated with siRNA knockdown (r² = 0.4905) but less clearly with DNA transfection (r² = 0.2427). - Degradation: PEAs 447 and 537 fully degraded within 24 h in aqueous buffer; at 72 h Mn was 450 ± 12 Da and 390 ± 47 Da, respectively. - Viability: Several formulations maintained >80% viability; Lipofectamine 2000 showed high toxicity.
Interpretation: The authors claim that synthetic PEA-siRNA self-assembled nanoparticles are effective for siRNA delivery to HUVECs, and end-modified PAAs can provide modest nucleic acid delivery. Different parameters are crucial for siRNA versus DNA delivery: high nanoparticle formation efficiency and a size distribution near 100 nm appear necessary but not sufficient. Some polymers deliver both nucleic acid types, while others are more specialized. These biodegradable nanoparticles may be useful for gene modulation in endothelial cells for tissue engineering and drug delivery applications.
Limitations: In vitro only: No in vivo validation, biodistribution, or therapeutic efficacy. - Single primary cell type: HUVECs only; generalizability to other endothelial or primary cells not established. - No targeting ligand: Delivery relies on nonspecific electrostatic interactions. - No long-term safety or toxicity data beyond 24 h MTS viability. - No mechanistic trafficking studies: Endosomal escape, intracellular transport, and nuclear entry not directly visualized. - siRNA required high polymer ratios: 60–150 w/w, which may raise toxicity concerns at higher doses. - Autofluorescence: Some polymer-scrRNA controls showed increased autofluorescence, complicating analysis. - No comparison with viral vectors and limited comparison with commercial reagents. - No disease model or therapeutic gene testing. - Citation details incomplete in the supplied file (journal/year/DOI not explicitly stated).
