Purpose: Non-viral polymeric gene delivery to human brain cancer cells is often limited by low efficacy and poor understanding of how polymer structure should be tuned for different nucleic acid cargos. Glioblastoma has a poor prognosis, and improved delivery of DNA and siRNA could enable new therapies.
Hypothesis: If poly(beta-amino ester) (PBAE) polymer structure, molecular weight, hydrophobicity, and degradation mechanism are systematically varied, then specific polymer properties can be identified that optimize either DNA or siRNA delivery to primary human glioblastoma cells; bioreducible PBAEs will improve siRNA delivery by enabling rapid cytosolic release, while hydrolytically degradable PBAEs will favor DNA delivery.
Aims: Synthesize an array of over 70 hydrolytically degradable and bioreducible PBAEs. - Formulate and characterize over 200 nanoparticles with different nucleic acid cargos. - Evaluate delivery of siRNA, linear DNA, and circular DNA of varying sizes (1.8–26 kb) to primary human glioblastoma cells. - Identify polymer properties that determine transfection vs. knockdown efficacy. - Investigate mechanistic steps: particle formation, stability, cellular uptake, pH buffering, and nucleic acid release. - Compare lead formulations with Lipofectamine 2000.
Delivery system:
Component: Polymer class; Details: Poly(beta-amino ester)s (PBAEs), hydrolytically degradable and bioreducible
Component: Monomers; Details: Backbone diacrylates: B3, B4, B5; side-chain amines: S3, S4, S5; end-caps: E3, E5, E6, E7, E10 (cystamine, reducible disulfide)
Component: Polymer naming; Details: B-S-E; e.g., 447 = B4-S4-E7; B:S molar ratios 1.05:1–1.2:1
Component: Nanoparticle type; Details: Polyplex nanoparticles formed by electrostatic self-assembly
Component: Payloads; Details: siRNA (21 bp); circular DNA: S-DNA (1.8 kb), M-DNA (4.7 kb), L-DNA (17.5 kb), con-DNA (26 kb); linearized M-DNA (4.7 kb)
Component: Targeting ligand; Details: None
Component: Key feature; Details: Bioreducible E10 end-cap for triggered siRNA release; hydrolytically degradable backbone for DNA release
Approach: In vitro only. No in vivo animal studies. - Cells: Primary human glioblastoma GB 319 cells; GFP-positive GB 319 (GFP-GB) cells for siRNA knockdown. - Transfection: 96-well plates; DNA dose 5 µg/mL; siRNA dose 60 nM (and lower doses in selected studies); 10% serum or serum-free conditions. - Controls: Lipofectamine 2000 and X-tremeGENE HP; untreated cells; scrambled siRNA (scrRNA) paired controls. - Replicates: n = 4 for most assays; statistical significance by t-tests, ANOVA with Dunnett’s tests. - Disease context: Glioblastoma; no animal tumor model.
Key methods: Polymer characterization: ¹H NMR, gel permeation chromatography (GPC), degradation rate by GPC at pH 5, 6, 7. - Nanoparticle characterization: Nanoparticle tracking analysis (NTA) for size; zeta potential; fluorescence quenching (Yo-Pro-1) for binding strength; gel electrophoresis for complexation and release. - Cellular uptake: Flow cytometry with Cy3-labeled DNA or siRNA; heparin washing to distinguish surface-bound vs. internalized. - DNA transfection: Flow cytometry for eGFP-positive cells; MTS assay for viability. - siRNA knockdown: Fluorescence plate reader and flow cytometry for GFP knockdown; MTS viability; knockdown duration over one month. - Statistics: Two-tailed t-tests with Bonferroni correction; one-way ANOVA with Dunnett’s post-test.
Key results: DNA delivery: Leading PBAE nanoparticles transfected up to 90 ± 2% of primary human glioblastoma cells with <10% nonspecific cytotoxicity, significantly better than Lipofectamine 2000 (p < 0.01). Top formulations included 433 1.1:1, 455 1.1:1, and 436 1.1:1. - siRNA delivery: Leading bioreducible PBAE nanoparticles caused up to 85 ± 0.6% knockdown after 5 days, with >90% knockdown up to 15 days and significant knockdown persisting over one month. Knockdown was higher than Lipofectamine 2000 (p < 0.01). - Molecular weight effects: For some base polymers (e.g., B3-S5), transfection correlated with molecular weight (r² = 0.63); for closely related B4-S5, no correlation (r² = 0.01). - E10 end-cap: Bioreducible cystamine E10 dramatically improved siRNA delivery but generally decreased DNA delivery compared with non-reducible end-caps. E10 polymers showed stronger nucleic acid binding and GSH-triggered release. - Particle size: Optimal nanoparticles for both DNA and siRNA delivery were 100–150 nm in mean diameter; smaller siRNA nanoparticles (<100 nm) showed high uptake but poor knockdown. - Serum effects: siRNA knockdown was lower in 10% serum, though E10-terminated polymers still achieved >50% knockdown. DNA transfection remained effective in 10% serum.
Interpretation: The authors claim that optimal PBAE polymer structure depends on the nucleic acid cargo: hydrolytically degradable PBAEs favor DNA delivery, while bioreducible PBAEs with reducible disulfide end-caps (E10) dramatically improve siRNA delivery by enabling rapid cytosolic release. The study provides a mechanistic framework for tuning polymer properties for DNA vs. siRNA delivery and demonstrates highly effective non-viral delivery to primary human glioblastoma cells, supporting potential future brain cancer therapy.
Limitations: In vitro only: No in vivo animal studies, biodistribution, tumor efficacy, or survival data. - Primary human GB cells only: Results may not generalize to other cancers or cell types. - No targeting ligand: Delivery relies on nonspecific electrostatic interactions. - No long-term safety or toxicity data beyond MTS viability. - Serum reduces siRNA efficacy: Knockdown in 10% serum was lower, with only E10 polymers exceeding 50%. - No direct mechanism visualization: Endosomal escape and intracellular trafficking inferred from functional assays, not directly imaged. - No comparison with viral vectors. - No dose–response optimization in vivo and no evaluation of repeated dosing. - No clinical translation data.
