Differentially Branched Ester Amine Quadpolymers with Amphiphilic and pH-Sensitive Properties for Efficient Plasmid DNA Delivery

Purpose: Nonviral gene delivery remains limited by low transfection efficacy, especially under physiological serum conditions and at low nanoparticle doses. Although linear poly(β-amino ester)s (PBAEs) are effective, branching structure had not been systematically evaluated in well-defined PBAE-like poly(ester amine) systems.

Hypothesis: Introducing controlled branching into amphiphilic, pH-sensitive poly(ester amine) quadpolymers will increase end-cap density, DNA binding, buffering capacity, serum stability, and transfection efficacy, with moderate branching being optimal. Increased branching was also hypothesized to improve cellular uptake via additional end-cap moieties.

Aims: Synthesize a series of Branched poly(Ester Amine) Quadpolymers (BEAQs) with well-defined degrees of branching via A2 + B2/B3 + C1 Michael addition. - Characterize polymer properties: molecular weight, branching, buffering capacity, solubility, and plasmid DNA binding. - Evaluate nanoparticle size, charge, cellular uptake, transfection efficacy, and viability in HEK293T and ARPE-19 cells. - Assess performance under high serum and low DNA dose conditions and identify structure–function relationships.

Delivery system: Polymer: Branched poly(Ester Amine) Quadpolymers (BEAQs) synthesized from: - B7: bisphenol A glycerolate (1 glycerol/phenol) diacrylate (diacrylate) - B8: trimethylolpropane triacrylate (TMPTA; triacrylate) - S4: 4-amino-1-butanol - E6: 2-(3-aminopropylamino)ethanol (end-cap) - Synthesis: Overall vinyl/amine ratio 2.2:1; 200 mg/mL in anhydrous DMF; 90 °C for 24 h; end-capped with E6 at room temperature for 1 h. Polymers named by triacrylate mole fraction: B8-0% (linear) through B8-90%. - Nanoparticle: BEAQ/DNA polyplexes formed in 25 mM sodium acetate buffer, pH 5.0, mixed 1:1 v/v; 5 min incubation. - Payload: eGFP-N1 plasmid DNA; Cy5-labeled plasmid DNA for uptake and confocal imaging. - Targeting ligand: None. - Key properties: Amphiphilic, pH-sensitive, biodegradable ester linkages; buffering in pH 5–7.4 range; zeta potential ~+15 mV; sizes ~50–100 nm at pH 5 and ~100 nm after dilution into PBS.

Approach: In vitro only. Cell lines: HEK293T (human embryonic kidney) and ARPE-19 (human retinal pigment epithelial). - 96-well plates: 12,000 cells/well; 384-well plates: 2,500 cells/well. - Nanoparticles incubated with cells for 2 h in complete media, then replaced. - Transfection assessed at 48 h by flow cytometry; viability assessed at 24 h by MTS. - Conditions tested: 10% serum, 50% serum, and low plasmid doses (5–10 ng/well; 16–256 pM; 0.25–4 pg/cell). - Controls: linear B8-0% polymer, branched PEI, linear PEI, JetPRIME, and Lipofectamine 2000. - n = 4 wells per condition; mean ± SEM.

Key methods: ¹H NMR and GPC for polymer structure and molecular weight. - Acid–base titration for buffering capacity and effective pKa. - Absorbance-based solubility limit assay. - Yo-Pro-1 iodide DNA binding assay and gel electrophoresis. - DLS, zeta potential, and TEM for nanoparticle characterization. - Flow cytometry for eGFP transfection (% positive, geometric mean expression), Cy5-DNA uptake, and viability. - Confocal microscopy with lysosome staining for lysosomal colocalization and nuclear localization. - Statistical analysis: one-way ANOVA with Dunnett/Bonferroni corrections.

Key results: Transfection efficacy: BEAQs achieved up to 99% transfection in HEK293T and 77% in ARPE-19 cells in 10% serum, exceeding linear PBAE and commercial reagents. - High serum: Under 50% serum, B8-50% transfected 98% of HEK293T and 65% of ARPE-19 cells, maintaining >70% geometric mean expression relative to 10% serum; linear polymers performed significantly worse. - Low dose: At 5–10 ng DNA/well, moderately branched B8-40% and B8-50% outperformed linear PBAE; optimal w/w ratio shifted to ~60 w/w. - DNA binding: At pH 5, linear and branched polymers bound DNA similarly; at pH 7.4, branched polymers bound DNA more strongly. Neutral-pH binding correlated with secondary amine content from end-caps; low-pH binding correlated with tertiary amines. - Nanoparticle properties: ~50–100 nm at pH 5; ~100 nm after dilution into PBS; zeta ~+15 mV; TEM diameters 30–60 nm. Linear B8-0% was smallest by TEM (32 ± 3 nm) vs B8-50% (54 ± 6 nm). - Uptake: >95% of cells were positive for DNA uptake for all polymers; branching did not significantly increase uptake over linear polymer. - Buffering: Increased with branching; effective pKa ~6.0–6.75. However, highly branched B8-80% and B8-90% had the lowest transfection despite high buffering, indicating buffering alone is not rate-limiting. - Lysosomal avoidance: B8-50% showed less lysosomal colocalization than linear B8-0%; at 24 h, <20% of internalized DNA was in lysosomes for B8-50% at 40 w/w vs 40–50% for linear polymer. - Structure–function: Optimal tertiary amine content was ~40 tertiary amines per base pair DNA across branching degrees; optimal secondary amines increased with branching. Buffering optimum ~20 nmol H⁺/µg DNA. Tertiary amine density best predicted viability-normalized expression (R² = 0.761 HEK293T; R² = 0.615 ARPE-19). - Viability: No notable cytotoxicity at tested doses; high serum and low dose conditions did not strongly reduce viability.

Interpretation: Moderately branched BEAQs outperform linear PBAEs and commercial reagents, especially under physiological serum and low-dose conditions. Branching improves DNA binding, buffering, serum stability, and lysosomal avoidance. Structure–function relationships identified here—particularly tertiary amine content and buffering capacity—can guide rational design of next-generation biodegradable polymers for nonviral gene delivery, including retinal gene therapy applications.

Limitations: Entirely in vitro; no in vivo animal or clinical validation. - Only two cell lines (HEK293T and ARPE-19) were tested. - No active targeting ligand; delivery specificity was not addressed. - Exact mechanism of endosomal escape and intracellular trafficking is not fully resolved. - Polymer degradation rate and long-term storage stability were not evaluated. - Synthesis uses DMF and ether purification, which may affect scalability.