Synthesis of Poly(â-amino ester)s Optimized for Highly Effective Gene Delivery

Purpose: Nonviral polymeric gene delivery vectors need systematic optimization of nonstructural factors—polymer molecular weight, chain end-group, and polymer/DNA ratio—that strongly influence transfection, cytotoxicity, and DNA complexation. These factors had not been comprehensively studied together for poly(β-amino esters).

Hypothesis: By varying amine/diacrylate stoichiometry, PBAE molecular weight and chain end-group can be controlled. Amine-terminated, higher-molecular-weight PBAEs at optimized polymer/DNA ratios will mediate efficient gene transfer, whereas acrylate-terminated polymers will be ineffective. Blending with poly-L-lysine (PLL) as a co-complexing agent will improve complex stability and reduce the required polymer dose.

Aims: Synthesize two PBAE structures, Poly-1 and Poly-2, with 12 stoichiometric variants each to vary molecular weight and end-group. - Characterize polymers by GPC and ¹H NMR. - Screen all polymers at nine polymer/DNA ratios for luciferase transfection in COS-7 cells. - Evaluate cytotoxicity, cellular uptake, and DNA binding as functions of molecular weight, end-group, and polymer/DNA ratio. - Test PLL co-complexation and GFP transfection across multiple cell lines, comparing with PEI and Lipofectamine 2000.

Delivery system: Polymers: - Poly-1: 1,4-butanediol diacrylate + 1-aminobutanol. - Poly-2: 1,6-hexanediol diacrylate + 1-aminobutanol. - Synthesized by solvent-free Michael addition at 100 °C for 5 h. - Amine/diacrylate mole ratios 0.6–1.4 produced amine-terminated (>1) or acrylate-terminated (<1) chains. - Molecular weights: 3,350–18,000 Da; PDI 1.55–2.20. - Nanoparticles: PBAE/DNA polyplexes formed in 25 mM sodium acetate buffer, pH 5. - Payload: pCMV-Luc (firefly luciferase) and pCMV-EGFP (GFP) plasmid DNA. - Formulation ratios: 10:1 to 150:1 polymer/DNA w/w. - Co-complexing agent: Poly-L-lysine (PLL) blended with PBAE. - Targeting ligand: None.

Approach: In vitro only. Main cell line: COS-7 green monkey kidney cells. Additional GFP transfections in CHO, NIH 3T3, and HepG2 cells. - Luciferase transfections: 96-well plates, 600 ng DNA/well, 1 h complex incubation, analyzed day 3; n = 4. - GFP transfections: 6-well plates, 5 µg DNA/well, 1 h incubation, analyzed after 2 days. - Controls: PEI 25 kDa and Lipofectamine 2000. - No in vivo model.

Key methods: GPC for molecular weight and PDI. - ¹H NMR for polymer structure. - Agarose gel electrophoresis for DNA binding/retardation. - PicoGreen dye exclusion assay with salt titration for complex stability. - Luciferase assay for cumulative transfection. - MTT assay for cytotoxicity. - Flow cytometry for Cy5-labeled DNA uptake and GFP expression. - GFP mean fluorescence and % positive cells.

Key results: Acrylate-terminated polymers: No appreciable transfection under any condition; poor cellular uptake and poor aqueous solubility. - Amine-terminated optimized polymers: - Poly-1 (Mw 13,100) at 150:1 achieved 60 ng luciferase/well. - Poly-2 (Mw 13,400) at 30:1 achieved 26 ng/well. - PEI (1:1 w/w) achieved 6 ng/well; Lipofectamine 2000 achieved 21 ng/well at 1 h and 27 ng/well at 4 h. - Cytotoxicity: Poly-1 was not cytotoxicity-limited; Poly-2 cytotoxicity limited transfection at high polymer/DNA ratios. - Uptake: Acrylate-terminated polymers showed poor uptake; Poly-1 was uptake-limited except at highest ratios; Poly-2 uptake increased above 30:1. - DNA binding: Low-molecular-weight amine polymers failed to retard DNA migration; Mw 13,100 and 13,400 polymers retarded DNA at 10:1. - PLL co-complexation: - Poly-1/PLL (60:0.1:1 w/w/w) yielded ~93% GFP+ COS-7 cells; mean fluorescence 6033. - Poly-2/PLL (15:0.4:1) yielded ~25% GFP+. - Lipofectamine 2000 ~90%; PEI ~77%. - Poly-1/PLL transfected CHO ~67%, NIH 3T3 ~25%, HepG2 ~30%.

Interpretation: Polymer molecular weight, chain end-group, and polymer/DNA ratio dramatically affect PBAE-mediated gene transfer. Only amine-terminated PBAEs were suitable; acrylate-terminated chains were ineffective. Optimized PBAEs surpassed PEI and Lipofectamine 2000 in vitro. PLL co-complexation improved complex stability and reduced the polymer dose required. These findings provide design rules for biodegradable polymeric gene delivery vectors, though in vivo testing remains future work.

Limitations: In vitro only: no animal or clinical validation; authors state optimization in serum is ongoing for in vivo testing. - No serum conditions: transfections were performed in Opti-MEM without serum, limiting physiological relevance. - High polymer/DNA ratios: effective formulations often required very high ratios, which may raise toxicity/translation concerns. - Limited polymer structures: only two base PBAE structures were tested. - Uptake assay sensitivity: lower than luciferase expression, complicating direct correlation. - No targeting ligand: delivery specificity was not addressed. - Long-term safety/stability: not evaluated.