Highly Branched Poly(#-amino esters) for Non-Viral Gene Delivery High Transfection Efficiency and Low Toxicity Achieved by Increasing Molecular Weight

Purpose: Nonviral gene delivery vectors suffer from an “efficacy–toxicity paradox”: high transfection efficiency usually comes with high cytotoxicity, while low-toxicity vectors often transfect poorly. For cationic polymers such as PEI, PAMAM, and PDMAEMA, increasing molecular weight typically improves transfection but also increases toxicity, limiting clinical translation.

Hypothesis: The branched topology of highly branched poly(β-amino esters) (HPAEs) will produce a different relationship between molecular weight and cytotoxicity than linear cationic polymers. Specifically, high-molecular-weight HPAEs can achieve both high transfection efficiency and low cytotoxicity, and the spatial configuration of linear versus branched segments will be pivotal.

Aims: Synthesize HPAEs with controlled molecular weights via an “A2+B3+C2” Michael addition strategy. - Compare transfection efficiency and biocompatibility of HPAEs with commercial/control vectors: branched PEI, PAMAM dendrimers, linear PDMAEMA, and Lipofectamine 2000. - Investigate mechanisms underlying the molecular-weight effect: DNA binding, polyplex size, zeta potential, morphology, and cell-membrane interactions. - Modulate the distribution of linear and branched segments in HPAEs and test how molecular configuration affects transfection and toxicity.

Delivery system: Polymer class: Highly branched poly(β-amino esters) (HPAEs). - Synthesis: “A2+B3+C2” Michael addition. - A2: 3-morpholinopropylamine (MPA) - B3: trimethylolpropane triacrylate (TMPTA) — branching monomer - C2: bisphenol A ethoxylate diacrylate (BE) - End-cap: 4-amino-1-butanol (S4) - Feed ratio TMPTA:BE:MPA = 0.5:1:1.46; [vinyl]:[NH] = 1.2:1; reacted at 90 °C; end-capped with S4. - Structural variants: HPAE-A, HPAE-B, HPAE-C with different spatial distributions of linear (MPA + BE) and branched (MPA + TMPTA) segments, synthesized via a two-step reaction. - Nanoparticle type: HPAE/DNA polyplexes formed by electrostatic self-assembly in 25 mM sodium acetate buffer, pH 5.2, mixed 1:1 by volume. - Payload: plasmid DNA — gWiz-GFP and pCMV-GLuc (Gaussia luciferase). - Formulation ratios: polymer:DNA w/w = 5:1, 10:1, 15:1, 20:1. - Targeting ligand: none. - Controls: branched PEI (1.8 and 25 kDa), PAMAM dendrimer (G2 and G5; ~3.5 and 13.9 kDa), linear PDMAEMA (5.8 and 16.7 kDa), Lipofectamine 2000.

Approach: In vitro only. Cell lines: HeLa cells and human SHSY-5Y astrocytes. - Cells seeded at 10,000 cells/well in 96-well plates and grown 24 h. - Polyplexes added to serum-containing medium; final DNA amount 0.5 µg/well; incubated 4 h; medium replaced; analysis 48 h post-transfection. - Viability assessed 48 h post-transfection by alamarBlue assay. - n = 4; one-way ANOVA; p < 0.05 considered significant. - No in vivo model.

Key methods: GPC for Mn, Mw, PDI, and Mark–Houwink alpha values. - ¹H NMR for chemical composition. - PicoGreen assay for DNA binding efficiency. - DLS for polyplex size and zeta potential. - TEM for polyplex morphology. - FITC labeling of HPAEs to assess cell-membrane adsorption/interaction. - Gaussia luciferase assay for transfection efficiency. - GFP fluorescence microscopy for reporter expression. - alamarBlue assay for metabolic viability/cytotoxicity.

Key results: High-MW HPAE achieves high transfection and low toxicity: HPAE-M21 (21.0 kDa) in HeLa cells at 20:1 w/w showed 1.85, 6.69, 10.7, 86.6, and 1723-fold higher Gaussia luciferase activity than lower-MW HPAE analogues, while maintaining 94.1% cell viability — up to 60% higher viability than counterparts. - Opposite trend vs conventional polymers: High-MW PEI, PAMAM, and PDMAEMA showed higher transfection but substantially higher cytotoxicity than their lower-MW versions. In contrast, high-MW HPAE exhibited much lower cytotoxicity than low-MW HPAE (91.2 ± 1.13% vs 57.6 ± 2.34% in the initial comparison). - Polyplex properties: At w/w ≥ 10:1, all HPAEs bound DNA well and formed positively charged polyplexes (+5 to +11 mV). At 5:1, HPAE-M5 binding was only 24%, and HPAE-M5/M7 polyplexes had negative zeta potentials (−7 and −6 mV). Size decreased with increasing MW and w/w; HPAE-M5/DNA at 20:1 was almost twice as large as HPAE-M21/DNA. TEM: HPAE-M5/DNA formed large irregular aggregates; HPAE-M21/DNA formed small uniform spheres. - Membrane interaction mechanism: FITC-labeled HPAE-M5/DNA showed 2.82-fold stronger cell fluorescence than HPAE-M21/DNA, indicating more low-MW polyplex adsorbed to cell membranes. Cells incubated with HPAE-M5/DNA showed abnormal morphology, whereas HPAE-M21/DNA preserved morphology. - Configuration matters: HPAE-A (more external linear segments) caused severe cytotoxicity and lower transfection. HPAE-B and HPAE-C preserved ≥85% viability at high w/w. HPAE-C achieved up to 17-fold higher gene transfection than HPAE-A.

Interpretation: HPAEs can overcome the conventional efficacy–toxicity trade-off: high-molecular-weight HPAEs achieve high transfection efficiency and low cytotoxicity simultaneously. The interaction between HPAE/DNA polyplexes and the cell lipid bilayer dictates cytotoxicity and transfection. Modulating the branched molecular configuration can circumvent high-MW-induced toxicity. These findings challenge the traditional view that higher cationic polymer molecular weight necessarily means higher toxicity, and provide design principles for next-generation nonviral gene delivery vectors.

Limitations: In vitro only: no animal or in vivo validation; only HeLa and SHSY-5Y cells tested. - No targeting ligand: delivery specificity was not addressed. - Mechanistic evidence is indirect: membrane interaction hypothesis is supported by FITC adsorption, morphology, size, and zeta data, but direct membrane disruption/endosomal escape assays were not performed. - Toxicity assessment limited: mainly alamarBlue metabolic activity and morphology; no comprehensive apoptosis/necrosis or long-term toxicity evaluation. - Polymer structural characterization: HPAEs are heterogeneous branched polymers; exact branching distributions are not fully resolved. - Storage/stability, scalability, and degradation kinetics were not evaluated. - No comparison across multiple serum concentrations or physiological barriers.