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Drug Delivery

Drug Delivery - Poster Session E

Poster X18 - Tri- and Tetrafunctional Branched Poly(Beta-Amino Ester) Nanoparticles Enable Efficient and Versatile Gene Delivery

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Poly(beta-amino ester) (PBAE) nanoparticles (NPs) hold great promise for nonviral gene delivery in a variety of contexts. Recent studies suggest branched PBAEs (BPBAEs) offer unique advantages over linear counterparts, but the effect of polymer structure has not been well investigated across many chemical constituents. Here, a library of novel BPBAEs was synthesized with tri- and tetrafunctional branching. We explored the effects of BPBAE branching and w/w on NP transfection efficacy to minimize the required polymer dose for efficacious transfection, characterized NP physiochemical properties, and assessed the broad applicability of these BPBAEs for nucleic acid delivery to diverse cell types (Figure 1).

Materials and

Methods:  BPBAEs were synthesized by combining a diacrylate linear monomer (bisphenol A ethoxylate diacrylate, B9), multiacrylate branched monomer, and amine sidechain (3-morpholinopropylamine, S91) in DMF and reacting at 85°C for 48 h. The polymers were then endcapped with 1-(2-aminoethyl)piperazine (E39) (37°C for 24 h in THF). The ratio of diacrylate monomer to multiacrylate monomer determined the degree of polymer branching, with “% branching” defined as the percentage of acrylate groups contributed by the branching monomer. Branching monomers included trimethylolpropane triacrylate (B8), pentaerythritol tetraacrylate (B12), and di(trimethylolpropane) tetraacrylate (B13) (Figure 2A). B8 generated BPBAEs with trifunctional branching, while B12 and B13 generated BPBAEs with tetrafunctional branching. Purified BPBAEs were combined with DNA, mRNA, or siRNA in sodium acetate buffer, and the resulting NPs were administered to various human and murine cell lines. In the case of siRNA delivery, NPs delivering siRNA against GFP were administered to cells engineered to stably express GFP. Transfection efficacy was assessed via flow cytometry. DNA binding efficiency, NP surface charge, NP size, and the approximate number of plasmids/NP were evaluated using gel electrophoresis, electrophoretic motility, dynamic light scattering, and nanoparticle tracking analysis, respectively.

Results, Conclusions, and Discussions: Transfection efficiency was assessed in B16F10 murine melanoma cells using B8-, B12-, and B13-based BPBAEs at 0% (linear equivalent) to 60% branching. NPs were complexed with GFP DNA at 10, 20, and 40 w/w (Figure 2B). Moderate branching (30%) allowed optimal transfection with 9,8-91-39, 9,12-91-39, and 9,13-91-39 BPBAE NPs at 20 w/w, effectively reducing the required polymer dose in half, as the linear equivalent (9-91-39) required 40 w/w for robust transfection. At 20 w/w, all BPBAEs led to complete DNA encapsulation (Figure 3A). These NPs demonstrated surface charges approaching +40 mV (Figure 3B), were ~50 nm in size (Figure 3C) and encapsulated 2-4 plasmids/NP (Figure 3D). Moderate branching (30%) also enabled significant improvements in transfection efficacy of 20 w/w NPs across human and murine cancer and noncancer cells (Figure 4A). Further, moderate branching of B8- and B13-based NPs significantly improved mRNA (Figure 4B) and siRNA (Figure 4C) transfection efficacy. Taken together, we observed that both tri- and tetrafunctional branching can confer significant improvements in PBAE transfection efficacy across diverse cell lines and with various nucleic acid cargoes. This work contributes to a rapidly developing space of BPBAE NPs, and these formulations present a promising platform for versatile and highly efficient gene delivery.

Acknowledgements (Optional):