Continuous Microfluidic Assembly of Biodegradable Poly(beta-amino ester)DNA Nanoparticles for Enhanced Gene Delivery

Purpose: Clinical translation of biomaterial-based gene delivery nanoparticles is limited by efficacy, safety, batch-to-batch consistency, scalable manufacturing, and long-term storage stability. Continuous microfluidic fabrication may overcome manufacturing and scale-up challenges for biodegradable PBAE/DNA polyplexes.

Hypothesis: Microfluidic 3D hydrodynamic flow focusing will produce PBAE/DNA nanoparticles with fewer DNA-free polymeric nanoparticles, and after lyophilization these nanoparticles will retain or improve transfection efficacy compared with bulk-mixed nanoparticles, while enabling scalable production and long-term storage.

Aims: Develop a continuous microfluidic device for scalable assembly of biodegradable PBAE 446/DNA polyplex nanoparticles. - Characterize nanoparticle size, concentration, DNA-containing fraction, and plasmids-per-particle using nanoparticle tracking analysis (NTA). - Compare fresh and lyophilized nanoparticles produced by bulk mixing, bulk drip freezing, and microfluidic mixing for transfection efficacy and cytotoxicity in multiple cancer cell lines. - Evaluate long-term storage stability at −20 °C and co-delivery of multiple plasmid DNAs.

Delivery system: Polymer: PBAE 446, synthesized by Michael addition from B4 (1,4-butanediol diacrylate) and S4 (4-amino-1-butanol) at 1.1:1, end-capped with E6 (2-(3-aminopropylamino)ethanol). Mn ≈ 7.4 kDa, Mw ≈ 14.0 kDa, PDI ≈ 1.89 by GPC; Mn ≈ 6.2 kDa, DP ≈ 21 by ¹H NMR. - Nanoparticle: PBAE/DNA polyplexes formed by electrostatic self-assembly in 25 mM sodium acetate buffer (pH 5.0). - Microfluidic device: PDMS/glass chip with 3D hydrodynamic flow focusing; flow ratio 3:4:1:1 for DNA/polymer primary/polymer pinch inlets; total flow rate 45 mL/h; complete mixing <10 ms. DNA at 0.09 mg/mL mixed 1:2 with polymer to final DNA 0.03 mg/mL. - Payload: Plasmid DNA encoding eGFP, DsRed, or dual reporters. Cy3-labeled plasmid DNA used for fluorescent NTA. - Formulation ratios: 10, 30, 60, and 90 w/w polymer:DNA. - Lyophilization: Sucrose added to 30 mg/mL; bulk frozen at −80 °C or microfluidic effluent drip-frozen into liquid nitrogen; lyophilized. - Targeting ligand: None.

Approach: In vitro only. Cell lines: patient-derived human glioblastoma GB319, murine melanoma B16-F10, and human triple-negative breast cancer MDA-MB-231. - Transfection: 96-well plates, 15,000 cells/well, 20 µL nanoparticle solution added to 100 µL complete media, 2 h incubation, media replaced. - Controls: Lipofectamine 2000 and 25 kDa branched polyethylenimine (PEI). - Groups: Fresh bulk, fresh microfluidic, lyophilized bulk, lyophilized bulk drip, lyophilized microfluidic; stored microfluidic nanoparticles at −20 °C for 3 months. - n: NTA ≥4 independently prepared samples; transfection/viability n = 4 wells.

Key methods: COMSOL simulation of laminar flow, convection, and diffusion. - Gel electrophoresis for DNA binding and shear testing. - Nanoparticle tracking analysis (NTA) with Cy3-labeled plasmid and fluorescent filtering to distinguish DNA-containing nanoparticles from total nanoparticles; size, concentration, plasmids per particle. - Flow cytometry for % transfected cells and geometric mean GFP expression. - MTS assay for metabolic viability/cytotoxicity. - Fluorescence microscopy for reporter expression and dual-plasmid co-expression.

Key results: DNA integrity: No plasmid DNA shearing up to 240 mL/h; PBAE 446 bound 100% of DNA at 10, 30, 60, and 90 w/w. - Nanoparticle properties: DNA-containing nanoparticle size distribution remained approximately constant across w/w ratios. Number-weighted average plasmids per nanoparticle: 8–12; volume-weighted average: 14–20. Total nanoparticle concentration increased with w/w, but DNA-containing nanoparticle concentration did not significantly change. - Fresh nanoparticles: Microfluidic mixing produced fewer DNA-free polymeric nanoparticles than bulk mixing. No significant difference in size distribution or transfection efficacy between fresh bulk and fresh microfluidic nanoparticles. - Lyophilized nanoparticles: Bulk lyophilization significantly reduced transfection efficacy in harder-to-transfect B16 and MDA-MB-231 cells (p < 0.05 vs fresh bulk). Microfluidic-lyophilized nanoparticles performed statistically better than bulk-lyophilized and bulk-drip-lyophilized nanoparticles (p < 0.05), especially at 60 and 90 w/w. GB319 transfection was generally unaffected by lyophilization. - Storage: Microfluidic-lyophilized nanoparticles stored for 3 months at −20 °C retained transfection efficacy in all three cell lines with no significant decrease. - Cytotoxicity: PBAE 446 nanoparticles showed minimal cytotoxicity under nearly all conditions; Lipofectamine 2000 was significantly toxic in all cell lines, and PEI was minimally effective with evident cytotoxicity. - Dual plasmid delivery: Microfluidic and bulk nanoparticles enabled co-expression of two reporter plasmids with similar trends; no significant differences between mixing methods.

Interpretation: Microfluidic assembly followed by lyophilization yields biodegradable PBAE/DNA nanoparticles that are efficacious, safe, robust, scalable, and stable during long-term storage. These properties support further development of continuous microfluidic manufacturing for therapeutic non-viral gene delivery, particularly for cancer applications.

Limitations: Entirely in vitro; no in vivo animal or clinical validation. - Only one PBAE polymer (446) was tested. - Long-term storage was evaluated for 3 months at −20 °C, not longer. - NTA requires dilution, which may not perfectly reflect in vivo nanoparticle behavior. - Scale-up demonstrated ~100 mg/h from a single device; larger clinical doses would require higher DNA concentrations, longer run times, or parallel devices. - No active targeting ligand; delivery relies on local or passive accumulation.