Purpose: Non-viral polymeric nanoparticles are promising for gene therapy and stem cell reprogramming, but the number of plasmids complexed per particle affects transfection efficiency and co-delivery of multiple plasmids. Existing methods for quantifying plasmids per particle have significant drawbacks, and few studies have characterized polymeric nanoparticles in physiologically relevant aqueous conditions.
Hypothesis: Nanoparticle tracking analysis (NTA) can be used to easily quantify the average number of plasmids encapsulated within polymeric nanoparticles in aqueous solution. The number of plasmids per particle depends on polymer structure and polymer-to-DNA weight ratio, and nanoparticles with higher plasmid/particle counts will enable greater co-expression when delivering multiple plasmids to the same cell.
Aims: Develop a novel NTA-based assay to quantify the number of plasmids per polymeric nanoparticle in aqueous solution. - Characterize PEI and six PBAE formulations for particle size, concentration, and plasmids per particle. - Determine how polymer structure and polymer-to-DNA weight ratio affect plasmid/particle counts. - Evaluate co-transfection and co-expression of EGFP and DsRed plasmids in primary human fibroblasts using same-particle versus separate-particle delivery.
Delivery system:
Component: Cationic polymers; Details: 25 kDa branched polyethylenimine (PEI) and six end-capped poly(β-amino ester)s (PBAEs): B4S4E7, B5S3E7, B4S5E7, B5S5E1
Component: Payload; Details: EGFP-N1 plasmid DNA (4.7 kbp); DsRed plasmid for co-transfection studies
Component: Nanoparticle formation; Details: Electrostatic self-assembly in 25 mM sodium acetate (pH 5) for PBAEs; 150 mM NaCl (pH 5.5) for PEI
Component: Polymer:DNA ratios; Details: PBAEs: 40, 60, or 100 wt/wt; PEI: 2 wt/wt (N/P 16)
Component: Targeting ligand; Details: None
Component: Key feature; Details: Aqueous-phase quantification of plasmids per nanoparticle using NTA
Approach: In vitro model: Human fetal fibroblasts (IMR90) seeded at 1.8 × 10⁵ cells/well in 6-well plates and transfected with 6 µg DNA per 100 µL particles per well. - Co-transfection conditions: 1. Same-particle co-delivery: EGFP and DsRed plasmids mixed before nanoparticle self-assembly; 4 h incubation. 2. Separate particles, same day: EGFP particles for 2 h, media change, DsRed particles for 2 h. 3. Separate particles, 24 h apart: EGFP particles for 4 h, then DsRed particles for 4 h after 24 h. - Controls: Lipofectamine 2000 as positive control. - Nanoparticle batches: At least duplicate independent batches; NTA with >500 completed tracks per replicate.
Key methods: Nanoparticle tracking analysis (NTA): Nanosight LM10 for number-averaged particle size and absolute particle concentration. - Dynamic light scattering (DLS): Malvern Zetasizer NanoZS for size confirmation. - Transmission electron microscopy (TEM): Dried nanoparticle imaging. - Gel electrophoresis: Confirmed 100% plasmid complexation at tested wt/wt ratios. - Fluorescence microscopy and flow cytometry: Co-expression of EGFP and DsRed in IMR90 cells.
Key results: PEI nanoparticles: 90 ± 10 plasmids/particle by NTA; theoretical maximum 267 plasmids/particle. - PBAE nanoparticles: 30 ± 2 to 120 ± 20 plasmids/particle; theoretical maximum 80–195. - Formulation-specific values: - B4S4E7 (40 wt/wt): 120 ± 20 plasmids/particle - B4S4E7 (60 wt/wt): 110 ± 10 plasmids/particle - B5S3E7 (60 wt/wt): 45 ± 9 plasmids/particle - B5S3E7 (100 wt/wt): 30 ± 2 plasmids/particle - B5S5E1 (100 wt/wt): 35 ± 2 plasmids/particle - Polymer structure effect: B4-based nanoparticles had ~3-fold higher DNA carrying capacity per particle than B5-based nanoparticles, despite similar ~100 nm size. - Weight ratio effect: Changing polymer:DNA wt/wt from 40 to 60 (B4S4E7) or 60 to 100 (B5S3E7) did not significantly change plasmids/particle. - Co-expression: Same-particle co-delivery produced more co-expressing cells than separate-particle delivery on the same day or 24 h apart. B4S4E7 nanoparticles achieved coexpression in 15.8 ± 0.5% of live cells; B5S3E7 achieved 3.3 ± 1%.
Interpretation: The authors claim this is the first study to characterize polymeric nanoparticles in terms of plasmids per particle in aqueous conditions. They state that NTA provides a quick, easy, and robust assay for quantifying plasmid/particle numbers, that plasmid/particle counts range from 30 to 120 depending on polymer structure, and that higher plasmid/particle counts lead to higher co-expression. This quantitative understanding may improve the design of non-viral vectors for co-delivering multiple plasmids for gene therapy and stem cell reprogramming.
Limitations: Assumes all DNA is complexed and nanoparticles are monodisperse; validated by gel electrophoresis but may not hold universally. - Reports average plasmids per particle; the distribution is broad, with particles having both higher and lower values. - Theoretical maximum estimates depend on assumptions about condensed plasmid size (e.g., 25.9 nm equivalent spherical diameter). - Only in vitro data in IMR90 fibroblasts; no in vivo validation. - Co-expression differences may also depend on other nanoparticle properties beyond plasmid/particle count. - NTA measures scattering centroids and may not capture all particle heterogeneity.
