Stabilization of Poly (β-Amino Ester) Nanoparticles for the Efficient Intracellular Delivery of PiggyBac Transposon

Purpose: Gene-editing tools are large plasmid constructs that cannot spontaneously enter mammalian cells, and viral vectors are limited by cargo size, immunogenicity, and manufacturing challenges. PBAE nanoparticles are promising nonviral carriers, but unmodified PEG-PDHA nanoparticles release encapsulated plasmid DNA too rapidly for efficient intracellular delivery of large gene-editing cargo such as piggyBac transposon.

Hypothesis: Stabilizing PEG-PDHA nanoparticles by covalent crosslinking, rather than by non-covalent layer-by-layer coating, will slow plasmid release, enhance intracellular delivery, and enable efficient transfection of the piggyBac transposon plasmid in human glioblastoma cells without a targeting moiety.

Aims: Synthesize and characterize PEG-PDHA block copolymer and nanoparticles. - Encapsulate PBCAG-eGFP piggyBac transposon plasmid at different polymer/plasmid molar ratios and identify optimal loading. - Compare non-covalent layer-by-layer stabilization versus covalent NHS/EDC crosslinking for controlling plasmid release. - Evaluate cellular uptake, transfection efficiency, and cytotoxicity in U87MG human glioblastoma cells.

Delivery system: Polymer: PEG-PDHA copolymer: poly(ethylene glycol)-block-poly(1,4-butanediol diacrylate-β-5-hydroxyamylamine)-block-poly(ethylene glycol). Synthesized via Michael addition polymerization. - Nanoparticle type: Self-assembled polymeric nanoparticles formed by complexation with plasmid DNA at low pH, then adjusted to physiological pH. - Payload: PBCAG-eGFP piggyBac transposon plasmid, 6.34 kb. - Formulation: Optimal polymer/plasmid molar ratio = 0.36. Unmodified PEG-PDHA NPs: ~150 nm, PDI 0.184, zeta −7.86 mV. PBCAG-loaded NPs: ~236.9 nm, zeta −6.72 mV. - Stabilization strategies: - Non-covalent: Layer-by-layer assembly of four biopolyelectrolyte layers of poly-L-lysine (PLL) and poly-γ-glutamate (PGA). - Covalent: NHS/EDC crosslinking of residual carboxylic acid end groups of PEG and amine groups of PDHA. - Targeting ligand: None. - Crosslinked NP properties: ~177.1 nm, PDI 0.126, zeta −19.2 mV.

Approach: In vitro only. Human glioblastoma U87MG cells cultured in EMEM + 10% FBS. - Cells seeded at 15,000 cells/well in 96-well plates. - Nanoparticles added at 5 or 10 µg/mL PBCAG-eGFP per well in Opti-MEM. - Positive control: Lipofectamine 3000. - Negative control: intact U87MG cells. - Uptake assessed at 0, 0.5, 1, 2, 5, and 24 h by flow cytometry and confocal microscopy. - Transfection assessed by GFP expression via flow cytometry and confocal microscopy. - Cytotoxicity assessed by MTT assay over 3 consecutive days. - n = 4 for transfection; no in vivo model.

Key methods: ¹H-NMR for polymer synthesis verification. - DLS for hydrodynamic diameter, PDI, and zeta potential. - TEM for nanoparticle morphology. - Gel electrophoresis and UV spectroscopy for plasmid encapsulation and release kinetics. - Flow cytometry for cellular uptake and GFP transfection efficiency. - Confocal microscopy for intracellular localization and GFP expression. - MTT assay for metabolic viability/cytotoxicity.

Key results: Encapsulation and release: Optimal polymer/plasmid molar ratio = 0.36. Unmodified PEG-PDHA NPs released essentially all PBCAG within 2 h. Layer-by-layer NPs showed no detectable release within 24 h but transfection was <1%. Crosslinked NPs released only ~40% of PBCAG after 10 days in PBS at 37 °C and no detectable release at pH 4 within 10 days. - Cellular uptake: PEG-PDHA NPs were taken up by or attached to U87MG cells within 1 h; confocal imaging showed localization in cytoplasm and nuclei without targeting moiety. - Transfection: Crosslinked PEG-PDHA NPs transfected ~55% of U87MG cells, compared with ~62% for Lipofectamine 3000. Unmodified PEG-PDHA NPs transfected only ~7%; layer-by-layer NPs <1%. - Cytotoxicity: No cytotoxic effects were observed for naked PBCAG, empty PEG-PDHA NPs, or crosslinked PEG-PDHA NPs. Lipofectamine 3000 caused abnormal spherical cell morphology and detachment, though MTT did not detect significant cytotoxicity. - Dose effect: Increasing PBCAG concentration from 5 to 10 µg/mL consistently decreased GFP-positive cells; mechanism not further investigated.

Interpretation: Covalent crosslinking is the most effective stabilization strategy for PEG-PDHA nanoparticles delivering large gene-editing plasmids. Crosslinked nanoparticles achieve transfection efficiency comparable to Lipofectamine 3000 in U87MG cells without cytotoxic effects or targeting ligands. This work supports further development of PBAE copolymer nanoparticles for nonviral intracellular delivery of gene-editing tools, including piggyBac transposon systems.

Limitations: In vitro only: no in vivo disease model or animal validation. - No transposase co-delivery: only the piggyBac transposon plasmid was delivered; the transposase protein required for genomic integration was not co-delivered in this study. - No targeting ligand: delivery relies on passive uptake; no active cell-type specificity. - Single cell line: only U87MG human glioblastoma cells were tested. - Mechanism unclear: nuclear translocation mechanism of PEG-PDHA NPs was not elucidated. - Dose-dependent decrease: increasing plasmid concentration from 5 to 10 µg/mL reduced transfection efficiency; reason not resolved. - Long-term expression and stability: not evaluated beyond the reported transfection time points. - Biocompatibility: only MTT metabolic activity was used; other cytotoxicity mechanisms and long-term safety were not assessed.