Non-Viral Gene Delivery Nanoparticles Based on Poly(β-amino esters) for Treatment of Glioblastoma

Purpose: Glioblastoma (GB) has poor survival and current therapies are insufficient. Viral gene therapy carries safety risks, while nonviral vectors often lack efficiency and practical storage stability. PBAEs had shown high transfection and low toxicity in other cell types but had not been tested in primary brain tumor cells or brain tumor stem cells (BTSCs). A stable, ready-to-use formulation was also needed for clinical translation.

Hypothesis: PBAE nanoparticles can efficiently deliver plasmid DNA to primary GB astrocytes and BTSCs with low toxicity. Polymer structure can confer specificity for tumor cells over healthy astrocytes and neural stem cells. Lyophilization with sucrose can produce stable, storable nanoparticles that retain transfection efficacy over months.

Aims: Synthesize and screen a PBAE library for DNA delivery to GB astrocytes and BTSCs. - Compare transfection efficiency and viability with commercial reagents and healthy fetal NSCs/astrocytes, including co-culture and 3D neurospheres. - Develop a lyophilized, sucrose-stabilized nanoparticle formulation and test storage stability up to 3–6 months. - Assess duration of transgene expression and long-term retention in tumor cells.

Delivery system: Polymer: Poly(β-amino esters) (PBAEs), synthesized by Michael addition from backbone diacrylates (B4, B5, B6), sidechain amino alcohols (S3, S4, S5), and end-capping small amines (E3–E9). Lead examples: 447, 457, 456, 455, 454, 453. - Nanoparticle: PBAE/DNA polyplexes formed in 25 mM sodium acetate (pH 5) at polymer:DNA w/w ratios of 30:1, 60:1, 90:1, or 120:1. - Payload: Plasmid DNA encoding EGFP or DsRed-Max; Cy3-labeled DNA for uptake tracking. - Targeting ligand: None; cell-type specificity attributed to polymer structure. - Lyophilization: Sucrose added (final 15–45 mg/mL; optimal 60 mg/mL in formulation), frozen at −80 °C, lyophilized 48 h, stored at 4 °C with desiccant.

Approach: In vitro only. Human primary GB astrocytes (GB319), human BTSCs (line 551, some EGFP-transduced), healthy human fetal NSCs (F34), and fetal astrocytes (F34 differentiated). - 24-well plates: 75,000 cells/well; 96-well plates: 15,000 cells/well. Transfection incubation 4 h, then replaced with complete medium. - Controls: Lipofectamine 2000 and FuGENE HD. Doses: 0.15–3.0 µg DNA/well. Serum-free and 10% serum conditions tested. n = 4 replicates unless noted. - Long-term expression monitored up to 70–90 days and after passaging.

Key methods: Fluorescence microscopy for GFP/DsRed expression and Cy3-DNA uptake; DAPI for viability. - ImageJ quantification of transfected cells and viability. - Flow cytometry for 3D neurosphere transfection and long-term expression. - Nanoparticle tracking analysis (NTA) for particle size and size distribution. - Lyophilization and storage stability assays. - One-way ANOVA with Dunnett post-test for statistics.

Key results: GB astrocytes: PBAE 447 at 30:1 achieved 60.6 ± 5% transfection in serum-free conditions and 40.0 ± 2% in 10% serum with 82.5 ± 4% viability. Lipofectamine 2000 at comparable viability transfected only 13.6 ± 2%. - BTSCs: Up to 43.0 ± 7% transfection with 61.4 ± 5% viability, or 37.6 ± 4% transfection with 85.7 ± 6% viability. In 3D neurospheres, 447 and 457 yielded 40–45% DsRed expression; 454 and 456 yielded 24.3 ± 7% and 30.8 ± 2%. - Specificity: PBAE 457 transfected nearly 20% of BTSCs but only 4.8% of F34 astrocytes and no apparent effect on F34 NSCs. PBAE 456 gave 13.4% BTSC transfection with little/no effect on F34 cells (0.3%, >85% viability). PBAE 447 transfected both tumor and healthy cells (19.1 ± 2% healthy NSCs). - Storage stability: Lyophilized particles with 60 mg/mL sucrose stored at 4 °C showed no significant difference vs. fresh particles at 3 months. At 6 months, approximately 50% of original transfection efficiency and total fluorescence per cell were retained. - Long-term expression: A small fraction (0.5–1.1%) of cells remained stably GFP+ after 70 days. DsRed expression in reformed BTSC neurospheres persisted at 45 days, with some cells still DsRed+ at 90 days after passaging.

Interpretation: PBAEs can deliver genes to GB astrocytes and BTSCs with high efficiency and low toxicity. Some polymers (e.g., 457, 456) show tumor-specific transfection with minimal effects on healthy neural cells, while others (e.g., 447) are broadly effective and may be useful for delivering secreted cytotoxic factors. Lyophilized PBAE/DNA nanoparticles retain function for at least 3 months, supporting practical clinical translation. This work presents a promising nonviral gene delivery platform for glioblastoma.

Limitations: In vitro only: no in vivo animal or clinical data; authors state future in vivo studies are needed. - Limited patient-derived lines: primary cells from a small number of patient samples (GB319, BTSC 551, F34). - Specificity not absolute: polymer 447 transfects healthy NSCs and astrocytes as well as tumor cells. - Storage decline: efficacy drops to ~50% after 6 months. - No active targeting ligand: specificity relies solely on polymer structure. - Mechanism not fully explored: uptake, endosomal escape, and intracellular trafficking were not investigated in detail. - Safety/immune response not evaluated: no in vivo toxicity or immunogenicity assessment.