Synthesis and application of poly(ethylene glycol)-co-poly(β-amino ester) copolymers for small cell lung cancer gene therapy

Purpose: Small cell lung cancer (SCLC) has high recurrence and mortality, and new therapies are needed. PBAE/DNA polyplexes are effective nonviral vectors but aggregate in physiological and acidic buffers, limiting therapeutic utility. PEGylation can improve stability but often reduces cellular uptake and transfection—the “PEG dilemma.”

Hypothesis: Blending PEGylated PBAEs with non-PEGylated end-capped PBAEs (ePBAEs) will overcome the PEG dilemma: the blend will maintain colloidal stability and low cytotoxicity while retaining efficient cellular uptake and transfection. When delivering HSV-tk plasmid followed by ganciclovir, the optimized PEG-PBAE polyplex will selectively kill SCLC cells more than healthy lung fibroblasts.

Aims: Synthesize and characterize PEG-PBAE copolymers and ePBAEs. - Formulate a combinatorial library of PEG-PBAE/ePBAE polyplexes and identify stable formulations. - Screen uptake, transfection, and cytotoxicity in hard-to-transfect H446 SCLC cells. - Evaluate therapeutic activity of HSV-tk/ganciclovir suicide gene therapy in H446 vs. healthy IMR-90 lung fibroblasts.

Delivery system: Polymer: PBAEs based on B4S4, B4S5, and B5S5 base polymers, end-capped with E4, E6, or E7. PEG-PBAEs synthesized by thiol-ene Michael addition of methoxy PEG-thiol (0.8 or 5 kDa) to B4S4 base polymer (4 or 13 kDa), using E7 as catalyst. - Nanoparticle: PEG-PBAE/ePBAE blend polyplexes formed by electrostatic self-assembly with plasmid DNA. - Payload: pEGFP, Cy3-labeled pEGFP, and pHSV-tk plasmid. - Formulation variables: ePBAE:PEG-PBAE mass ratios 1:2, 1:1, 2:1; total polymer:DNA 30, 60, 90 w/w. - Targeting ligand: None. - Lead formulation: ePBAE 457 blended with PEG-PBAE 5k-13k at 1:1 w/w, total polymer:DNA 30 w/w.

Approach: In vitro only. Human SCLC H446 cells and healthy human lung fibroblast IMR-90 cells. - 96-well plates, 15,000 cells/well. Polyplexes incubated 4 h in serum-containing medium, then washed and replaced with fresh medium. - Uptake assessed at 4 h; transfection at 48 h; viability by MTS at 24 h. - HSV-tk/ganciclovir: 4 h polyplex incubation, 24 h fresh medium, then 10 or 20 µg/mL ganciclovir, repeated after 48 h; cell death measured 24 h later. - Controls: untreated cells and PEI 2 w/w. n = 4 for most assays. - No in vivo model.

Key methods: ¹H NMR for polymer synthesis confirmation. - Nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) for size and 24 h stability. - Zeta potential for surface charge. - Flow cytometry for Cy3-DNA uptake and EGFP transfection. - Fluorescence microscopy for qualitative transfection. - MTS assay for metabolic activity/cytotoxicity. - Statistical analysis: one-way ANOVA with Dunnett post-test; Student’s t-test for H446 vs. IMR-90 killing.

Key results: Non-PEGylated PBAE polyplexes aggregated over 24 h in acidic and physiological buffers. - Of 36 PEG-PBAE formulations, four maintained small size; lead formulation 457 + 5k-13k 1:1 30 w/w stayed below 300 nm over 24 h in PBS. - Lead PEG-PBAE formulation transfected ~40% of H446 cells; cellular uptake was ~30%. - ePBAE 457 alone at 60 w/w transfected ~73% of H446 cells but caused ~50% cytotoxicity in healthy IMR-90 cells. - With HSV-tk + ganciclovir, PEG-PBAE polyplex killed 35% of H446 cancer cells vs. 15% of IMR-90 healthy fibroblasts (p < 0.01). - Increasing ganciclovir from 10 to 20 µg/mL had negligible additional effect, indicating HSV-tk expression was limiting. - PEG-PBAE polyplexes prevented polymer-mediated cytotoxicity in IMR-90 cells, with near 100% viability in pEGFP and pHSV-tk + 0 µg/mL ganciclovir controls.

Interpretation: PEG-PBAE blended polyplexes can overcome the PEG dilemma by maintaining colloidal stability without compromising transfection. They enable safe, efficient delivery of HSV-tk/ganciclovir suicide gene therapy to SCLC cells with apparent cancer-cell selectivity, supporting further development of PEGylated PBAE nanoparticles for SCLC gene therapy.

Limitations: In vitro only: no animal or in vivo validation. - No active targeting ligand: cancer selectivity relies on intrinsic polymer/cell differences and higher proliferation of cancer cells. - Limited cell panel: only H446 SCLC and IMR-90 fibroblasts were tested. - Trade-off in efficacy: PEG-PBAE transfection (~40%) was lower than non-PEGylated 457 (~73%). - Short stability window: particle stability assessed over 24 h, not long-term storage or systemic circulation. - No pharmacokinetics, biodistribution, or immune/safety evaluation. - Ganciclovir bystander effect and long-term therapeutic durability were not assessed.