Purpose: DNA-based therapeutics require an intracellular delivery system to be effective. For diseases like brain cancer—where local access to residual tumor is often available following surgical resection—an implantable device that enables both controlled release and intracellular delivery of DNA could open new complementary treatment avenues. While cationic poly(β-amino ester)s (PBAEs) have shown superior transfection efficiency and low toxicity in solution, they had not been previously used for DNA delivery from a substrate surface in a layer-by-layer (LBL) system.
Hypothesis: Combining traditional LBL assembly with electrospinning—specifically, using PBAE 447 (1-(3-aminopropyl)-4-methylpiperazine end-capped poly(1,4-butanediol diacrylate-co-4-amino-1-butanol)) for its transfection ability and PBAE 4P (poly(1,4-butanediol diacrylate-co-4,4'-trimethylenedipiperidine)) for its structural properties—can create thin films that enable both controlled release and intracellular delivery of DNA from a surface.
Aims: Primary aim: Develop biodegradable polyelectrolyte multilayer films for sustained DNA release and high exogenous gene expression in primary human glioblastoma cells. - Secondary aims: - Evaluate DNA release kinetics and DNA loading efficiency of LBL films with varying numbers of bilayers. - Assess the transfection efficacy of DNA released from films (both pre-released and direct from surface). - Explore electrospinning of PBAE 447 alone and in combination with 4P/PCL blends. - Combine electrospun fibers with PBAE 447/DNA multilayers to enhance DNA release and intracellular delivery. - Assess cytotoxicity of PBAE 447 alone and in complex with DNA.
Delivery system:
Component: Cationic polymer (transfection agent); Details: PBAE 447: 1-(3-aminopropyl)-4-methylpiperazine end-capped poly(1,4-butanediol diacrylate-co-4-amino-1-butanol)
Component: Structural polymer (for electrospinning); Details: PBAE 4P: poly(1,4-butanediol diacrylate-co-4,4'-trimethylenedipiperidine)
Component: Supporting polymer; Details: Poly(ε-caprolactone) (PCL), 70–90 kDa
Component: Payload; Details: pEGFP-N1 plasmid DNA (encoding green fluorescent protein)
Component: Delivery platform; Details: (1) LBL multilayer films via iterative dipping; (2) electrospun nanofibrous scaffolds; (3) combination of electrospun fibers + LBL multilayers
Component: Substrate; Details: Glass coverslips (15 mm diameter)
Component: Polymer:DNA ratio; Details: 60:1 (w/w) for transfection; 10:1 or above for complete DNA complexation
Component: Bottom layer; Details: PBAE 447 (20 or 50 mg/mL in DMSO) or PDL (poly-D-lysine, 20 or 5 mg/mL)
Component: DNA concentration; Details: 0.75 mg/mL in 100 mM sodium acetate buffer
Component: Number of bilayers tested; Details: 3, 4, 6, 9, and 12
Approach: Model system: Primary human glioblastoma (GB) cell line GB 319, derived from intraoperative patient samples. - Culture conditions: DMEM/F-12 (1:1), 10% FBS, 1% antibiotic-antimycotic; seeded at 1.5 × 10⁵ cells/mL (7.5 × 10⁴ cells per LBL film) or 1.5 × 10⁴ cells/well in 96-well plates. - In vitro only: No in vivo animal studies were performed. - Controls: Fresh DNA (not previously used for coating) as positive control; PDL as alternative bottom layer; no-treatment groups for viability. - Group structure: n = 4 for transfection treatments; multiple time points for release studies (0–24 hr).
Key methods:
Technique: Absorbance at 260 nm (Synergy 2 Multiplate Reader); Purpose: Quantify DNA content in LBL films after each bilayer
Technique: NanoDrop 2000; Purpose: Measure DNA concentration in release supernatants
Technique: Gel electrophoresis (1% agarose, ethidium bromide); Purpose: Assess plasmid integrity before and after layering/release; gel retardation assay for complexation
Technique: Flow cytometry (FlowJo 7); Purpose: Quantify GFP⁺ cells (transfection efficiency)
Technique: Fluorescence microscopy; Purpose: Visualize GFP expression
Technique: MTS assay (CellTiter 96® AQueous One Solution); Purpose: Assess cell viability
Technique: GPC (Waters system, Breeze 2 software); Purpose: Polymer molecular weight analysis
Technique: NMR (Bruker 400, TopSpin 2.0); Purpose: Polymer structure confirmation
Technique: Electrospinning; Purpose: Fabricate nanofibrous scaffolds (7 kV, 8 cm distance, 260 rpm rotating plate)
Technique: Ethanol precipitation; Purpose: Isolate DNA from release samples for transfection
Key results: DNA loading: Films with 447 as the bottom layer had higher DNA loading than PDL-based films. DNA mass increased linearly with number of bilayers (up to ~7 μg for 4 bilayers with 447 vs. ~4 μg with PDL). - DNA release: Sustained release over ~20 hours, correlating with dissolution/degradation of PBAE/DNA film in PBS. Higher number of bilayers → greater cumulative release (up to ~170 μg for 12 bilayers at 20 hr). - Transfection efficiency (pre-released DNA): DNA released from LBL films and complexed with fresh 447 achieved up to ~90% transfection in GB 319 cells—comparable or higher than fresh DNA positive control. The 5–10 hr release window was most effective (>90% transfection even after 4× dilution). - Direct transfection from LBL films: Successful GFP transfection with additional 447 polymer (either as top layer before seeding or added to medium after seeding). Viability: 91 ± 2.1% (polymer added to medium) and 82 ± 3.4% (polymer as top layer). - Cytotoxicity of 447: Dose-dependent decrease in viability for both free polymer and polymer/DNA NPs (60 w/w). Free polymer: r² = 0.8037; polymer/DNA NPs: r² = 0.9015. Viability of cells transfected directly from LBL films fell between the two predicted values. - Electrospun fiber morphology: 447 fibers: ~1 μm diameter; 4P fibers: ~2.5 μm; 4P+PCL (layered): ~6 μm; 4P/PCL (blend): ~2.5 μm. Cell viability on fibers: 447 ~5%, 4P ~30%, 4P+PCL ~40%, 4P/PCL ~85%, no fibers ~100%. - Combined system (electrospun 4P/PCL + 4 bilayers of 447/DNA): Transfection efficiency by released DNA: 0–1 hr: ~75%; 1–5 hr: ~92%; 5–10 hr: ~93%; 10–15 hr: ~60%; 15–20 hr: ~10%; 20–25 hr: ~5%. - Gel electrophoresis: Fresh DNA primarily supercoiled; after deposition and release, an additional band of nicked/linear plasmid appeared but did not significantly impact transfection. Complete complexation by 447 at 10 w/w or above.
Interpretation: The authors claim that PBAE 447 is a promising material for constructing polyelectrolyte multilayers for gene delivery, and that electrospun 4P/PCL fibers combined with 447-based multilayers can enable gene transfer. They state that combining electrospinning and LBL dipping is effective for DNA loading and release over 24 hours, that released DNA performs as well as fresh DNA in transfection studies, and that 447 mediates highly effective gene delivery to human brain cancer cells. They conclude that electrospun PBAEs and their combination with polyelectrolyte PBAE multilayers are promising for DNA release and delivery from a surface, with potential application in local treatment of brain cancer after surgical resection.
Limitations: In vitro only: No in vivo animal studies or tumor models were used to validate efficacy or safety. - Additional polymer required: Successful transfection from LBL films required supplemental 447 polymer (either as a top layer or added to medium), meaning the film alone was insufficient for transfection. - DNA nicking: Gel electrophoresis showed an additional band of nicked/linear plasmid after deposition and release from films, indicating some DNA damage during processing, though transfection was not significantly impaired. - Short release window: DNA release and transfection were evaluated over only 24 hours; longer-term release kinetics and stability were not assessed. - Room-temperature instability: 447 substrates were viscous liquids at room temperature and easily deformed upon addition of aqueous media, limiting their standalone use as electrospun scaffolds. - No mechanistic studies: Endosomal escape or intracellular trafficking mechanisms were not directly investigated in this system. - Limited cell types: Only one primary glioblastoma cell line (GB 319) was used; generalizability to other cell types or cancer models remains to be established.
Report prepared from the Just Accepted Manuscript. Note that this version may differ from the final published article.
