Purpose: Suspension lymphoma/leukemia cells are notoriously hard to transfect with non-viral vectors. Existing methods such as Lipofectamine give poor plasmid DNA delivery in these cells, and viral methods raise safety concerns. A simple, efficient, biodegradable cationic polymer nanocomplex is needed for gene delivery to hard-to-transfect hematopoietic cancer cells.
Hypothesis: If poly(β-amino ester) (PBAE) polymers are formulated with plasmid DNA into a nanocomplex, then the nanocomplex will deliver plasmid DNA into hard-to-transfect lymphoma/leukemia cells. Polybrene pretreatment will further enhance transfection efficiency. The nanocomplex will be stable at physiological pH but dissociate under acidic pH to release DNA intracellularly.
Aims: Synthesize and fractionate PBAE polymers into >7 kDa and <7 kDa fractions. - Formulate and characterize PBAE–plasmid DNA nanocomplexes. - Evaluate pH-dependent stability and dissociation of nanocomplexes. - Test transfection efficiency in Jurkat, K562, and Karpas 299 lymphoma/leukemia cells. - Optimize polybrene concentration, pretreatment time, polymer:DNA ratio, and DNA dose. - Assess cytotoxicity and effects on cell-surface biomarker expression and proliferation. - Compare performance with Lipofectamine and PEI.
Delivery system:
Component: Polymer class; Details: Poly(β-amino ester) (PBAE)
Component: Synthesis; Details: 5-amino-1-pentanol + 1,4-butanediol diacrylate at 1.2:1 amine/diacrylate ratio, 95 °C, 24 h
Component: Fractionation; Details: Dialysis with 7 kDa MWCO membrane into >7 kDa and <7 kDa fractions
Component: Payload; Details: pMax-GFP plasmid DNA (~3.5 kbp) encoding green fluorescent protein
Component: Nanocomplex formation; Details: Electrostatic self-assembly at pH 5.2; polymer:DNA mass ratio typically 40:1
Component: Size / stability; Details: ~200 nm at pH 7.4; stable for at least 12 h; dissociates/enlarges at pH <7
Component: Targeting ligand; Details: None
Component: Key feature; Details: Biodegradable PBAE; pH-triggered dissociation; polybrene pretreatment enhances transfection
Approach: In vitro only. No in vivo animal studies. - Cell lines: Jurkat (human T-cell lymphoma/leukemia), K562 (human myeloid leukemia), Karpas 299 (human anaplastic large cell lymphoma). - Transfection protocol: Cells pretreated with polybrene (0–8 µg/mL) for 5 min, then incubated with PBAE–plasmid nanocomplex for 4 h in serum-free RPMI-1640, then cultured in complete medium. - Analysis: GFP expression by flow cytometry and fluorescence microscopy at 48 h. - Optimization: Polybrene concentration (0–8 µg/mL), pretreatment time (5–120 min), polymer:DNA ratio (10:1–60:1), DNA dose (0.25–2.0 µg/mL). - Controls: Plasmid DNA alone; Lipofectamine–plasmid; PEI; polybrene–plasmid complexes. - Cytotoxicity: Trypan blue and MTT assays; surface biomarkers CD2, CD3, CD5, CD45; proliferation rate.
Key methods: Dynamic light scattering (DLS): Nanocomplex size and pH-dependent stability. - Transmission electron microscopy (TEM): Nanocomplex morphology at pH 7.4 and pH 5.0. - Flow cytometry: GFP-positive cells; surface biomarker expression. - Fluorescence microscopy: Confirmation of GFP expression. - Trypan blue and MTT assays: Cell viability and proliferation. - Statistics: At least three repeats; similar results.
Key results: Nanocomplex properties: ~200 nm at pH 7.4, stable for 12 h. At pH <7, nanocomplexes rapidly dissociated and enlarged, consistent with proton-sponge–type release. - Transfection without polybrene: PBAE >7 kDa nanocomplex gave 3% GFP-positive Jurkat cells, similar to Lipofectamine (2%). - Transfection with polybrene: Polybrene pretreatment (6 µg/mL, 5 min) increased transfection to 32% in Jurkat, 19% in K562, and 19% in Karpas 299. Lipofectamine gave 2–4% in these cells. - Optimization: Optimal polybrene = 6 µg/mL; optimal pretreatment = 5 min; optimal polymer:DNA = 40:1; optimal DNA dose = 1 µg/mL gave 28% GFP-positive cells. - Polybrene–plasmid alone: Little or no transfection (0.03–0.20%). - Cytotoxicity: PBAE >7 kDa had much lower toxicity than PEI. No effect on surface biomarkers CD2, CD3, CD5, CD45 or on proliferation rate.
Interpretation: The authors claim that a simple PBAE–plasmid nanocomplex enables efficient gene delivery into hard-to-transfect suspension lymphoma/leukemia cells, especially with polybrene pretreatment. The nanocomplex is biodegradable, stable at physiological pH, dissociates at acidic pH, and has minimal cytotoxicity. This approach may be useful for in vitro gene manipulation and potentially for in vivo gene delivery, including to other hard-to-transfect cells such as stem cells and hematopoietic cells.
Limitations: In vitro only: No in vivo validation, biodistribution, or therapeutic efficacy. - Polybrene required for high efficiency: Without polybrene, transfection was low (~3%). - Mechanism unclear: How polybrene enhances PBAE nanocomplex transfection was not determined. - Limited cell panel: Three suspension lymphoma/leukemia cell lines only. - No targeting ligand: Delivery relies on nonspecific electrostatic interactions. - No long-term expression data: GFP measured at 48 h only. - No comparison with electroporation or viral vectors. - No therapeutic gene or disease model. - Cytotoxicity assessed in vitro only; no systemic toxicity or immune response data. - DOI/link not provided in the supplied file.
