Polyethylenimine Linear (PEI, MW 40,000): Mechanism, Benchmarks, and Transfection Best Practices

Executive Summary: Polyethylenimine Linear (PEI, MW 40,000) is a cationic polymer widely validated as a DNA transfection reagent in vitro, achieving 60–80% efficiency in HEK-293 and related cell lines under serum-compatible conditions (ApexBio K1029). It acts by condensing negatively charged DNA molecules into nanoscale complexes, promoting rapid cellular uptake via endocytosis (Polyethylenimine Linear Mechanistic Review). PEI MW 40,000 supports both small-scale and large-scale (up to 100 L) transient gene expression workflows. It is validated in nanoparticle formulations for mRNA delivery and is compatible with serum-containing media. Common misconceptions include overestimating its suitability for in vivo delivery and for all primary cell types (Roach 2024).

Biological Rationale

Linear polyethylenimine (PEI) is a synthetic cationic polymer with a molecular weight of 40,000 Da. It is used extensively as a DNA transfection reagent in molecular biology and cell biology research (ApexBio K1029). Its structure consists of repeating ethylenimine units, providing a high density of protonatable amines. These amines facilitate electrostatic interaction with negatively charged nucleic acids such as DNA and mRNA. The resulting complexes are crucial for efficient delivery into mammalian cells. PEI-based transfection is especially important for transient gene expression studies, recombinant protein production, and functional genomics. The reagent is compatible with a wide range of adherent and suspension cell lines, notably HEK-293, HEK293T, CHO-K1, HepG2, and HeLa cells. It is an essential tool for applications needing high protein yields or rapid gene function analysis (Mechanistic Review).

Mechanism of Action of Polyethylenimine Linear (PEI, MW 40,000)

PEI MW 40,000 operates through charge-based condensation. The positively charged polymer binds to negatively charged phosphate groups on DNA, resulting in nanoscale polyplexes (typically 100–200 nm) (Mechanistic Nuances). These polyplexes exhibit a net positive zeta potential. This feature facilitates interaction with anionic cell surface proteoglycans and glycosaminoglycans. Upon contact, complexes are internalized predominantly via clathrin-mediated endocytosis. PEI’s “proton sponge” effect—arising from its high amine density—promotes osmotic swelling and endosomal rupture, enabling efficient cytosolic release of the DNA cargo. Linear PEI is less cytotoxic and more effective than branched variants at equivalent molecular weights, likely due to superior complexation and release kinetics (Roach 2024, Table 3).

Evidence & Benchmarks

• PEI MW 40,000 achieves 60–80% transfection efficiency in HEK-293 cells in serum-containing media (ApexBio technical documentation, K1029 datasheet).
• Polyplexes formed with PEI (N/P ratio 6–10) exhibit mean hydrodynamic diameters of 120–180 nm and zeta potentials of +20 to +35 mV, stable for at least 4 hours at 37°C (Mechanistic Review).
• Functional delivery of mRNA and plasmid DNA via PEI is validated in kidney-targeted mesoscale nanoparticle systems, maintaining particle size <200 nm and high encapsulation efficiency (>85%) (Roach 2024, Table 2).
• Linear PEI supports scalable transfection from 96-well plates to 100-liter bioreactors without loss of efficiency, enabling both screening and manufacturing workflows (Scalability Review).
• PEI MW 40,000 is serum-compatible, with no significant loss in transfection efficiency in 10% FBS compared to serum-free conditions (Serum Compatibility Analysis).
• Transient protein expression using PEI transfection can yield up to 200 mg/L recombinant protein in HEK293T suspension cultures (48–72 hours post-transfection, 37°C, 5% CO₂) (High-Yield Protocols).

Applications, Limits & Misconceptions

PEI MW 40,000 is widely used for:

• DNA transfection in adherent and suspension mammalian cell lines.
• Transient gene expression for recombinant protein production.
• Functional genomics, reporter assays, and CRISPR/Cas9 delivery.
• Nanoparticle formulation for mRNA and gene delivery platforms (Roach 2024).

However, its use is constrained in several contexts.

Common Pitfalls or Misconceptions

• Not suitable for in vivo systemic delivery without additional formulation: Naked PEI-DNA complexes are rapidly cleared and can induce toxicity in vivo.
• Primary cells and some sensitive lines (e.g., neurons) show reduced viability and efficiency: Cytotoxicity risk is higher compared to liposomal reagents in certain primary cells (Mechanistic Review).
• Optimal N/P ratio is critical: Suboptimal nitrogen/phosphate ratios markedly reduce transfection efficiency and increase toxicity.
• Repeated freeze-thaw cycles degrade performance: Store at -20°C long-term, and 4°C for frequent use to maintain reagent integrity (ApexBio K1029).
• Not all applications permit direct translation of transfection conditions: Protocols may require optimization for cell type, culture format, and expression goals.

This article extends the mechanistic scope of previous analyses by integrating new nanoparticle data and clarifying context-dependent limitations. For a deeper dive on clinical and translational perspectives, see From Mechanism to Medicine: Polyethylenimine Linear (PEI, MW 40,000), which is updated here with direct experimental evidence from kidney-targeted platforms.

Workflow Integration & Parameters

• Working concentration: Supplied at 2.5 mg/mL; typical usage is 1–3 μg PEI per μg DNA, diluted in 150 mM NaCl or PBS (pH 7.4).
• Complex formation: Mix PEI and DNA at room temperature for 10–20 minutes to allow polyplex assembly (N/P ratio 6–10 optimal for most cell lines).
• Cell seeding density: 60–80% confluence at time of transfection yields maximal efficiency and viability.
• Serum compatibility: Medium may contain up to 10% FBS; no media change required post-transfection for most protocols (Serum Compatibility Analysis).
• Scale: Applicable from 96-well (<100 μL) to 100 L bioreactor formats (Scalability Review).
• Storage: -20°C for long-term; 4°C for frequent use; avoid freeze-thaw cycles.
• Quality assurance: Hydrodynamic diameter and zeta potential should be periodically verified by DLS and electrophoretic light scattering (Roach 2024).

For the full product specification and ordering details, refer to the Polyethylenimine Linear (PEI, MW 40,000) product page. This workflow guidance updates and contextualizes the in-depth mechanistic dissection available at Polyethylenimine Linear Mechanistic Review.

Conclusion & Outlook

Polyethylenimine Linear (PEI, MW 40,000) remains a gold-standard DNA transfection reagent for in vitro studies, combining high efficiency, serum compatibility, and scalability. Its mechanism is now validated in advanced nanoparticle and kidney-targeted mRNA delivery platforms (Roach 2024). However, limitations in primary cell types and in vivo use require careful consideration. Ongoing refinements in nanoparticle design and hybrid formulations may further extend its utility for therapeutic gene delivery. For detailed guidance and updates, practitioners should consult both product documentation and emerging application-specific literature.