Transfection is the technique of introducing foreign nucleic acid, such as plasmid DNA or RNA, into eukaryotic cells to modify their function or study gene expression. Non-viral methods are commonly employed for this purpose, and Polyethylenimine (PEI) stands out as a highly effective, synthetic delivery vector. PEI is a cationic polymer, meaning it carries a positive charge, which allows it to naturally associate with the negatively charged genetic material. This interaction makes PEI a widely adopted, versatile, and cost-effective reagent for transient gene delivery in numerous cell lines.
The Mechanism of PEI Transfection
Polyethylenimine’s function as a gene delivery agent is entirely dependent on its positive charge density, which facilitates the creation of a specialized nanoparticle. The PEI polymer, rich in amine groups, readily interacts with the phosphate backbone of DNA or RNA, which carries a negative charge. This electrostatic attraction causes the nucleic acid to condense tightly into a compact, nanometer-sized structure known as a polyplex. The formation of this condensed particle shields the genetic cargo from degradation by cellular enzymes while maintaining a net positive surface charge, which promotes uptake by the cell.
Once the polyplexes are introduced to the cell culture, their positive surface charge encourages binding to the negatively charged components on the cell membrane, initiating uptake primarily through endocytosis. The step for successful transfection involves the polyplexes escaping the endosome, the cellular vesicle that encapsulates them upon entry. Without this escape, the cargo would be trafficked to the lysosome and destroyed by hydrolytic enzymes.
PEI achieves endosomal escape through the “proton sponge effect.” As the endosome matures, proton pumps (V-ATPases) actively acidify the internal environment by pumping protons (H+) inside. The amine groups within the PEI polymer act as a buffer, absorbing these incoming protons, which causes the cell to pump even more protons into the endosome in an attempt to reach the target acidic pH.
To maintain electrochemical neutrality, negatively charged counter-ions (such as chloride) are drawn into the endosome along with the protons. The resulting high concentration of ions triggers a significant influx of water due to osmotic pressure. The swelling and eventual rupture of the endosomal membrane releases the PEI-DNA polyplexes into the cytoplasm, allowing the genetic material to travel to the nucleus for gene expression.
Essential Reagent Preparation and Cell Handling
Before initiating the transfection procedure, meticulous preparation of both the PEI stock solution and the target cells is necessary. The PEI polymer (typically 25 kDa linear PEI) must be dissolved in high-quality, endotoxin-free water to a stock concentration, often 1 mg/mL. Since PEI is a weak base, the solution is highly viscous and requires heating and the addition of hydrochloric acid (HCl) to fully dissolve and adjust the pH to a neutral range, typically around 7.0.
Once dissolved and pH-adjusted, the stock solution must be filter-sterilized using a 0.22 µm syringe filter to eliminate microbial contamination and remove insoluble aggregates. The working stock is then aliquoted and stored, often at -20°C, to maintain stability and prevent repeated freeze-thaw cycles. The plasmid DNA or RNA to be delivered must also be of high purity and concentration, often requiring an endotoxin-free preparation kit to minimize cellular toxicity.
The recipient cells must be in a state of optimal health and activity. For adherent cells, the ideal confluency at the time of transfection is between 60% and 80%, as actively dividing cells are more receptive to nucleic acid uptake. Cells should be passaged at least 24 hours prior to transfection to allow recovery and ensure they are in the logarithmic growth phase. Using low-passage cells (fewer than 30 passages) helps maintain consistent transfection efficiency.
Step-by-Step Transfection Procedure
The core of the PEI transfection method lies in the precise, sequential formation of the polyplex nanoparticles. To begin the procedure, the nucleic acid and the PEI stock solution must first be diluted separately into a serum-free, reduced-serum, or plain buffer, such as Opti-MEM. A total volume is prepared for each component, ensuring the final ratio of PEI mass to DNA mass is appropriate for the cell line being used, often falling in the range of 3:1 to 6:1 (PEI:DNA, w/w).
The next step involves the controlled mixing of the two diluted solutions, which is when the polyplexes form. The PEI solution must always be added to the DNA solution, rather than the reverse, to ensure the positively charged polymer completely encapsulates the negatively charged DNA and prevents the formation of large, ineffective aggregates. This mixing should be done quickly and gently, often by flicking the tube or using a brief vortex pulse, to encourage the formation of small, uniform nanoparticles.
Following the mixing, the solution must be incubated at room temperature for a specific period, typically ranging from 5 to 20 minutes, to allow the electrostatic complexation reaction to reach completion. This incubation time dictates the size and stability of the resulting polyplexes, which directly affects cellular uptake efficiency. After the complex formation period, the completed polyplex solution is ready to be added to the cells.
The polyplex solution is then added dropwise to the cell culture media, ensuring it is distributed evenly across the surface of the cultured cells without disturbing the cell monolayer. The cells are typically incubated with the polyplexes for a defined period, ranging from four to eight hours, before a media change is performed. Replacing the transfection media with fresh, complete growth media limits the cell’s exposure to potentially toxic free PEI, improving cell viability while allowing the gene expression process to continue.
Troubleshooting and Optimization Factors
Achieving high transfection efficiency with minimal cellular toxicity requires careful optimization of several interdependent factors. The most significant variable is the Nitrogen to Phosphate (N:P) ratio, which is the molar ratio of the protonatable nitrogen atoms in the PEI polymer to the anionic phosphate groups in the nucleic acid. This ratio dictates the net positive charge of the polyplexes and is directly proportional to transfection efficiency, but also to cytotoxicity.
A higher N:P ratio means a larger excess of positively charged PEI, which increases the likelihood of polyplex formation and enhances membrane binding and endosomal escape. However, excess free PEI molecules that do not bind to the DNA can interact nonspecifically with the cell membrane, leading to increased cell death. Optimization involves titrating the PEI amount against a fixed quantity of DNA to find a specific ratio (e.g., N:P of 12 or mass ratio of 4:1) that maximizes gene expression while maintaining cell viability above 80%.
The influence of serum and the choice of cell line are also important considerations. Complex formation often occurs in a serum-free buffer, like Opti-MEM, because proteins in fetal bovine serum (FBS) can bind to the cationic PEI. This binding interferes with polyplex formation and reduces transfection efficiency by competing for PEI binding sites.
The cell line itself is a major factor; for instance, HEK293 cells are highly transfectable, while primary or stem cells often require significantly higher PEI concentrations and different N:P ratios. Linear 25 kDa PEI is generally the preferred polymer choice due to its superior transfection efficiency compared to lower molecular weight or branched forms. Managing the post-transfection incubation time is also a form of toxicity management; shortening the exposure time to as little as four hours before a media change can reduce cell death in sensitive cell types.