Non-viral Vectors for Gene Transfection: Polyethyleneimine

Product Manager: Harrison Michael

Polyethyleneimine (PEI) is a widely used non-viral vector for gene transfection. Its cationic nature enables it to effectively bind DNA and facilitate its entry into cells. However, the efficiency of PEI-mediated transfection is influenced by a variety of factors, including molecular weight, N/P ratio (the molar ratio of nitrogen atoms in PEI to phosphate groups in DNA), cell type, culture conditions, and the physicochemical properties of the transfection complexes. This article will explore in detail the impact of these key parameters on PEI-mediated gene delivery and summarize optimization strategies to enhance transfection efficiency while reducing cytotoxicity.

1. Basic Mechanism of PEI-mediated Gene Transfection

As the core carrier of a gene delivery system, PEI undergoes several key steps during the transfection process:

1.1 Formation of PEI-DNA Complexes

PEI binds to negatively charged DNA through electrostatic interactions, forming nanoscale complexes known as polyplexes. The ideal characteristics of these complexes include:

·Particle size: Controlled within 100-200 nm, which ensures stability and facilitates cellular uptake.

·Zeta potential: Typically ranging from +20 to +40 mV, which enhances the interaction between the complexes and the cell membrane, thereby improving transfection efficiency.

The formation of PEI-DNA complexes is influenced by the N/P ratio. An appropriate N/P ratio can enhance the stability of the DNA complexes while reducing cytotoxicity caused by excess PEI.

1.2 Cellular Uptake and Endocytosis

PEI-DNA complexes primarily enter cells through endocytosis, which includes the following pathways:

·Macropinocytosis: Suitable for larger PEI complexes (>200 nm).

·Clathrin-mediated endocytosis: Suitable for complexes ranging from 50 to 200 nm, which is most favorable for DNA delivery.

The efficiency of cellular uptake is influenced by the size, charge, and cell type of the PEI-DNA complexes. Optimizing these parameters can improve transfection efficiency.

1.3 Endosomal Escape and Nuclear Delivery

Successful endosomal escape is a critical step in gene transfection. PEI exhibits the "proton sponge effect," which means:

·During the acidification of endosomes, PEI absorbs protons, causing chloride ions and water molecules to enter the endosomes, leading to their swelling and rupture.

·The ruptured endosomes release PEI-DNA complexes into the cytoplasm, promoting gene expression.

For non-dividing cells, the entry of DNA into the nucleus is a limiting step. This can be enhanced by adding nuclear localization signals (NLS) or optimizing cell culture conditions.

2. Key Factors Affecting PEI-mediated Gene Transfection Efficiency

2.1 Influence of PEI Molecular Weight

Different molecular weights of PEI affect DNA complexation ability, transfection efficiency, and cytotoxicity:

·Low molecular weight PEI (<10 kDa): Low cytotoxicity, but weak DNA binding ability and lower transfection efficiency.

·Medium molecular weight PEI (10-25 kDa): Stronger DNA complexation ability while maintaining moderate cytotoxicity, widely used for gene delivery.

·High molecular weight PEI (>50 kDa): Strong DNA binding ability, but prone to forming larger complexes, leading to significantly increased cytotoxicity.

2.2 Optimization of N/P Ratio

The N/P ratio determines the stability of PEI-DNA complexes and transfection efficiency:

·N/P<4:1: Complexes are unstable, DNA may dissociate, resulting in low transfection efficiency.

·N/P 6:1-10:1: Formation of uniform and stable nanocomplexes, optimal for transfection efficiency.

·N/P >12:1: Excess PEI leads to increased cytotoxicity, affecting cell viability.

2.3 Influence of Cell Density

Cell density affects the adsorption and uptake of PEI-DNA complexes:

·Below 30%: Poor cell proliferation status, low transfection efficiency.

·50%-70%: Optimal transfection efficiency with good cell condition.

·Above 90%: Cell-to-cell contact may interfere with transfection, potentially reducing gene expression.

2.4 Influence of Cell Culture Medium

The composition of the culture medium also has a significant impact on transfection efficiency:

·Serum-free medium: Should be used 4-6 hours before transfection to enhance the uptake of DNA complexes.

·Complete medium: Returned after 6 hours to reduce PEI cytotoxicity and improve cell viability.

Additionally, supplementing with Hepes buffer can enhance cell tolerance to PEI complexes and increase transfection success rates.

3. Optimization Strategies for PEI-mediated Gene Transfection

3.1 Selection of Linear vs. Branched PEI

·Linear PEI (L-PEI): Lower cytotoxicity, higher stability, suitable for both in vitro and in vivo gene delivery.

·Branched PEI (B-PEI): Higher transfection efficiency, but relatively higher cytotoxicity.

3.2 Complex Modification Strategies

To further improve the transfection performance of PEI, the following modifications can be made:

·PEGylation: Reduces PEI cytotoxicity and enhances biocompatibility.

·Lipid modification: Enhances cell membrane penetration ability and improves transfection efficiency.

·Surface ligand modification (e.g., RGD peptides, chitosan): Enhances targeting to specific cell types and improves transfection outcomes.

3.3 Combined Delivery Strategies

·PEI-liposome complexes: Combines the efficient cellular uptake of liposomes with the gene delivery advantages of PEI to improve transfection efficiency and reduce toxicity.

·Electroporation combined with PEI transfection: Enhances the ability of DNA to enter the nucleus, suitable for difficult-to-transfect cells.

4. Applications of PEI in Gene Therapy

PEI-mediated gene transfection is not only applicable to basic research but also holds significant value in gene therapy and biomedical applications:

·CAR-T cell gene editing: By delivering CRISPR-Cas9 components via PEI, T cells can be effectively modified to improve the efficiency of cancer immunotherapy.

·Vaccine development: PEI serves as a DNA vaccine delivery system, enhancing antigen expression levels and boosting immune responses.

·Protein expression: Used for transient expression of recombinant proteins in mammalian cells, such as antibody and enzyme production.。

5. Conclusion

As an efficient non-viral gene carrier, the transfection efficiency of PEI is influenced by multiple factors, including molecular weight, N/P ratio, cell density, and culture conditions. Optimizing these parameters can significantly improve the success rate of gene transfection and reduce cytotoxicity. Through surface modification, combined delivery, and targeting optimization strategies, the application prospects of PEI will be further expanded, providing better solutions for gene therapy, biomedical research, and vaccine development.

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