Linearized polyethyleneimine

Linearized polyethyleneimine (PEI) is a water-soluble, high cationic charge density synthetic polymer. The linear PEI with CAS number 26913-06-4 is a high-quality reagent designed specifically for gene delivery and cell transfection, with common molecular weights of 25000 Da (PEI 25K) or 40000 Da (PEI 40K). As one of the "gold standards" in the field of non viral vectors, it is widely used in basic life science research, recombinant protein production, antibody process development, and gene therapy virus vector preparation.

1. Chemical and physical properties

Nature description

Chemical name: Polyvinylimine, linear type

CAS number 26913-06-4

The molecular formula (C ₂ H ₅ N) ₙ, n is approximately 580-930 (corresponding to 25k-40k Da)

Appearance: White to off white solid or freeze-dried powder, or sterile clear solution (depending on supplier)

Soluble in water, methanol, and ethanol, insoluble in acetone and ether

PH (1% aqueous solution) about 10-11 (alkaline)

The charge density is extremely high, with each monomer unit containing about one protonated nitrogen atom

The main chain of linear PEI is a straight chain structure, which is different from branched PEI. All protonated nitrogen atoms are located on the backbone chain, resulting in more regular binding with DNA, more stable complexes, and relatively lower cytotoxicity.

2. Working principle

The core function of linear PEI is to serve as a cationic polymer transfection reagent, efficiently delivering nucleic acids (plasmid DNA, mRNA, siRNA, etc.) into eukaryotic cells.

Mechanism steps:

Electrostatic recombination: PEI with a strong positive charge spontaneously forms nanoscale "PEI nucleic acid complexes" (plexes) with a negatively charged nucleic acid phosphate backbone through electrostatic interactions.

Cellular uptake: Excess positive charges on the surface of the complex bind to negatively charged proteoglycans on the cell membrane, and enter the cell through endocytosis mediated by clathrin or caveolin.

Introverted body escape: PEI's "proton sponge effect" is its key advantage. In the acidic endosome formed by endocytosis, the amino group of PEI absorbs a large amount of protons, accompanied by the influx of chloride ions, resulting in an increase in osmotic pressure and the rupture of the endosome, releasing nucleic acids into the cytoplasm.

Nuclear delivery: DNA further relies on nuclear localization signals or the disintegration of the nuclear membrane during cell division to enter the nucleus, achieving the expression of exogenous genes.

3. Main purpose

3.1 Transient transfection production of recombinant proteins and antibodies

Application areas: Biopharmaceutical industry, such as the production of monoclonal antibodies, recombinant antigens, cytokines, and enzymes.

Typical hosts: suspended HEK293 cells, CHO cells.

Advantages: It can be directly used in serum-free culture medium, and the target protein can be harvested 4-6 days after transfection. The process is simple and the cost is much lower than that of liposome transfection reagents.

3.2 Preparation of viral vectors

Application: Production of adeno-associated viruses (AAV), lentiviruses, and adenoviruses for gene therapy and vaccine research.

Features: PEI transfection can achieve high packaging efficiency and is easy to linearly scale up to the scale of a bioreactor (several thousand liters).

3.3 Basic scientific research

Gene overexpression and knockdown experiments.

Report gene analysis (luciferase, GFP, etc.).

Research on protein interactions and promoter functions.

Suitable for various adherent cell lines: HEK293, HEK293T, HeLa, HepG2, NIH/3T3, Vero, etc.

4. Product features and advantages

Characteristic Description

The high transfection efficiency can reach 80% to 95% in HEK293 and other cells, comparable to top commercial liposome reagents

The low cytotoxicity linear structure significantly reduces the toxicity compared to branched PEI, and under optimized conditions, the cell survival rate is>90%

Serum compatibility can be operated in complete culture medium containing 10% fetal bovine serum (FBS) without changing the culture medium

Extremely cost-effective compared to liposomes, the cost per batch is reduced by 1-2 orders of magnitude, making it particularly suitable for large-scale production

The process scalability can be used from 96 well plates to 1000 L bioreactors, and the transfection conditions are linearly adjustable

The chemical definition is clear and there are no animal derived ingredients, which meets the quality control requirements of biopharmaceuticals

5. Typical usage plan (taking PEI 25K transfection of HEK293 adherent cells as an example)

The following is a reference process, and specific conditions need to be optimized for cell lines and nucleic acids.

Solution preparation: Prepare 1 mg/mL PEI storage solution with sterile water for injection (WFI), adjust the pH to 6.8-7.2, sterilize with 0.22 µ m filter membrane, and store at -20 ℃ for a long time after packaging.

Preparation of DNA-PEI complex:

Dilute DNA and PEI separately using serum-free culture medium (such as Opti MEM).

Recommended quality ratio: DNA: PEI=1:3 (e.g. 1 µ g DNA+3 µ g PEI).

Mixing: Quickly add the diluted PEI solution to the diluted DNA solution, vortex and mix well, and let it stand at room temperature for 15-20 minutes.

Transfection: Drop the complex directly into cells containing complete culture medium (including serum) and gently shake well.

Detection: Gene expression is detected 48-72 hours after transfection.

For large-scale transfection of suspended cells, serum-free medium should be used, and the cell density should be controlled at 2-3 × 10 ⁶ cells/mL, with a DNA dosage of 0.5-1 µ g/mL in the culture volume.

6. Storage and stability

Solid powder: can be stored for 2 years at room temperature (15-25 ℃) when dry, away from light.

Aseptic solution (1 mg/mL, pH 7.0): can be stably stored for 6 months at 4 ℃; -It can be stored at 20 ℃ for more than 1 year to avoid repeated freezing and thawing.

Working fluid (diluted): It is recommended to use immediately and should not be left for more than 1 hour.

7. Precautions

PEI has strong positive charge and should not be directly mixed with negatively charged polymers such as heparin, dextran sulfate, and some proteins.

Wear gloves and goggles during operation to prevent inhalation of dust or contact with skin and mucous membranes (which can be irritating).

Do not dilute PEI storage solution with bicarbonate or phosphate buffer solution, as precipitation may occur. Recommend using 150 mM NaCl or pure water.

The optimal DNA/PEI ratio varies greatly among different cell types (commonly 1:1.5 to 1:4), and it is recommended to perform gradient optimization before formal experiments.