Physical Gene Transfer or Transfection Methods

While chemical (viral and non-viral) delivery systems have attracted significant attention for delivering nucleic acids to cells, non-viral physical gene transfer methods hold promise for hard-to-transfect cells. In physical gene transfer methods, nucleic acids are directly introduced into the cells.

Chemical approaches utilize synthetic or natural compounds as carriers to deliver genes into cells, some of which may be toxic to cells. In contrast, physical or mechanical techniques have the advantage of preventing the entry of foreign materials, such as chemicals or viruses, into target cells or tissues. They are therefore presented as an alternative approach.

Various types of physical gene delivery methods include: Microinjection, Gene gun, Electroporation, Sonoporation, Optoporation, Liposome-mediated delivery, Hydroporation with hydrodynamic delivery, Virus-mediated delivery, Magnetofection, and Laser irradiation. These methods disrupt the cell membrane by applying physical force and facilitate the transfer of intracellular genes.

Microinjection

One of the most widely used direct transfer methods is microinjection, which was first reported about 30 years ago. In this method, glass micropipettes with excellent tips (less than 0.5 micrometers) are used to inject the desired sample into the nucleus or cytoplasm of adherent cells. Microinjection has several advantages, including high transfer efficiency and a near 100% survival rate, which enables the injection of a wide range of molecules. Furthermore, the injection of entire organelles has also been reported. Additionally, the cell cycle and culture conditions can be altered before, during, or after injection.

Physical gene transfer methods are primarily employed to avoid complications associated with viral and chemical strategies. Specifically, the use of Biolistic gene transfer methods is notable due to their wide application and low toxicity. Biolistic gene transfer has been used for many years, mainly for studying and producing transgenic plants.

However, microinjection also has disadvantages, including being technically challenging and requiring a lengthy training period to achieve reproducible results. Another drawback of classical microinjection methods is that only a limited number of cells (approximately 100 to 200 cells) can be injected in a single experiment. This method also has limitations for specific cell types.

Gene Gun

Micro-particle bombardment, also known as the gene gun or biolistic gene transfer, is considered a popular delivery method due to its reduced dependence on the characteristics of the target cell. Biolistic technology enables efficient transfection under laboratory conditions, even in cells that are difficult to transfect.

This method will aid in designing the gene gun device and bring further improvements to in vitro and in vivo transfection studies, including gene therapy and vaccination. Some cells, tissues, and intracellular organelles, especially plant cells, are impermeable to foreign DNA. In this method, the plasmid is mixed with gold or tungsten particles of various sizes (from nanometers to micrometers), and an electrical discharge or plasma is used to deliver the plasmid/particle complexes to tissues or cell cultures.

The gene gun is part of the biolistic gene transfer method (also known as bioballistic bombardment or particle bombardment). In this method, DNA or RNA is attached to biologically neutral particles such as gold or tungsten. The DNA particle complex is placed above the target tissue under vacuum conditions, and with a high-powered shot, the DNA is effectively delivered into the target cells. Uncoated metal particles can also be shot through a DNA-containing solution surrounding the cell, picking up the genetic material and delivering it into living cells.

The efficiency of gene gun transfer depends on several factors, including cell type, cell growth status, culture medium, type of gene gun ammunition, gene gun settings, and practical experience.

Electroporation

Electroporation is the most commonly used physical gene transfer method due to its speed, low cost, and simplicity. Pulsed electric fields can be employed to introduce DNA into animal, yeast, plant, and bacterial cells. Factors affecting transfection efficiency by electroporation include:

The strength of the applied electric field
The length of the electrical pulse
Temperature
DNA composition
DNA concentration
Ionic composition of the transfection medium

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Electroporation is the application of controlled, pulsed electric fields to biological systems. Upon delivery of the electroporation pulse, pores form on the cell membrane (40 to 120 nanometers in diameter). The target molecules enter the cells before these pores reseal, and after pore closure, they integrate inside the cell. Cell membrane electroporation is used as a tool for injecting drugs and DNA into cells. The plasma membrane of the cell separates the molecular contents of the cytoplasm from the external environment.

Cell membrane electroporation is used as a tool for injecting drugs and DNA into cells. The plasma membrane of the cell separates the molecular contents of the cytoplasm from the external environment. This membrane consists of a phospholipid bilayer, where each phospholipid has a hydrophobic head and a hydrophilic tail. Polar molecules, such as DNA and proteins, cannot freely cross this membrane; however, a strong external electric field can disrupt the lipid matrix, allowing these molecules to pass through. This disruption leads to increased membrane conductivity and diffusive permeability, resulting from the formation of aqueous pores in the membrane.

Electroporation occurs when lipid molecules in the bilayer membrane reorient to form hydrophilic pores. Termination of the external pulse causes the reorientation of lipid molecules and closure of the membrane pores within seconds.

Steps of Gene Transfer Using an Electroporation Device

The steps for gene transfer using an electroporation device are as follows:

Harvest cells in the mid-to-late logarithmic growth phase.
Centrifuge them at 4°C for 5 minutes at 2000 rpm.
Resuspend the cells in growth medium or a specialized electroporation buffer.
Transfer the cell suspension to the electroporation cuvette and add the DNA.
Set the electroporation device settings according to the cell type and apply the pulse.
Transfer the electroporated cells to a culture dish and culture the cells.

In a study on gene transfer to E. coli by electroporation, 80% of the cells received foreign DNA. The amount of DNA required in this method is less than in other methods, and it can be used in vivo for vaccine injection or disease treatment (electrochemotherapy).

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Sonoporation

Sonoporation is the use of ultrasound waves aided by encapsulated microbubbles (EMB), which can temporarily open the cell membrane and deliver macromolecules into cells. Ultrasound increases transfer efficiency in animal cells, laboratory tissues, and protoplasts. However, reports indicate that ultrasound can damage cells and completely disrupt their membrane. The application of this method in DNA delivery leverages the remarkable ability of ultrasound to generate cavitation activity.

Cavitation refers to the formation and/or activity of gas-filled bubbles in a medium exposed to ultrasound waves. Sound waves cause microbubbles to expand and then collapse. When microbubbles burst, microjets are emitted. If the collapsing microbubble is near the cell membrane, these microjets can rupture the cell membrane. The ruptured membrane creates a pore that allows cells to become temporarily more permeable to plasmid DNA (gene transfer).

An image depicting pore formation in the cell membrane by microbubble cavitation, allowing exogenous nucleic acids to diffuse into the cytoplasm passively.
There are two types of cavitation: inertial and non-inertial. Gas bubbles expand under low pressure and contract under high pressure. If the bubble oscillation size remains relatively stable (repeatable over many cycles), it is referred to as stable or non-inertial cavitation. In any case, the cell membrane opens briefly, allowing foreign molecules or DNA to enter the cells.

Advantages of sonoporation include:

In theory, this method can deliver DNA or RNA to any cell type
It is a non-invasive method that does not require direct physical contact
It can potentially be used in vivo.

However, the transfection efficiency of sonoporation has been reported to be relatively low under laboratory and in vivo conditions.

Sound waves cause microbubbles to expand and then collapse. When microbubbles burst, microjets are emitted; if the collapsing microbubble is near the cell membrane, it can rupture the cell membrane. The ruptured cell membrane forms a pore that allows cells to become temporarily more permeable to plasmid DNA (gene transfer).

Laser Irradiation or Optoporation

Lasers are efficient for introducing foreign DNA into cultured cells. Cells undergo changes in plasma membrane permeability or form pores in the membrane at the irradiation site after laser exposure. It has also been reported that a cavity created by a laser beam on a cultured cell repairs within a short time. Different wavelengths are used to create pores in the plasma membrane or alter its permeability through effects such as heat, absorption, photochemical effects, or the generation of reactive oxygen species.

Advantages:

Laser irradiation provides the advantage of targeted transfection, which is not feasible with chemical or viral methods (which affect all cells in the sample population). Consequently, desired cells in a mixed population can be identified and selectively targeted for treatment.
This method also enables direct penetration not only into the cell’s plasma membrane but also into the nuclear membrane. This feature is essential for transfecting slow-growing, non-dividing, or primary cell lines such as neurons.

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Disadvantages:

The transfection rate is low.
Significant increase in cell mortality resulting from the use of high-energy pulses that enhance transfection
This method is limited for clinical use because focusing electrical energy is complex and highly destructive.

Magnetofection

Magnetofection is a transfection method (gene transfer) where nucleic acids or other vectors are associated with magnetic nanoparticles coated with cationic molecules. The resulting molecular complexes are then targeted towards cells and assisted by a suitable magnetic field. The magnetic force accelerates the delivery of nanoparticles, shortens process times, and simultaneously significantly improves transfer rates. The structure and integrity of the membrane remain intact compared to other physical gene transfer methods. Magnetic nanoparticles are made of iron oxide, which is fully biodegradable and non-toxic at recommended doses.

Advantages:

The incubation time required to achieve high transfection is short.
Gene transfer to non-permissive, hard-to-transfect, primary, and non-dividing or slow-dividing cells is possible.
The method is cost-effective.

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Combining magnetic nanoparticles with gene vectors of any type leads to a significant increase in the uptake of these vectors and transfer efficiency. These advantages make magnetofection an ideal tool for ex vivo gene therapy approaches. For in vivo gene and nucleic acid-based therapies, magnetofection may be a suitable choice, particularly when localized treatment is required.

Hydroporation

Hydroporation causes hydrodynamic DNA delivery. In this method, transient pores open in the cell membrane, allowing DNA to enter the cytoplasm, and they typically close within 10 minutes after injection. The combined effect of large volume and high injection speed determines the gene transfer efficiency in this hydrodynamic-based method.

Advantages: It is the most straightforward and most convenient method for in vivo gene transfer. Hydroporation efficiency is also high.

Disadvantages: Besides the liver, its application in other tissues was not thoroughly investigated in the past. Recently, hydroporation has also been used in muscle and the kidney.