Nanoinjection

Nanoinjection is the process of using a microscopic lance and electrical forces to deliver DNA to a cell. It is claimed to be more effective than microinjection because the lance used is ten times smaller than a micropipette and the method uses no fluid. The nanoinjector mechanism is operated while submerged in a pH buffered solution. Then, a positive electrical charge is applied to the lance, which accumulates negatively charged DNA on its surface. The nanoinjector mechanism then penetrates the zygotic membranes, and a negative charge is applied to the lance, releasing the accumulated DNA within the cell. The lance is required to maintain a constant elevation on both entry and exit of the cell.

Nanoinjection results in a long-term cell viability of 92% following the electrophoretic injection process with a 100 nm diameter nanopipette, the typical diameter of nanoinjection pipet.

Single cell transfections are used to virtually transfer any type of mammalian cell into another using a syringe which creates an entry for DNA to be released. A nano needle is used as a mechanical vector for plasmid DNA. The method can be improved further with Atomic Force Microscopy or AFM. In order to avoid causing permanent damage to the cell or provoke cellular leaking of intracellular fluid, AFM is a tool of choice, as it allows for precise positioning of the DNA, allowing for tip penetration into the cytosol, which is critical for viable DNA transfer into the cell.

Reasons to use nanoinjection include the insertion of genetic material into the genome of a zygote. This method is a critical step in understanding and developing gene functions.

Nanoinjection is also used to genetically modify animals to aid in the research of cancer, Alzheimer’s disease, and diabetes.

Fabrication
The lance is made using the polyMUMPs fabrication technology. It creates a gold layer, and two structural layers that are 2.0 and 1.5 μm thick respectively. It is a simple process, which makes it good as a platform to prototype polysilicon MEMS devices at a low commercial cost of fabrication. The lance has a solid, tapered body, that is 2 μm thick, with a tip width of 150 nm. The taper is set at 7.9°, coming to a maximum width of 11 μm. Two highly folded electrical connections provide an electrical path between the lance and two equivalent bond pads, with a gold wire connecting one of the bond pads to an integrated circuit chip carrier’s pin. The carrier is then placed into a custom built electrical socket.

In the situation of fertilizing eggs, the lance is incorporated into a kinematic mechanism consisting of a change-point parallel-guiding six-bar mechanism and a compliant parallel-guiding folded-beam suspension.

Electrophoretic Injection
Electrophoretic injection remains the most common form of nanoinjection. Just as with the other methods, a lance ten times smaller than that of microinjection is used. Preparing the lance for injection, a positive charge is applied, attracting the negatively-charged DNA to its tip. After the lance has reached a desired depth within the cell, the charge is reversed, repelling the DNA into the cell. The typical injection voltages are ±20 V, but can be as low as 50-100 mV.

Diffusion
A manual force is applied to a center fixture of the injection device, moving the lances through cell membranes and into the cytoplasm or nucleus of adhered cells. The magnitude of the force is measured using a force plate on a small number of injections to obtain an estimate of the manual force. The force plate is arranged to measure the force actually applied to the injection chip (that is, not including the stiffness of the support spring). After holding the force for five seconds, the force is released and the injection device is removed from the cell. The diffusion protocol presented data for comparison against other variations in the injection process.

Applications
By delivering certain particles into cells, diseases can be treated or even cured. Gene therapy is possibly the most common field of foreign material delivery into cells and has great implications for curing human genetic diseases.

For example, two monkeys colorblind from birth were given gene therapy treatment in a recent experiment. As a result of gene therapy, both animals had their color vision restored with no apparent side effects. Traditionally, gene therapy has been divided into two categories: biological (viral) vectors and chemical or physical (nonviral) approaches. Although viral vectors are currently the most effective approach to delivering DNA into cells, they have certain limitations, including immunogenicity, toxicity, and limited capacity to carry DNA.

One factor critical to successful gene therapy is the development of efficient delivery systems. Although advances in gene transfer technology, including viral and non-viral vectors, have been made, an ideal vector system has not yet been constructed.

Alternatives
Microinjection is the predecessor to nanoinjection. Still used in biological research, microinjection is useful in the examination of non-living cells or in cases where cell viability does not matter. Using a glass pipette 0.5-1.0 micrometers in diameter, the cell has its membrane damaged upon puncture. As opposed to nanoinjection, microinjection uses DNA-filled liquid driven into the cell under pressure. Depending on factors such as the skill of the operator, survival rates of cells undergoing this procedure can be as high as 56% or as low as 9%.

Other methods exist that target groups of cells, such as electroporation. These methods are incapable of targeting specific cells, and are therefore not usable where efficiency and cell viability are a concern.