Image from Chiappini et. al.
Gene therapy, the delivery of genetic material to cells in the form of DNA or RNA, has been explored as a means to treat illnesses. These treatments hinge on ourability to getDNA and RNA inside cells, where they can interact with the cell’s machinery. Currently, successful delivery of nucleic acids has been stalled by inefficient insertion of the molecules into cells, safety concerns, limited accessibility of the target cells, and poor scalability.
In order to overcome these limitations, a team of scientists has proposed nanoinjection through a carefully designed array of tiny needles, which they’re calling nanoneedles. Nanoinjection provides a more uniform delivery due to the high density of nanoneedles that can occupy a givensurface area. The researchers fabricated biodegradable, porous nanoneedles from silicon with a geometry that was optimized for intracellular delivery.
The nanoneedles had a 5 m length, 50 m width at the sharp end, and 600 nm base diameter, providing a 300-fold increase in surface area for delivery compared to a non-porous wireof equivalent diameter. The porosity of the needles could also be tailored to modulate things likepayload volume, mechanical strength, and how long the needle persists inside cells.
Characterization of the needles demonstrated they could withstand the force required to penetrate cells and were effective in delivering either DNA or RNA. The nanoneedles progressively dissolved over 36 hours in physiological conditions; after 72 hours only the solid stump remained.
Nanoinjection was tested by either placing nanoneedles beneath or on top of a layer of cells. In both cases, nanoinjection did not induce significant toxicitycells continued tofunction and grow normally over the course of five days. Thenanoneedle constructs were also able to load, retain, and deliver nucleic acids over a 12-18 hour period, achieving uniform RNA spread inside the cellafter 48 hours.
The nanoneedles could deliver two separate types of nucleic acids using a RNA strand and a fluorescently labeled DNA strand. These constructsdemonstrated that the molecules were active once inside the cell; they couldmodulate gene activityby either expressing a gene carried on the DNAor silencing expression of genes via RNA interference.
The efficacy of the device was also tested in rats. The nanoneedles could be used for a localized injection, as shown by their ability to carry fluorescent dyes into the skin and muscle of test rats. Nanoinjection was also assessed on ear and muscle to demonstrate that tissue architecture does not influence the delivery process. Injection of fluorescent dyes did not induce local inflammation withina 24 hour period, and imagingof the skin and muscles revealed that the tissue maintained its normal appearanceafter nanoinjection.
Finally, the researchers compared efficiency of nanoinjection todirect injection of DNA. They tested this usingVEGF165, which is a gene that influences the development of blood vessels. While both injections resulted in expression of human VEGF165 for up to one week, the blood vessels formed were very different.Nano injectionpromoted the formation ofhighly interconnected blood vessels near the surface of the skin and a six-fold increase in overall blood perfusion; direct injection did not.
These nano needles could provide a new route totargeted delivery of RNA and DNA, which could lead to major improvements in efficient gene therapy strategies.
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