This article details a method for intranasal delivery of a neuron-targeting peptide-siRNA complex in anesthetized mice. The procedure aims to facilitate gene silencing in neuronal tissues through targeted delivery.
Begin with anesthetized mice placed on a positioning device with their backs parallel to the chair's back support and perpendicular to the seat.
The mice are positioned in a head-down-and-forward posture and secured using chair belts.
Using a micropipette loaded with a neuron-targeting peptide-siRNA complex, place a drop near one nostril, allowing the mouse to inhale the solution naturally.
Gently press the mouse's head downward during inhalation to prevent the solution from draining into the lungs.
Pause briefly, then repeat the administration in the other nostril.
Repeat the procedure for the remaining mice.
The complex penetrates the nasal mucosa, from where it enters the olfactory and trigeminal nerves and travels through them to reach the brain.
Inside the brain, the complex binds to neuronal receptors, undergoes endocytosis, and facilitates intracellular siRNA delivery.
The siRNA becomes part of the RISC complex, which, upon binding to the target mRNA, induces gene silencing.
To begin this procedure, assess the anesthesia level of the mice bipedal reflex in order to maintain the surgical plane. Next, place the positioning device at an appropriate distance and height to allow convenient access to all required reagents.
Then apply ointment on the animal's eyes. Strap one mouse with chair belts, making sure the mouse's forelimbs provide natural support while it is in a relaxed position without any discomfort. Repeat the procedures for the remaining mice. Gently place each mouse on the designated chair by laying its back parallel to the back support of the chair, and at 90 degrees to the chair seat.
Let the animal lie naturally in the head down and forward position without pushing or pressing. Once all the mice are strapped, immediately start the intranasal inoculation. To perform intranasal delivery, load the micropipette with 2 microliters of RVG9R-siRNA complex solution. Hold the pipette in the dominant hand, and support it with the other hand to avoid uncontrolled movements while administering siRNA.
Place about a 2-microliter droplet very close to one nostril so that the mouse can directly inhale it. If a tiny drop is not easily formed, replace the pipette tip with a new one and repeat the operation. Gently push the mouse head down with index finger for 5 seconds while it is inhaling, and maintain the position.
To prevent drainage of drug into the lungs, it is important to slightly push the mouse head down with the index finger for 5 seconds while the mouse is inhaling.
Start a stopwatch to clock three to four minutes after this to time inoculation into the second nare of the first mouse. This time interval is necessary for the mouse to complete inhalation of the first dose and restore normal breathing. In the three to four-minute interval between inoculation of alternate nares on the first mouse, complete intranasal inoculation of one nare in each of the remaining three mice placed on the device.
When the stopwatch sounds, inoculate the second nostril on the first mouse and repeat the procedure for the other mice as above. Repeat the procedure until the 20 to 30 microliters is delivered for all four mice. It will take about 30 to 45 minutes to complete the entire inoculation procedure. Return the animals back to their designated cages. Do not leave the mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.
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