jetPEI and siRNA were diluted separately in a 10% glucose solution. The solutions containing siRNA and PEI nanoplexes were mixed and incubated for 15 min at room temperature. The formulation corresponds to a N/P ratio of 7. Intranasal administration was performed on lightly anesthetized mice. Each mouse was placed on a sterile surgical pad and lightly stretched out to better hold the scruff. With a firm grip on the scruff, the mouse was turned on its back while still allowing the mouse to breathe and be comfortable. With the neck and chin flat and parallel to the pad, the tip of the pipettor containing the sample was placed near the left nostril of the mouse at a 45 degree angle, and about 5 μL of sample was administered to the nostril with a 2–3 sec interval in between for a total of 10 μL/nostril. The mouse was held in this position for 5 sec or until it regained consciousness, then the administration step was repeated for the other nostril for a total of 20 μL/mouse. After the mouse had received all drops, the animal was kept restrained on its back until the material disappeared into the nares and then returned back to its cage. After 4, 24, 48, 72 and 120 hours mice were sacrificed and half of the brain hemisphere, liver, lungs, heart, spleen and kidneys were cryopreserved.

We previously reported that activation of the host autophagic protein, Beclin1, by HIV-1 infection represents an essential mechanism in controlling HIV replication and viral-induced inflammatory responses in microglial cells. Existing antiretroviral therapeutic approaches have been limited in their ability to cross the blood-brain barrier effectively and recognize and selectively eliminate persistent HIV-infected brain reservoirs. In the present study and for the first time, the bio-distribution and efficacy of noninvasive intranasal delivery of small interfering RNA (siRNA) against the Beclin1 gene using the cationic linear polyethylenimines (PEI) as a gene carrier was investigated in adult mouse brain. Fluorescein isothiocyanate (FITC)-labeled control siRNA delivered intranasally was found in the cytoplasm of neurons and glial cells of the prefrontal cortex at 4 and 24 hours post-delivery, with no major adverse immune reaction encountered. Intranasal delivery of the siRNA targeting Beclin1 significantly depleted the target protein expression levels in brain tissues with no evidence of toxicity. Binding of siRNA to PEI-polymer was characterized and confirmed by Raman spectroscopy. These results indicate that the intranasal drug delivery allows for the direct delivery of the PEI-siRNA nano-complex to the central nervous system, which could potentially offer an efficient means of gene silencing-mediated therapy in the HIV-infected brain.