(T1330-04-24) Characterization of Particle Disposition following Intranasal Inoculation with a 3D Printed Device

Purpose: A significant challenge in treating central nervous system (CNS) diseases is achieving effective drug concentrations in CNS tissues including the brain. A potential solution to this problem is the use of intranasal delivery (IND) as a route of administration, which has the potential to bypass the blood-brain barrier via olfactory and trigeminal nerve pathways in the posterior nasal cavity. Our strategy for achieving nose-to-brain (N2B) uptake is delivery of a penetration-enhancing, oil-in-water nanoemulsion (NE) to the posterior nasal cavity. The purpose of this study was to evaluate the in vivo performance of a custom aerosolization device for IND and the effect of NE formulations on uptake. We hypothesize that uptake of a fluorescent compound in the posterior nasal cavity will be greater when using NE vehicle compared to a saline vehicle control

Methods: To prepare the fluorescent compound for delivery, NeutriAvidin-Labeled FluoSpheres (0.04 μm, 505/515 nm, Molecular Probes) were mixed with either NE or physiological saline at a 1:10 volumetric ratio. Three formulations of NE were tested containing an 8% peceol base with either 2% labrasol and 0.5% capryol (NE-LC); 5% gelucire and 0.5% capryol (NE-GC); or 2% gelucire and 0.5% labrafac60 (NE-GL). Batches were prepared by mixing excipients in DI water and sonicating for 10 minutes. The average size and polydispersity index (PDI) was measured at three concentrations for each NE using a Zetasizer Nano-ZS. To inoculate fully anesthetized adult male Sprague-Dawley rats (250-500 g) intranasally with fluorophore in NE or saline vehicle, a custom aerosolizing IND device was used. The device, which was adapted from the work of Piazza et al. and 3D-printed from polylactide polymer, features a nozzle end which fits into the rat nasal cavity and a connecting end to which aerosolizing pressure from an external air compressor is supplied. Using this device, 20 μL of fluorophore-containing solution was delivered into a single nostril of each animal using a delivery pressure of 20 psi and 200 millisecond air duration. An additional rat was inoculated with saline only as a negative control. Rats were awoken and allowed to roam freely for thirty minutes prior to sacrifice via transcardiac perfusion under anesthesia. Rat heads were placed in formalin for 48-96 hours, then placed in Leica Decalcifier I solution for 7-10 days. Heads were divided into four pieces which were frozen in OCT and sectioned at 5 μm thickness on a Leica CM1900 cryostat. Cover-slipped slides were mounted in Vectashield Vibrance Antifade Mounting Medium and imaged using a Leica TCS SP8 Resonant-scanning confocal microscope. For each section of each head, 7-11 slides were sampled for fluorescence. For six of the 44 sections examined, only 1-6 slides could be sampled due to difficulty cutting the tissue. A representative anterior (section 1 or 2) and posterior (section 3 or 4) scan was collected for each nasal cavity at 5x magnification.

Results: NE-LC exhibited the lowest average particle size (8.9 ± 5.8 nm) and polydispersity index (0.26 ± 0.05), followed by NE-GC (22.5 ± 6.1 nm, 0.37 ± 0.02), and lastly NE-GL (44.4 ± 36.1 nm, 0.53 ± 0.25) (Table 1).

In the anterior nasal cavity, fluorescence was not observed in any rats inoculated with saline but was observed in three of six rats inoculated with NE vehicle. In the posterior nasal cavity, fluorescence was observed in one of four rats inoculated with fluorophore in saline as well as four of six rats inoculated with fluorophore in NE. A representative scan of the posterior nasal cavity containing fluorescence is depicted in Figure 1. The fraction of slides with visible fluorescence was calculated for each section of each head. The percentage for each section was averaged within treatment groups to produce the bar graph in Figure 2. For sections two through four of the nasal cavity, more fluorescence was observed in two of the three nanoemulsion vehicles compared to the saline vehicle. The highest overall percentages of fluorescence in sections one, three, and four corresponded to NE-GL, whereas the highest overall percentage for section two corresponded to NE-GC (Fig. 2). No fluorescence was observed in either the negative control or NE-LC treatment group.

Conclusion: Using both saline and NE as the vehicle, fluorophore was observed in the posterior nasal cavity, suggesting that N2B delivery may be possible using the custom 3D-printed aerosolizer. In all four sections of the nasal cavity, at least one NE vehicle formulation resulted in greater fluorescence accumulation compared to the saline vehicle control, suggesting that formulation with NE could be promoting delivery of fluorophore to the nasal tissues. This feasibility study demonstrates proof-of-concept for N2B delivery using NE vehicle with an aerosolizer for small animal intranasal delivery. Work is ongoing to characterize IND with the prototype aerosolizer and NE in a larger group of animals, and perform accompanying immunohistochemistry to better understand the correlation between deposition patterns and rat nasal anatomy.

References: J. E. Piazza et al., “A Novel Intranasal Spray Device for the Administration of Nanoparticles to Rodents,” Journal of Medical Devices, vol. 9, no. 4, p. 041001, Dec. 2015, doi: 10.1115/1.4029907.

Acknowledgements: Animal studies were conducted in accordance with institutional guidelines under an approved protocol by the IU School of Medicine (IUSM) IACUC (#20118, approval date 17 February 2021). Nanoemulsion excipients were provided by Gattefosse Incorporated, USA. Funding was provided by the Undergraduate Research Opportunity Program at Indiana University-Purdue University Indianapolis (IUPUI). Facility access was provided by the Nanoscale Characterization Facility at IUPUI, and the Laboratory Animal Resource Center and Indiana Center for Biological Microscopy at IUSM.

Table 1: Size and polydispersity index of each nanoemulsion (NE) formulation.

Figure 1: Representative scan demonstrating fluorescence in the posterior nasal cavity. Image shown is from section four of a rat inoculated with fluorophore in NE-GL vehicle.

Figure 2: Average fraction of slides exhibiting fluorescence for each section (1-4) of the nasal cavity when delivering fluorophore with saline or one of three nanoemulsions (NE) as the vehicle. Saline only (no fluorophore) is included as a negative control.