Aerosolized In Vivo 3D Localization of Nose-to-Brain Nanocarrier Delivery Using Multimodality Neuroimaging in a Rat Model—Protocol Development

The fate of intranasal aerosolized radiolabeled polymeric micellar nanoparticles (LPNPs) was tracked with positron emission tomography/computer tomography (PET/CT) imaging in a rat model to measure nose-to-brain delivery. A quantitative temporal and spatial testing protocol for new radio-nanotheranostic agents was sought in vivo. LPNPs labeled with a zirconium 89 (89Zr) PET tracer were administered via intranasal or intravenous delivery, followed by serial PET/CT imaging. After 2 h of continuous imaging, the animals were sacrificed, and the brain substructures (olfactory bulb, forebrain, and brainstem) were isolated. The activity in each brain region was measured for comparison with the corresponding PET/CT region of interest via activity measurements. Serial imaging of the LPNPs (100 nm PLA–PEG–DSPE+89Zr) delivered intranasally via nasal tubing demonstrated increased activity in the brain after 1 and 2 h following intranasal drug delivery (INDD) compared to intravenous administration, which correlated with ex vivo gamma counting and autoradiography. Although assessment of delivery from nose to brain is a promising approach, the technology has several limitations that require further development. An experimental protocol for aerosolized intranasal delivery is presented herein, which may provide a platform for better targeting the olfactory epithelium.


PLA-DSPE-PEG nanoparticle preparation
PLA-DSPE-PEG NPs were prepared as previously reported with modifications [2]. Briefly, 5 mg of PLA, 8 mg of DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000), 2 mg of DSPE-PEG2000-NH2, and 0.1 mg of DiR were dissolved in 0.5 ml dichloromethane, and then dropped into 3 ml doubledistilled water. The mixed solution was emulsified over an ice bath for 1 min using a microtip probe sonicator (XL-2000, Misonix) at 7 W output. The dichloromethane was removed by rotary evaporation to harden NPs. After centrifugation at 13,500 xg for 10 min, pellets were discarded, and NP suspension was washed three times with 10% sucrose solution by 30 kD MWCO ultrafiltration (Vivaspin 6, GE Healthcare). The purified NPs were lyophilized and stored at -20⁰C. The materials used in nanoparticle handling and their sources are listed in Table 1. The physicochemical characteristics of these NPs were determined and are summarized in Table   Table S2. PLA-PEG NP physiochemical characterization.

Transmission electron microscopy
Images of NPs were obtained using TEM (Hitachi model H-7650) using an acceleration potential of 100 kV. Samples were prepared by layering the nanoparticles suspension on a copper grid followed by negative staining for 10 sec with freshly prepared and sterilefiltered 2% (w/v) uranyl acetate solution. The TEM results are depicted in Fig 1 and Table  3.

Particle size and zeta potential measurements
Hydrodynamic diameter and zeta potential of PLA-PEG nanoparticles were measured using a particle size analyzer (NanoBrook 90Plus, Brookhaven Instruments Corp., Holtsville NY) and zeta potential analyzer (NanoBrook ZetaPALS, Brookhaven Instruments Corp.) equipped with a 660-nm laser. The measured delay time correlation functions were fitted to a non-negative least squares (NNLS) model to calculate particle size distribution. The DLS results are depicted in Fig 1 and Table 3.

Quantitative determination of DSPE-PEG
DSPE-PEG was measured calorimetrically with ammonium ferrothiocyanate method [3]. Samples were dissolved in 1 ml chloroform and mixed with 1 ml of ammonium ferrothiocyanate reagent (30 mg/ml ammonium thiocyanate and 27 mg/ml iron chloride hexahydrate). The mixed solution was shaken for 3 min and centrifuged at 1,000 xg and the red lower layer was collected. The DSPE-PEG derivative was determined at 470 nm absorbance using a spectrophotometer (DU800, Beckman Coulter).

PLA quantification
Lyophilized sample was dissolved in stabilized tetrahydrofuran and 20 μl aliquot was analyzed. Chromatographic separation was performed on a gel permeation chromatography system connected to a refractive index detector (Agilent 1100 series) with a PLgel MIXED-D column (300 mm×75 mm, 5 μm, Agilent). PLA was eluted by 100% tetrahydrofuran at a 1 ml/min of flow rate and PLA content was determined by peak area of refractive index signal.

Radiolabeling of PLA-PEG NPs with 89 Zr.
Zirconium 89 ( 89 Zr) was produced with a cyclotron at Washington University at St. Louis and overnight shipped to our institution for nanoparticle tagging. For 89 Zr-labeling, PLA-PEG NPs were first conjugated with a derivative of desferrioxamine (Distearoylphosphatidylethanolamine: DFO-Bz-NCS) through amide formation. Specifically, 1.55 mg PLA-PEG NPs were stirred with 0.02 mg DFO-Bz-NCS in water for an hour. Purification (molecular weight cut-off (MWCO) 100kD) was done at 8,000 rpm with a centrifugal concentrator (Vivaspin 500 GE HealthCare) and washed with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer. Before radiolabeling, the PLA-DSPE-PEG NPs were neutralized with 89 Zr-oxalate by using 1M NaOH in HEPES buffer and a final pH of 7.4 was obtained. Radiolabeled PLA-PEG NPs, 89 Zr (1 mCi) were added to 0.4 mg of PLA-DFO and incubated in pH 7.4 HEPES buffer for 30 minutes. Radiolabeled PLA-DSPE-PEG NPs were then purified by centrifugation and the labeling efficacy was measured with an instant thin layer chromatography (ITLC) autoradiogram. The radiolabeling activity was 650 µCi per 1 mg of PLA-PEG NPs. Details of zirconium tagging of NPs are listed in Table 4.