- Oral presentation
- Open Access
Protein aerosol for intranasal nose to brain (N2B) delivery
© Stützle et al. 2015
- Published: 14 December 2015
- Dispersion Effect
- Cloud System
- Nasal Epithelium
- Nasal Epithelial Cell
- Central Nervous System Drug
For the treatment of metabolic and neurodegenerative diseases, drug delivery to the central nervous system (CNS) has gained considerable recent interest[1, 2]. But many concepts for the delivery of therapeutic agents have failed by the natural blood-brain barrier (BBB). The N2B route for drugs is able to provide an adequate alternative of CNS drug delivery -bypassing the BBB[3, 4]. N2B drug transport takes mainly place at the olfactory cleft, an area that is well hidden in the skull base . In a previous study, we determined the characteristics of aerosols for optimal deposition for N2B delivery by a computational approach and validated those data with experimental deposition in a human nose model generated by rapid prototyping [manuscript in preparation]. With the determined criteria, different aerosol generators were evaluated for protein dispersion. Moreover, a surrogate method was developed that imitates the dispersion effect was used for formulation screening to maintain stability and function and to avoid shear stress during dispersion. The most promising protein formulations were tested in an in vitro exposition system for passage through a nasal epithelium in a transwell culture. This test system can be used to develop an N2B platform approach.
The Carleton-civic standardized human nose model was constructed from CT scans and displays the complex 3D geometry of the human nose and its olfactory cleft. The deposition of liquid airborne aerosols was simulated numerically by computational fluid and particle dynamics (CFPD). Those data were validated experimentally in a positive rapid prototyped human nose model. A surrogate agitation method, imitating the dispersion effect was applied for formulation screening within a Design of Experiment (DoE) approach using trehalose (0.5-6 % (w/v)), sorbitol (0.5-5 % (w/v)), arginine (0.5 - 5 % (w/v)), tween (0.005-0.05 % (v/v)) and cyclodextrin (0.35-3.5 % (v/v)). Protein stability was evaluated applying SE-HPLC and photometry to determine monomer content before and after dispersion with a vibrating mesh nebulizer (Aeroneb Pro, Aerogen 112 Inc., Galway, Ireland). Identified suitable formulations were exposed to a nasal epithelial cell line (RPMI 2650) growing on transwell inserts to investigate transport ratios. Therefore, various seeding densities and transwells were tested under submers and air-liquid interface (ALI) conditions. The confluence of a monolayer was investigated with confocal microscopy by staining adhesion and tight junction proteins and a valid transport model was established with FITC-dextran in the Vitrocell® Cloud System (Waldkirch, Germany). Finally, two formulations were further analyzed in detail to study molecular weight, concentration and formulation dependencies on transport ratios.
In the present study, we found that aerosol parameters had to be defined very precisely to enable deposition at the anatomically hidden olfactory cleft. Validation of CFPD simulation by the experimental nose model determined a particle size of 5-10 µm and a flow rate of 5-20 L/min as optimal conditions for improved deposition.
Formulation and stability: Fab, IgG A and IgG B were dispersed and analyzed via SE-HPLC comparing monomer content before and after dispersion (n = 3, ± SD).
103.21 ± 3.62
90.27 ± 6.87 (ns)
100.00 ± 0.53
77.86 ± 2.44 (**)
103.17 ± 0.12
101.27 ± 2.79 (ns)
100.00 ± 0.28
86.92 ± 2.99 (**)
92.72 ± 4.51
74.88 ± 5.15 (**)
100.00 ± 3.09
77.15 ± 4.12 (***)
In conclusion, shear stress and dispersion effects had a strong impact on protein aggregation when generating micrometer-sized particles. Here, we present the establishment of a surrogate method imitating the dispersion effect and the subsequent formulation screen that led to improved protein stability after dispersion. Moreover, an in vitro exposition system was optimized to study transport ratios of different molecular weight sized proteins, concentrations and formulations. As aggregated proteins appear and cause a proinflammatory reaction, the nasal-epithelial cells will be co-cultivated with macrophages to study signs of immunogenicity after dispersion. These results will be verified in in vivo.
In summary, this test system is suitable to be used asN2B platform for protein aerosols. That includes particle size, deposition in experimental and in silico nose model, protein stability, in vitro and later on in vivo studies.
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