• Citer

Combining 3D-printed nasal cast and pharmaceutical formulation towards N2B delivery

par Deruyver, Laura
Président du jury Amighi, Karim
Promoteur Goole, Jonathan
Publication Non publié, 2024-09-18

Thèse de doctorat

• Accès en ligne
• Détails
• Contenu
• Statistiques

Résumé :

The use of the nose as a route of administration is extensively known for local treatment (e.g., decongestants, allergic rhinitis, or chronic rhinosinusitis). For thirty years, the interest in the nasal route as a way to reach the brain (called “Nose-to-Brain” delivery) has been steadily growing. The interest in this route began in 1989 with the works of William H. Frey which was the first to discuss a method to reach the brain via the olfactory nerves. Targeting the olfactory region in the nasal cavity is the key to optimising the brain bioavailability. Indeed, from there, there are two possibilities: following the olfactory nerve (the drug reaches the olfactory bulb and then the brain by the axonal transport) or by the olfactory epithelium (the drug reaches the cerebrospinal space by cellular uptake and then the brain via cerebrospinal fluid). The olfactory pathway allows a fast passage in the brain (1.5 to 6 hours) and permits to have a non-invasive pathway to treat neurological diseases.The objectives of the present work are divided into two different parts: (i) the development of a formulation optimized for N2B delivery and (ii) the development of a new in vitro tool for studying the deposition of nasal formulation in a 3D-printing nasal cast.We chose cubosomes as our nanoparticle strategy intended for nose-to-brain delivery. Cubosomal nanoparticles is composed of a bicontinuous lipid cubic phase (commonly generated by monoolein) with a single lipid bilayer that forms a continuous periodic membrane lattice structure with two interwoven water channels forming the pores (i.e. a gyroid structure). They are biocompatible, bioadhesive and biodegradable, and they have a broader lipidic internal structure than liposomes.The formulation part started with the study of the capacity of the cubosomal nanoparticles to encapsulate several drug candidates for nose-to-brain delivery. Indeed, the literature described this nanocarrier as able to encapsulate hydrophilic, hydrophobic, and amphiphile drugs. We focused our research on neurological disease treatments and thus we selected three potential candidates: paliperidone palmitate to treat schizophrenia and L-dopa and pramipexol to treat Parkinson’s disease. In this way, the first step was the study of the encapsulation efficiency and the nanoparticle integrity of cubosomes with those different drug candidates by using a “standard” cubosomal nanoparticles formulation developed by von Halling Laier et al. We completed the formulation strategy part by the improvement of the powder properties. Indeed, the work was based on the development of powder formulation thanks to its numerous advantages for nose-to-brain delivery. The spray-drying method was selected to produce powder because it is a well-known method which converts a solution, suspension or emulsion into a dried powder in a single step by passing an atomized spray through a warm gaseous medium and which permits a precise control of particle size, production yield, and residual moisture content. We added l-leucin (a hydrophobic amino acid) to our formulation to improve powder properties during the spray-drying process. Consequently, we studied the influence of the l-leucine content on powder properties (particle size, production yield, and residual moisture). After we selected the suitable percentage of l-leucine, we studied the influence of the spray-drying parameters on these powder properties. Furthermore, the study of these influences was realised using a Design of Experiment, which gave the optimal parameters for a suitable powder formulation for nose-to-brain delivery.After the first part of formulation development, paliperidone palmitate was selected. The cubosomes loaded with it showed a significantly higher encapsulation efficiency (91.9 ± 1.9%) compared to the two others (45.8 ± 8.2 and 41.4 ± 6.4% for L-dopa and pramipexol, respectively). They also demonstrated suitable nanoparticle characteristics for nose-to-brain delivery with a mean z-average of 159.3 ± 10.4 nm, a PDI of 0.341 ± 0.094 , and a zetapotential of + 8.2 ± 2.8 mV. We obtained great powder particle properties with the addition of 25% m/m of l-leucin with a median diameter of 13.74 ± 3.06 m, and a span value of 2.81 ± 0.32. Moreover, the production yield reached 78.53 ± 1.31 % with a residual moisture content of 1.838 ± 0.078 %.The last part of the formulation consisted of developing an optimised cubosomal nanoparticle formulation. The literature describes that the use of cholesterol consolidates the lipid bilayer structure and reduces thermal fluctuations. Also, charged lipids electrostatically swell the internal structure of the cubosomes and increase the water channel diameters. In this way, we determined the suitable proportion of monoolein, cholesterol, and DOTAP (positively charged phospholipid) for the lipid mixture intended for stable cubosome formation. We chose to produce positively charged nanoparticles because of the negative charge of the nasal mucosa, caused by the main proteins in mucus (the mucins). Thus, positively charged nanoparticles improve the mucoaffinity by electrostatic interaction. Parallelly, we also developed a negatively charged cubosomes with DOPS as the charged lipid compound. Indeed, the final strategy was to develop a hybrid nanoparticle system (lipid and polymer compound). The hybrid systems for improving nose-to-brain delivery recently emerged but none of them uses cubosomes as lipid compounds. Thus, we developed a negatively charged cubosomes composed of cholesterol and DOPS that permitted us to coat them with positively charged chitosan (as the polymer compound) and generate a final positively charged hybrid system. Chitosan is widely exploited to improve nose-to-brain delivery. Indeed, this polymer has both mucoadhesive and cell-penetrating properties. Consequently, the last experimental part was focused on the development and the characterisation (NP stability, morphology, mucoaffinity, cell permeation, deposition testing, storage stability) of a hybrid system composed of cubosomes and chitosan intended for nose-to-brain delivery. The chitosan-coated cubosomal formulation seems to be the most promising formulation for nose-to-brain delivery. Indeed, it has a high mucoaffinity and a significantly higher apparent permeability coefficient than the standard and the cubosomes with DOTAP. Finally, it reaches well the olfactory region. Indeed, the selected chitosan-coated cubosomal nanoparticles loaded with paliperidone palmitate had a size of 305.7 ± 22.54 nm, their polydispersity index was 0.166 ± 0.022, and their zeta potential was +42.4 ± 0.2 mV. This formulation had a drug loading of 70% and an encapsulation efficiency of 99.7 ± 0.1 %. Its affinity with mucins was characterized by a ZP of 20.93 ± 0.31. Its apparent permeability coefficient thought the RPMI 2650 cell line was 3.00E-05 ± 0.24E-05 cm/s. After instillation in a 3D-printed nasal cast, the fraction of the injected powder deposited in the olfactory region reached 51.47 ± 9.30 % in the right nostril and 41.20 ± 4.59 % in the left nostril, respectively. Moreover, the stability study demonstrated in promising results for the storage stability in time.The part concerning the 3D-printed nasal cast development is crucial for better comprehension of nasal product deposition in a nasal cavity. Despite a growing interest in nasal products, particularly those intended for nose-to-brain delivery, specific in vitro tools or procedures to study them do not exist. On the contrary, for inhaler products, standardised in vitro procedures with the multistage cascade impactor or Next Generation Impactor devices were well established. Moreover, while the lower respiratory tract is roughly uniform across the population, the nose can differ widely between people. So, in the case of treatment requiring precise targeting, such as nose-to-brain delivery, formulation efficiency should be assessed independently for each patient.Thanks to the expertise of the Transferts, Interfaces, and Processes (TIPs) department of the Polytechnic School of Brussels combined with specialists of Ear, Nose and Throat and Cervico-Facial Surgery department of CUB Erasme Hospital a protocol to create a 3D model from CT scan was developed. We decided to segment this 3D model of the nasal cavity into five pieces corresponding to the nostrils, lower turbinates, middle turbinates, olfactory region, and nasopharynx.After an analysis of the surface rugosity and a solvent resistance study, the stereolithography 3D printing with Formlabs resin material and fused deposition modelling with polylactic acid material were selected for more detailed studies.To stay as close to reality as possible, we developed an artificial nasal mucus to coat the walls of the nasal cast was with a thin uniform film. A preliminary deposition test which compared the deposition profile by using stereolithography with fused deposition modelling technologies with or without mucus coating was conducted. It revealed a significant difference between the variance of results with fused deposition modelling nasal cast in comparison to stereolithography nasal cast. In this way, we selected the stereolithography technology for further analysis. Then, we validated this in vitro procedure with a first study of the parameters influencing the olfactory deposition. We selected five parameters: a “normal” patient with and without a septal perforation, a unidirectional and a bidirectional device, the insertion angle (direct aim of the olfactory region or the centre of the nasal valve), the insertion side (left or right), and the inspiratory flow (0, 15, and 60 L/min). This part validated the used of the nasal cast as an in vitro tool for deposition study combined with the use of a Design of Experiments to obtain a valid and robust deposition protocol. Indeed, the statistical analyses selected optimised parameters to target the olfactory region and the olfactory deposition with optimal parameters showed an amount of powder reaching this zone almost twice higher compared to the average deposition. This study also highlighted the influence of the anatomical diseases on olfactory deposition. Consequently, we realised the importance of a further study of the anatomical influence on this olfactory deposition. Thus, in the last part of this work we used 3D printing to produce nasal replicas based on 11 different CT scans presenting various anatomical traits such as turbinates hypertrophy, septum perforation, septum deviation, paediatric, and normal anatomy. Then, for each anatomy and using the Design of Experiments methodology, we characterised the amount of a powder deposited in the olfactory region of the replica as a function of multiple parameters. We found that, for each anatomy, the maximum amount of powder that can be deposited in the olfactory region is directly proportional to the total area of this region. More precisely, the results show that, whatever the instillation strategy, if the total area of the olfactory region is below 1500 mm2, no more than 25% of an instilled powder can reach this region. On the other hand, if the total area of the olfactory region is above 3000 mm2, the deposition efficiency reaches 50% with the optimal choice of parameters, whatever the other anatomical characteristics of the nasal cavity. Finally, if the relative difference between the areas of both sides of the internal nasal valve was higher than 20%, it becomes important to carefully choose the side of instillation. This work, by predicting the amount of powder reaching the olfactory region, provides a tool to evaluate the adequacy of nose-to-brain treatment for a given patient. While the conclusions should be confirmed via in vivo studies, it is a first step towards personalised treatment of neurological pathologies.