Purpose: Intra nasal route of drug administration is a non-invasive route which can potentially carry the drug directly to the CNS. Nanoparticles are of great interest for use in drug delivery systems due to their potential to enhance the drug bioavailability and higher stability as drug carriers. Owing to their small particle size, drug-loaded nanoparticles can provide better targeting and transport through the nasal mucosa. This study focuses on preparation of formulations for the poorly soluble drug- DBM and further characterizations for the formulations. This formulation is intended for intranasal administration to treat neurodegenerative diseases anticipating improved brain delivery via nasal-route.
Methods: DBM was formulated in Brain-Homogenate (BH), as a 7%w/v coarse polymeric suspension in HPMC and as polymeric lipid nanoparticles (7%w/v). As studied previously, BH has the ability to cross the nasal mucosa into the underlying lamina propria. BH was prepared from crude hamster brains and diluted to 10% w/v suspension with Dulbecco’s phosphate buffered saline (DPBS) that were mechanically homogenized using a glass homogenizer. 50mg of DBM was dissolved in 500µL of brain homogenate at room temperature and stirred constantly on magnetic stirrer (IKA® Works, Inc) at the motion speed of 400rpm for 30minutes. For coarse polymeric suspension, FDA approved polymers such as polyvinyl pyrrolidone (PVP), hydroxy propyl β cyclodextrin (HPβCD), and hydroxy propyl methylcellulose (HPMC) were screened and HPMC was selected. For nanoparticles, Gelucire50/13 was used as lipid; Tween80 as surfactant; and HPβCD was used as the polymer. Various amounts of DBM (50mg and 100mg) along with the polymer were incorporated in various lipids to form oil in water emulsions which were then probe sonicated (MISONIX Sonicator 3000) and freeze-dried. Particle size analysis was done on Zeta plus Particle Sizing Software (Brookhaven Instruments Corp.). DBM nanoparticles were optimized for a size range below 250 nm. UV method for DBM (342nm) was developed. Other characterizations include X-ray diffraction (XRD), IR spectroscopy, Thermogravimetric analysis (TGA), Scanning Electron Microscopy (SEM), Transmission electron microscopy (TEM) and 1H-NMR. Physical stability was tested for a period of 30 days with respect to particle size. Release studies using UV Spectroscopy was also conducted.
Results: DBM was uniformly distributed in BH but started settling within 5 minutes of standing. As for coarse dispersions, HPMC inhibited precipitation of DBM for 3 days at room temperature. As expected, concentration dependent viscosity increase was observed in these coarse dispersions. On the other hand, analysis of nanoparticles showed a particle size of 138.7± 1.8 nm and 216.6 ± 4.5 nm for nanoparticles loaded with 50mg and 100mg of DBM respectively. No change in particle size was observed for 3 days. XRD results (Figure 1) showed that DBM-HPMC system was crystalline while the lyophilized nanoparticles were amorphous but with two unique peaks which will further be investigated. TGA data shows the formulation to be stable upto more than 150˚C and percentage weight loss calculated was found to be less than 1% for the nanoparticles. In IR analysis (Figure 2), the peak at 1592.15 cm-1 of dibenzoylmethane indicating aromatic carbons were significantly depressed in IR spectra of the dispersion in BH, micro particulate and nanoparticulate formulations, thereby suggesting possible interactions and bonding between DBM and other components of the system. Characteristic peaks such as at 3262 cm-1 for OH stretch of HPβCD, 2989 cm-1 for BH and 3269 cm-1 for OH stretch in HPMC were seen. SEM images showed irregular, porous morphology for the nanoparticles. TEM images indicated dissociation of the particles to aggregates less than 50 nm size in size upon sonication.
Conclusion: DBM coarse, micro-particulate and nano-particulate formulations were designed and characterized to carry out intranasal studies on Hamster to understand and distinguish effectiveness, pharmacokinetic and pharmacodynamic profile and histological characteristics and thus prove its nose-to-brain delivery.