Purpose: The intranasal route is an attractive site for drug delivery because of its ease of administration; rapid absorption of most molecules; the opportunity to provide both local and systemic drug effects; avoidance of the harsh gastrointestinal environment and first-pass hepatic metabolism; and the potential to deliver drugs directly to the brain. Nanoparticles may provide unique therapeutic opportunities when included in nasal drug delivery systems, yet the primary uptake and transfer pathways for these particles in the nasal mucosa are currently unknown. Endocytosis plays an important role in nanoparticles uptake into the nasal mucosa. The endo-lysosomal uptake pathways, including clathrin-mediated endocytosis and macropinocytosis, may present additional drug delivery challenges requiring lysosomal escape, but transcytosis can be accomplished through caveolae-mediated endocytosis which may offer better delivery opportunities for nasally-administered nanoparticles. The objectives of this study were to synthesize, characterize, and study the endocytic pathways involved in the uptake of fluorescently-labelled solid lipid nanoparticles (SLNs) across the nasal mucosa.
Methods: SLNs were synthesized using an emulsion/solvent evaporation method. The particles were labeled with Nile Red, a fluorescent dye that can be measured spectrophotometrically, in order to determine the concentration of nanoparticles in tissues or in other media. The nanoparticles were characterized for their size, shape, and release behavior. The uptake of the SLNs was investigated in both the nasal olfactory and respiratory mucosae using Navicyte® vertical diffusion chambers (Harvard Apparatus). Inhibition of specific endocytic pathways was accomplished by the inclusion of chemical inhibitors. Chlorpromazine (10 µg/mL) was included to inhibit clathrin-mediated endocytosis; amiloride (26 µg/mL) was used to inhibit macropinocytosis; and methyl-ß-cyclodextrin (5 mg/mL) was used to inhibit caveolae-mediated endocytosis. The nasal respiratory or olfactory mucosae were equilibrated with Krebs Ringer buffer (KRB) (pH=7.4) containing one of the pharmacologic inhibitors for 60 min prior to exposure to the SLNs. The inhibitor-containing media was removed and replaced with an SLN dispersion and blank media in the donor and receiver chambers, respectively. Tissues were incubated with a 0.1% (w/v) SLN dispersion for either 30 or 60 min. At the end of the uptake period, tissues were rinsed with KRB and the exposed region was excised and weighed. In order to quantify the amount of nanoparticles reaching different regions of the nasal mucosa, trypsin-EDTA was used to separate the epithelial cell layer from the underlying submucosa. The recovered epithelial cells and the remaining submucosal tissues were placed into separate tubes containing Cellosolve® acetate to dissolve the SLNs and release the incorporated dye. Histopathological studies were also carried out to assess the effects of the inhibitors on the morphology of the nasal tissues. Briefly, tissues were treated in a similar manner as in the transport experiments. At the end of the experimental period (2hr), the exposed region of the nasal mucosa was excised and fixed in zinc formalin for 48 hr. Each tissue sample was placed in a graded series of 10%-30% sucrose solutions, each for 24 hr. The samples were cryo-frozen and sectioned (10 µm), placed on Surgipath® adhesive coated slides, stained using hematoxylin and eosin at room temperature, and examined using brightfield microscopy with an Olympus BX-61 motorized light microscope.
Results: The SLNs were spherical in shape with a diameter of ~150 nm. No burst release of Nile Red was observed, and ~50% of the loaded dye was released in 24 hr. The amiloride pretreated nasal tissues showed a lower uptake of SLNs than non-treated tissues indicating that macropinocytosis plays a significant role in the uptake of the SLNs in the nasal olfactory and respiratory tissues. Chlorpromazine pretreated tissues showed no difference in the uptake of the SLNs compared to non-treated tissues, and this suggests that clathrin-mediated endocytosis plays a more limited role in the uptake of these SLNs in the nasal tissues. Methyl-β-cyclodextrin pretreated tissues exhibited higher uptake than non-treated tissues which may be attributed to the direct effects of methyl-β-cyclodextrin on the permeability of the cell membranes following the removal of cholesterol and lipids. Histologic analysis showed that the use of pharmacological inhibitors did not significantly affect the overall integrity of the nasal tissues.
Conclusion: Macropinocytosis, an endocytic process capable of transferring large (up to 5µm) particles, plays a significant role in the uptake of the ~150 nm SLNs in both nasal respiratory and olfactory tissues. Inhibition of clathrin-mediated endocytosis did not significantly reduce SLN uptake, yet smaller (<150 nm) SLNs may experience improved uptake based on the known size capacity (<120 nm) of the clathrin endosome.
Maureen Donovan– University of Iowa, Iowa