Purpose: The nasal mucosa provides a non-invasive route for drug administration to the systemic circulation and potentially directly to the CNS. Nanoparticles made from biodegradable polymers, including PLGA, are of great interest for use in drug delivery systems due to their relative safety and ease of surface modification. Owing to their small size, nanoparticles may provide improved targeting and transport through the nasal mucosa, and drug-loaded nanoparticles may enhance the delivery of drugs or vaccines via the intranasal route. An improved understanding of the mechanisms and pathways of nanoparticle transfer across the nasal mucosa is needed to design effective new nasal delivery systems. This study focuses on the preparation of PLGA nanoparticles and the in vitro investigation of their mechanisms of endocytosis and exocytosis in the nasal mucosa. Identifying controlling mechanisms for nanoparticle uptake and transfer will provide a much-needed understanding of nanoparticle trafficking and contribute to the design of effective particulate delivery systems.
Methods: PLGA nanoparticles (60 nm or 125 nm) containing the lipophilic fluorescent dye, Nile Red, were prepared using a surfactant-free nanoprecipitation method. The nanoparticles were evaluated using light scattering techniques to measure their particle size and zeta potential. Nanoparticles were further characterized using scanning electron microscopy (SEM) to visually evaluate their size and shape. Nanoparticle uptake into the nasal mucosa was determined by exposing excised nasal mucosal segments to nanoparticle dispersions for 30 or 60 minutes using Navicyte® vertical transport chambers (Warner Instruments). The number of nanoparticles translocated into the nasal mucosa was quantified by extracting the Nile Red from the nanoparticles located in the tissues. The number of nanoparticles corresponding to the amount of released dye was calculated based on a previously determined correlation between particle mass and dye entrapment.
Endocytosis mechanisms involved in the uptake of PLGA nanoparticles were studied using chemical inhibitors including chlorpromazine (30 mM), amiloride (300 mM) and methyl-beta-cyclodextrin (4 mM) to evaluate clathrin-mediated endocytosis, macropinocytosis and caveolae mediated endocytosis, respectively.
In order to investigate the exocytic mechanisms involved in nanoparticle trafficking in the nasal epithelium, RPMI-2650 cells were cultured on collagen-coated polytetrafluoroethylene (PTFE) membrane filter inserts. Cells were grown in an air-liquid interface (ALI) configuration where the medium was in contact with only the basolateral surface. The cultured layer was examined to assess the cell layer integrity and functionality by evaluating of the expression of the tight junction protein, ZO-1, and the transport of atenolol and propranolol were evaluated as a paracellular and transcellular markers, respectively. The uptake of 60 nm Nile Red-loaded PLGA nanoparticles was evaluated following 30 and 60 min incubations and the transfer of the nanoparticles across the cell layer into the receiver medium was measured.
Results: Two sizes of PLGA nanoparticles 60 nm and 125 nm (average polydispersity index (PDI) = 0.112) were prepared. Both sizes possessed a slightly negative surface charges (average zeta potential of -27 ¬¬to -35 mV), and SEM images showed spherical nanoparticles with sizes range similar to those obtained from dynamic light scattering. In-vitro uptake of the nanoparticles by the nasal respiratory and olfactory tissues revealed that the Nile Red-loaded PLGA nanoparticles were transported across the epithelial layer and accumulated in the sub-mucosal connective tissues. Nanoparticle uptake in the full thickness tissues was time-dependent, where 2 % of the total load of nanoparticles exposed to the tissues was transferred to the mucosal tissue after 30 minutes and 4 % were transferred to the tissues after 60 minutes. Studies probing the role of energy-dependent endocytic mechanisms involved in nanoparticle internalization revealed that nanoparticle uptake in the nasal mucosa is an energy dependent process utilizing multiple mechanisms, including clathrin-mediated endocytosis and micropinocytosis.
RPMI-2650 cells showed good expression of ZO-1, and the measured apparent permeability coefficients for atenolol and propranolol HCl were 15 *10-6 and 39 *10-6 cm/s, respectively, confirming the barrier integrity of the cultured cell layer. Nanoparticle uptake studies showed that 60 nm particles were able to transfer into the cell layer in a time dependent manner similar to the excised nasal mucosal tissues but no nanoparticles were detected in the receiver chamber, indicating limited exocytosis of the particles across the basolateral membrane.
Conclusion: The measurable transfer of PLGA nanoparticles into the nasal mucosal tissues indicate that they may be useful delivery vehicles for drugs with either local or systemic activities. The multiple endocytotic mechanisms involved in nanoparticle uptake can assist to increase the total number of PLGA nanoparticles transferred into the nasal mucosa. Nanoparticle uptake into RPMI-cells was comparable to that observed in excised nasal mucosal tissues model, but few nanoparticles underwent exocytosis from these cells. The accumulation of nanoparticles in the submucosal tissues in light of the observation of limited exocytosis from the epithelial cells indicates that paracellular transfer of nanoparticles through the nasal epithelium may be the primary pathway for the nanoparticle transfer to the submucosal region.
Mohammed Albarki
– Graduate Student Researcher, University of Iowa, Iowa City, IowaMohammed Albarki
– Graduate Student Researcher, University of Iowa, Iowa City, IowaMaureen Donovan
– University of Iowa, IowaMohammed Albarki
– Graduate Student Researcher, University of Iowa, Iowa City, Iowa335 Views