Mucopenetration properties and interaction with airway epithelial cells of solid lipid nanoparticles containing magneto sensitive iron oxide

Chronic respiratory diseases, whose hallmarks are oxidative stress and persistent inflammation, need novel therapeutic tools and pharmaceutical agents. We have previously demonstrated the ability in reducing oxidative stress and inflammation by natural substances obtainable from the red grape seed (polyphenols) encapsulated in solid lipid nanoparticles (SLNs), in airway epithelial cells in vitro [1]. These studies revealed that SLN were taken up in a dose-dependent fashion and persisted into cells at detectable levels up to 16 days. Since in most chronic respiratory diseases excessive viscous airway secretions oppose a formidable permeation barrier to drug delivery systems limiting their therapeutic efficacy in vivo, one of the possible strategies pursued to overcome mucus clearance is based on nanoparticles with magnetic cores pulled by external magnetic forces through the mucosal barrier. Thus, we evaluated whether penetration of SLNs through airway sputum may be improved using a magnetic field. For this purpose, we tested magneto-sensitive iron oxide containing SLNs (mSLNs) based on Gelucire® 50/13 [2] on mucus samples derived from three different sources: i) a sputum obtained from chronic obstructive pulmonary disease (COPD) outpatients and ii-iii) aspirates from high and low airways of COPD patients, respectively, during their admission in the intensive care unit (ICU). Based on the FEV1/FVC ratio (Tiffenau index) >0.7, COPD oupatients were in mild condition, whereas COPD patients in ICU required mechanical ventilation for severe traumatic brain injury and acute respiratory insufficiency. Briefly, fluorescent mSLNs were prepared with the melt-emulsification method in presence of magnetic iron oxide (II, III) and an ethanolic solution of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt (Liss Rhod PE). As control, plain fluorescent SLN (pSLNs) were produced in absence of magnetic iron oxide. The resulting SLNs were characterized for their size, zeta potential and polydispersion index (PDI). SLNs characterization revealed that incorporation of iron oxide induced a significant increase in mean diameter value (313 ±14 nm for mSLNs vs 141 ± 11 nm for pSLNs). In general, Liss Rhod PE adsorption resulted in a shrinking of mean diameter for both SLNs. Concerning zeta potential values, all SLNs resulted negatively charged (with and without Liss Rhod PE), indicating a good colloidal stability which should prevent their aggregation. Moreover SLNs exhibited wide PDI values, particularly for both mSLNs and pSLNs adsorbing Liss Rhod PE. Sputum diffusion studies were performed as previously described [3]. Briefly, one-hundred µL of pooled mucus/sputum was stratified on the upper chamber of a semipermeable filter (Transwell, 0.33 cm2, porosity 3 µm), while 600 µL of PBS were placed on the bottom chamber. Either fluorescently-labelled magnetic SLNs (mSLNs) or plain SLNs (pSLNs) diluted in 100 µL PBS at a final concentration of 16 µg/mL were placed onto the mucus/sputum layer or in PBS in absence of mucus/sputum as control, the transwells were placed on top of a magnetic plate at 37°C, and the fluorescence in the basolateral medium was measured at different times, up to 30 hours. The results of mucopenetration in presence of a magnetic field, show that mSLNs are more permeable in COPD sputum than pSLNs, while high and low secretions are still difficult to overcome. Moreover, in order to study mSLNs interaction with differentiated airway epithelial cells in presence of magnetic field, primary bronchial epithelial cells were grown on transwells in air-liquid interface (ALI) conditions for two weeks producing mucins and expressing cilia. We observed a dose-dependent rate of adhesion with a strong effect of the magnetic field both after 2 h and 4 h of incubation. Moreover, mSLNs did not affect the organization of tight junctions. The results highlight that mSLNs can achieve mucopenetration properties in the presence of a magnetic field, depending essentially on the viscoelastic features of the mucus/sputum sample, suggesting that the delivery of therapeutic agents with this strategy might be more successful when the patients are considered in a milder condition. References 1. Castellani, S., et al., Nanoparticle delivery of grape seed-derived proanthocyanidins to airway epithelial cells dampens oxidative stress and inflammation. J Transl Med, 2018. 16(1): p. 140. 2. Trapani, A., et al., Protection of dopamine towards autoxidation reaction by encapsulation into non-coated- or chitosan- or thiolated chitosan-coated-liposomes. Colloids Surf B Biointerfaces, 2018. 170: p. 11-19. 3. De Leo, V., et al., Preparation of drug-loaded small unilamellar liposomes and evaluation of their potential for the treatment of chronic respiratory diseases. Int J Pharm, 2018. 545(1-2): p. 378-388.