Transport Mechanisms Of Solid Lipid Nanoparticles Across Intestinal Epithelial Cell Monolaver And Vector Construction

Nanoparticulate drug delivery system has made considerable progress in improving oral bioavailability of some drugs with poor solubility and membrane permeability. Compared with traditional oral dosage forms, nanoparticulate drug delivery system has some irreplaceable advantages, such as the distribution on a larger surface area in gastrointestinal tract, greater protection of the encapsulated drugs from degradation, more controlled drug release, and more site-specific targeting. In the past few years, solid lipid nanoparticles (SLNs) have been widely investigated and developed as a potential nanocarrier for oral drug delivery. SLNs are prepared by biodegradable and biocompatible materials and have been considered to be the most promising nano-scale drug delivery system to be developed for clinical use. SLNs have shown great advantages in oral drug delivery, but the related transport mechanisms of SLNs in gastrointestinal tract have rarely been reported. A comprehensive investigation and advanced understanding of the mechanism involved in the transport of SLNs across gastrointestinal epithelial cell monolayer is significant for guiding the fabrication of SLNs with good absorption and high oral bioavailability. In the present study, the molecular transport mechanisms of SLNs crossing simulative intestinal epithelial cell monolayer were studied. SLNs with various structures were prepared and the transport mechanisms crossing simulative intestinal epithelial cell monolayer were compared. Via these comparisons, the structure characteristic of SLNs with high transcytosis efficiency was obtained.SLNs were prepared using glycerol monostearate (GMS) by a solvent diffusion method in an aqueous system. FITC-labeled ODA (ODA-FITC) was synthesized to be used as a fluorescence marker to be incorporated into SLNs. Au nanoparticles (5nm) were incorporated into SLNs to prepare the Au nanoparticle-loaded SLNs (Au-SLNs) for the transmission electron microscopy (TEM) analysis of cell samples. The results indicated that the particle size of these three kinds of nanoparticles was approximately 100nm, with uniform distribution. The quality of SLNs dispersion was not altered significantly during one week storage period.In this study, Madin-Darby canine kidney (MDCK) cells were selected to form a simulative intestinal epithelial cell monolayer to investigate the transport mechanisms of SLNs crossing the epithelial cell monolayer. The results showed that approximately 20% of the SLNs added in the apical side of Transwell device was endocytosed into MDCK cell monolayer, and only 2.5% of the SLNs was transported to the basolateral side. The TEM results showed that SLNs remained intact after their transcytosis across MDCK cell monolayer. The transport of SLNs across MDCK cell monolayer exhibited no effect on tight junctions as well as surface morphology of apical side. Endocytosis of SLNs by MDCK cells was time and energy dependent. Adhesion of SLNs to the cell membrane is the prerequisite step for endocytosis, and the adhesion by SLNs could significantly reduce the cell membrane fluidity. The endocytosis of SLNs was found to be a vesicle-mediated process validated by TEM results. The mechanisms of SLNs transport across MDCK cell monolayer were demonstrated to be mediated by lipid raft/caveolae- and clathrin-related pathways, and to some extent dependent on actin filaments. Endoplasmic reticulum, Golgi complex and microtubules were important organelles for SLNs transcytosis. The transcytosis results indicated that there may be direct pathways that transport SLNs to the basolateral side.Furthermore, Caco-2 cells were employed to investigate the molecular mechanisms of SLNs trafficking across the epithelial cell monolayer, and compared with that found in MDCK cell monolayer. First, the cytotoxicology of SLNs in Caco-2 cells was systematically studied, and the results demonstrated that SLNs had low cytotoxicity, had no effects on the integrity of cell membrane, did not induce oxidative stress, and could significantly reduce cell membrane fluidity. For the first time, the endocytosis and exocytosis of SLNs in Caco-2 cells was directly verified to be a vesicle-mediated process using total reflection fluorescence microscopy. The transport mechanisms of SLNs in Caco-2 cell monolayer were similar as that found in MDCK cell monolayer, except for the certain roles of macropinocytosis pathway played in the transport of SLNs across Caco-2 cell monolayer. The amount of SLNs transcytosed by Caco-2 cell monolayer was less than that by MDCK cell monolayer. Additionally, the transport of SLNs in Caco-2 cell monolayer was a more energy-dependent procedure compared with that in MDCK cell monolayer.SLNs with various structures were fabricated, such as using solid lipid materials with different carbon chain length and modifying the particle surface with different amount of SA-PEG2000. MDCK cell monolayer was employed to compare the transcytosis of these different SLNs and investigated the related molecular mechanisms. The results demonstrated that the amount of SLNs endocytosed into the cell monolayer from apical side as well as SLNs transcytosed to basolateral side decreased with the increase of carbon chain length. The related mechanisms indicated that SLNs prepared by solid lipid materials with low carbon chain length was inclined to be transcytosed via endoplasmic reticulum and Golgi complex mediated pathways, and with the increase of carbon chain length the proportion of transcytosed SLNs by these two organelles decreased. Furthermore, a certain amount of PEG modification (PEG-20%) not only can increase the amount of SLNs endocytosed into cell monolayer, but also can significantly increase the amount of transcytosed SLNs. The mechanism study showed that a certain amount of PEG modification can increase the amount of SLNs endocytosed into cell monolayer via lipid raft/caveolae- and clathrin-mediated pathways, and also can transport SLNs to basolateral side directly via the more effective pathways that not mediated by endoplasmic reticulum and Golgi complex. Moreover, rats were employed to conduct the in vitro and in situ intestinal absorption experiments to compare the difference of SLNs with various structures across the intestinal epithelial cell monolayer. The results found in in vitro and in situ experiments were consistent with that found in simulative intestinal epithelial cell monolayer. In conclusion, structure characteristic of SLNs with high transcytosis efficiency was obtained, i.e. the SLNs prepared by solid lipid with medium carbon chain length and endowed with a certain amount of hydrophilic modification on particle surface, and with these features SLNs can transport across intestinal epithelial cell monolayer efficiently. Our findings may provide certain instructions on designing promising SLNs systems with high transport efficiency and excellent bioavailability.