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Transport Mechanisms of Solid Lipid Nanoparticles across Caco‑2 Cell Monolayers and their Related Cytotoxicology Gui-Hong Chai,† Yingke Xu,‡ Shao-Qing Chen,† Bolin Cheng,† Fu-Qiang Hu,† Jian You,† Yong-Zhong Du,† and Hong Yuan*,† †
Institute of Pharmaceutics, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China Key Laboratory of Biomedical Engineering of Ministry of Education, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China
‡
S Supporting Information *
ABSTRACT: Solid lipid nanoparticles (SLNs) have been extensively investigated and demonstrated to be a potential nanocarriers for improving oral bioavailability of many drugs. However, the molecular mechanisms related to this discovery are not yet understood. Here, the molecular transport mechanisms of the SLNs crossing simulative intestinal epithelial cell monolayers (Caco-2 cell monolayers) were studied. The cytotoxicology results of the SLNs in Caco-2 cells demonstrated that the nanoparticles had low cytotoxicity, had no effect on the integrity of the cell membrane, did not induce oxidative stress, and could significantly reduce cell membrane fluidity. The endocytosis of the SLNs was time-dependent, and their delivery was energydependent. For the first time, the transport of the SLNs was directly verified to be a vesicle-mediated process. The internalization of the SLNs was mediated by macropinocytosis pathway and clathrin- and caveolae (or lipid raft)-related routes. Transferrinrelated endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus were confirmed to be the main destinations of the SLNs in Caco-2 cells. As for the transport of the SLNs in Caco-2 cell monolayers, the results demonstrated that the SLNs transported to the basolateral side were intact, and the transport of the nanoparticles did not destroy the structure of tight junctions. The transcytosis of the SLNs across the Caco-2 cell monolayer was demonstrated to be mediated by the same routes as that in the endocytosis study. The ER, Golgi apparatus, and microtubules were confirmed to be important for the transport of the SLNs to both the basolateral and apical membrane sides. This study provides a more thoroughly understand of SLNs transportation crossing intestinal epithelial cell monolayers and could be beneficial for the fabrication of SLNs. KEYWORDS: solid lipid nanoparticles, oral drug delivery, cytotoxicology, vesicle-mediated process, Caco-2 cell monolayer, transport mechanisms
1. INTRODUCTION The oral drug delivery system is the most widely used and most readily accepted manner among the various routes of administration, as it offers many advantages, such as being painless and easily self-administered, resulting in a comparatively high patient compliance.1 The gastrointestinal tract (GIT), however, presents various chemical and enzymatic barriers that affects the oral delivery of unstable drugs.2 Poor gastrointestinal permeability, due to the epithelial cell monolayer, is another factor that significantly affects the oral bioavailability of numerous drugs.3,4 Consequently, an increasing number of highly innovative and sophisticated drug delivery systems have been designed to overcome these hindrances.5,6 © XXXX American Chemical Society
Nanoparticulate drug delivery systems, which have been employed to improve oral bioavailability, have gained more and more attention recently.7 Nanocarriers not only protect a drug from degradation by the harsh environment of the GIT but also provide an approach to overcome the issue of intestinal epithelial cell monolayers.8 Compared with naked drugs, nanoparticles could change the cellular internalized routes of the nanoparticle-loaded drugs and improve the delivery of these drugs across the intestinal epithelial cell monolayers.9 Received: January 21, 2016 Accepted: February 10, 2016
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DOI: 10.1021/acsami.6b00821 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 1. Schematic diagram and characterization of ODA-FITC-labeled and Au nanoparticle-loaded SLNs. (A1) Schematic diagram of ODA-FITClabeled SLNs. (A2) TEM results of ODA-FITC-labeled SLNs. (B1) Schematic diagram of Au nanoparticle-loaded SLNs. (B2) TEM results of Au nanoparticle-loaded SLNs.
were used to study the transport mechanisms of the SLNs across intestinal epithelial cell monolayers. These inhibitors used in our study have been validated elsewhere as exerting negligible cytotoxicity to cells compared with cell culture medium alone,15 confirming the safety of the inhibitors used during the process of investigation.
In the past several years, solid lipid nanoparticles (SLN) have been extensively investigated and developed as a potential nanocarrier for oral drug delivery.10 Similar to liposomes, lipid nanoemulsions, and micelles, SLNs could improve oral absorption of many drugs because of their encapsulation.11 Generally, SLNs were prepared by biodegradable and biocompatible ingredients and have been developed as an oral drug delivery vector to load various fragile active pharmaceutical ingredients. Orally administered drug loaded into SLNs has numerous benefits, such as preferable physicochemical stability, better constant plasma drug concentration, more controlled drug release, and superior targeting therapeutic effects.12,13 Because of the variable ingredients, surface character, or particle size, the mechanisms of various nanoparticles crossing intestinal cell monolayers have been reported to be significantly different.14 Therefore, a detailed understanding of such an excellent nanocarrier for drug delivery across intestinal epithelial cell monolayers is significant for fabrication of SLNs to improve the oral bioavailability of the incorporated drugs. Few studies have been reported referring to the related transport mechanisms of the SLNs in intestinal epithelial cells to date. The delivery of nanoparticles across intestinal epithelial cell monolayers is very complicated, and this process includes the nanoparticles endocytosed via the apical membrane, endocellularly delivered, and eventually exocytosed from the basolateral membrane (namely, transcytosis).15 An intensive study of the transport process involved in the delivery of SLNs across intestinal epithelial cell monolayers will give us some useful guidance for developing optimized SLNs with good absorption and superior oral bioavailability. In our previous research using SLNs as a drug delivery system, cellular internalization,16,17 delivery mechanisms in MDCK cells,18 in vivo pharmacokinetic characteristics,12 and treatment effects19 were systematically studied. However, an intensive investigation of the transport mechanisms of SLNs across the gastrointestinal epithelial cell monolayer had not yet been performed. In this work, the SLNs (including ODAFITC-labeled and Au nanoparticle-loaded SLNs) were fabricated, and Caco-2 cells were selected to form simulative intestinal epithelial cell monolayers to study transport mechanisms related to the crossing process. First, cytotoxicology of the SLNs in Caco-2 cells was systematically investigated. Subsequently, different inhibitors and several novel techniques
2. RESULTS AND DISCUSSION 2.1. Properties of ODA-FITC-Labeled and Au Nanoparticle-Loaded SLNs. Octadecylamine-fluorescein isothiocyanate (ODA-FITC) has previously been validated to be an excellent fluorescent marker for the SLNs,20 and a schematic diagram of an ODA-FITC-labeled SLNs is illustrated in Figure 1A1. Au nanoparticles were loaded into the SLNs to make them detectable under transmission electron microscopy (TEM), and a schematic diagram of a Au nanoparticle-loaded SLNs is illustrated in Figure 1B1. The characterization of ODAFITC-labeled and Au nanoparticle-loaded SLNs was conducted using dynamic light scattering (DLS). The diameter and polydispersity index (PI) of these two types of nanoparticles were 86.7 nm (PI: 0.128) and 91.6 nm (PI: 0.143), respectively. The Zeta potential of these two types of nanoparticles was −28.78 and −27.34 mV, respectively (Figure S1). The TEM results are illustrated in Figure 1A2 and B2. These results indicated that the particle sizes of these two types of nanoparticles were identical (
