A common strategy to improve transmembrane transport in polarized epithelial cells based on sorting signals: Guiding nanocarriers to TGN rather than to the basolateral plasma membrane directly

Abstract

The intestinal barrier has always been the rate-limiting step in the oral administration process. To overcome the intestinal barrier, researchers have widely adopted nanocarriers, especially active-targeting nanocarriers strategies. However, most of these strategies focus on the ligand decoration of nanocarriers targeting specific receptors, so their applications are confined to specific receptors or specific cell types. In this study, we tried to investigate more common strategies in the field of transmembrane transport enhancement. Trans-Golgi network (TGN) is the sorting center of biosynthetic route which could achieve polarized localization of proteins in polarized epithelial cells, and the basolateral plasma membrane is where all transcytotic cargos have to pass through. Thus, it is expected that guiding nanocarriers to TGN or basolateral plasma membrane may improve the transcytosis. Hence, we choose sorting signal peptide to modify micelles to guide micelles to TGN (named as BAC decorated micelles, BAC-M) or to basolateral plasma membrane (named as STX decorated micelles, STX-M). By incorporating coumarin-6 (C6) or Cy5-PEG-PCL in the micelles to indicate the behavior of micelles, the effects of these two strategies on the transcytosis were investigated. To our surprise, BAC-M and STX-M behaved quite differently when crossing biological barriers. BAC-M showed significant superiority in colocalization with TGN, transmembrane transport and even in vivo absorption, while STX-M had no significant difference from blank micelles. Further investigation revealed that the strategy of directly guiding nanocarriers to the basolateral plasma membrane (STX-M) only caused the stack of vesicles near the basolateral plasma membrane. So, we concluded that guiding nanocarriers to TGN which related to secretion may contribute to the transmembrane transport. This common strategy based on the physiological function of TGN in polarized epithelial cells will have broad application prospects in overcoming biological barrier.

Introduction

Biological barriers developed during the long-term evolution of organisms to maintain normal body activities and prevent body from foreign objects [1], which also hinders the transport of therapeutic drugs. Nanodrug delivery systems are widely used to deliver drugs to cross biological barriers [[2], [3], [4]]. In order to further improve the absorption efficiency, some researchers adjusted the physical and chemical properties of nanocarriers [5,6], such as changing particle size or shape [7] and surface charge [8]. Also, researchers decorated ligands on the surface of nanocarriers to make them be recognized by receptors on cell surface [9,10]. Although these methods all showed significantly enhanced cell uptake, only few nanocarriers could pass through the epithelial or endothelial cell layer. This phenomenon is called “Easy Entry, Hard Transcytosis” [[11], [12], [13], [14]]. Due to this phenomenon, the transport efficiency of nanocarriers is quite low, which limits the therapeutic possibilities of nanomedicines. Recently, some studies have focused on improving the transmembrane transport by designing nanocarriers to target intestinal transport receptors [15], by using the pathways of intestinal nutrient transport [16,17] or by enhancing the chylomicron pathway [18], etc. These new strategies did significantly increase the transmembrane transport, but they mostly focused on ligand decoration of nanocarriers for specific receptors, so their application would be limited to specific receptors or specific cell types. To overcome these shortcomings, in this study, we tried to investigate common strategies to improve the transmembrane transport by designing nanocarriers according to characteristics of polarized epithelial cells.

Epithelial cells are polarized structurally and functionally to direct specific molecules in and out of cells to maintain transepithelial barrier. This selective transport is achieved by dividing the plasma membrane into different apical and basolateral domains, which have different lipid compositions and specific proteomes [19]. In general, apical membrane is rich in cholesterol, sphingolipids, and glycolipids, and contains a variety of specific, highly glycosylated proteins, while basolateral plasma membrane is rich in E-cadherin, integrin molecules and growth factor receptors [20], etc.

Polarized localization of proteins to apical or basolateral plasma membrane in epithelial cells is achieved through a variety of transport routes, including biosynthetic route, endocytic recycling route, and transcytosis route. In these routes, trans-Glogi network (TGN) and common recycling endosomes (CRE) are two important sites for sorting proteins to corresponding membrane fields. In the biosynthetic route, the transport of newly synthesized apical and basolateral plasma membrane proteins starts from endoplasmic reticulum (ER), successively to cis-Golgi network (CGN), TGN, and then be sorted into different vesicles and transported to correct membrane fields, or sorted into different endosomes and then transported to apical or basolateral plasma membrane field to be secreted [[21], [22], [23]]. In the endocytic recycling route, some proteins are internalized and enter sorting endosomes and transfer to corresponding surface via recycling endosomes or CRE [24], while in the transcytosis route, some proteins transport from apical plasma membrane to basolateral plasma membrane, or vice versa [25,26].

Syntaxins are members of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. With the polarization of epithelial cells, the t-SNARE protein Syntaxin3 is transferred to apical plasma membrane, while Syntaxin4 is transferred to basolateral plasma membrane. Researchers found that the region between the N-terminal residues 24–29 (ALVVHP) is essential for the basolateral sorting of Syntaxin4 [27,28]. So we considered this domain as a signal peptide that guides syntaxin4 to be transported to the basolateral plasma membrane.

Based on these general characters of polarized epithelial cells, we planned to use two strategies to increase the transmembrane transport of nanocarriers: (1) guiding nanocarriers to TGN and then be transcytosed through the biosynthetic route, and (2) guiding nanocarriers directly to the basolateral plasma membrane then be transcytosed. Researches revealed that phosphorylated BACE-1 (a β-secretase playing a key role in the pathogenesis of Alzheimer's disease [29,30]) is located in TGN and the cytoplasmic domain (DDIDLL) is essential for correct positioning of BACE-1 [[31], [32], [33]]. Thus, we considered modifying it (both ends were extended by one amino acid residue, which was ADDIDLLK, named as BAC) to nanocarriers' surface as a tool to explore the feasibility of guiding nanocarriers to TGN to be sorted through the biosynthetic route, and achieve the transcytosis improvement by secretion of TGN. In addition, we considered modifying signal peptide of Syntaxin4 (both ends were extended by one amino acid residue, which was VALVVHPG, named as STX) to nanocarriers' surface as a tool to explore whether guiding nanocarriers directly to the basolateral plasma membrane could improve transcytosis.

Poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL) was selected as the nanocarrier material because of its good hydrophilicity, biocompatibility and strong ability to encapsulate poorly soluble drugs. BAC and STX decorated PEG-PCL micelles in different modification density were prepared to find out how the sorting signal peptide's density affect the endocytosis and transcytosis pathway of micelles. Blank micelles were referred as Blank-M, BAC decorated micelles were referred as BAC-M and STX decorated micelles were referred as STX-M. All micelles had coumarin-6 (C6) entrapped in them or had Cy5-PEG-PCL added during preparation process as fluorescence label. First, blank micelles and functional micelles were prepared by film hydration method and were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Then, the uptake, endocytic mechanism and colocalization with organelles were studied. Next, we used Caco-2 monolayer cell model to investigate transcytosis and intracellular pathway of micelles. Also, TEM was used to visualize vesicles in Caco-2 cell monolayer. Last, we tested C6 concentration in blood after oral administration in SD rats to reveal whether our strategies could improve oral bioavailability of micelles in vivo. As shown in Scheme 1, our study revealed that guiding micelles to basolateral plasma membrane directly is not conducive to transmembrane transport, while guiding micelles to TGN is an effective way to improve transcytosis of micelles, which provides a common strategy to improve the transmembrane transport of nanocarriers.