Elsevier

Biomaterials

Volume 34, Issue 3, January 2013, Pages 794-806
Biomaterials

Transferrin receptor specific nanocarriers conjugated with functional 7peptide for oral drug delivery

https://doi.org/10.1016/j.biomaterials.2012.10.003Get rights and content

Abstract

In an attempt to increase the interaction of a nanocarrier system with gastrointestinal epithelial cells, a transferrin receptor (TfR) specific 7peptide was conjugated to PEG-b-PCL copolymer and the functional nanocarriers were constructed and characterized. The endocytosis, intracellular trafficking and transcytosis of such nanocarriers loaded with coumarin 6 (7pep-M-C6) in a human colon carcinoma cell line (Caco-2) were investigated, followed by the in vivo intestine distribution study. The real-time imaging of live cell, three dimensional reconstruction of confocal image, quantitative colocalization analysis and other techniques were applied. First, the TfR expression was confirmed in Caco-2. Then, 7pep-M-C6 exhibited higher intracellular uptake compared with unmodified nanocarriers. In a live cell study, 7pep-M-C6 demonstrated faster uptake kinetics especially in the surface of cells. Together with a competition study using TfR antibody, it was proved that the increased cellular uptake was due to a receptor-mediated mechanism. Besides the unspecific endocytosis pathway, 7pep-M-C6 was found to enter the cells through a specific clathrin-mediated mechanism, related to the expression of TfR on Caco-2 cells. Possibly for this reason, 7pep-M-C6 tended to colocalize more with late endosomes and lysosomes than the control micelles. Also for the same mechanism, the increased transport of 7pep-M-C6 across Caco-2 monolayer was found, through a transcellular but not a paracellular pathway, while an increased in vivo intestinal distribution of 7pep-M-C6 was observed. In conclusion, the functional nanocarriers could specifically interact with gastrointestinal endothelial cells, increase their transport and alter their pathway as a result.

Introduction

Epithelial cells form the covering of various tissues, including the skin, urinary tract and intestines. They function as the main biological barrier in human body and are involved in a number of important physiological events like nutrient absorption and ion transport. These cells are highly polarized with their two surfaces (apical surface and basolateral surface) facing different physiological environments [1]. Due to the presence of small intestinal epithelium, the bioavailability of many orally administrated drugs is hindered due to their improper solubility and permeability across the cellular membrane. Several strategies have been employed to tackle this obstacle, such as synthesis of prodrug or reduction of drug particle size [2], [3]. In recent years, with the booming of nanotechnology, the application of nanocarriers has been proposed as a new approach to enhance the oral absorption of drugs with low solubility and permeability [4], [5], [6].

Nanocarriers such as nanoparticles, liposomes and micelles could enhance the oral bioavailability of drugs through the high degree of dispersion [7], [8], [9]. Micelles can be constructed from self-assembling of amphiphilic block copolymers and they offer several advantages in the delivery of therapeutic agents [9], [10]. Generally speaking, they are composed of a hydrophilic shell which provides steric stability and aqueous solubility, and a hydrophobic core which serves as a reservoir for poorly water-soluble drugs [11]. Among polymers commonly used to construct micelles, poly (ethylene glycol)-block-poly (ε-caprolactone) (PEG-b-PCL) has manifested a number of attractive features, including lower toxicity, biodegradability and biocompatibility [12].

The successful application of nanocarriers as the oral drug delivery system is highly dependent on their interaction with gastrointestinal (GI) epithelial cells [13]. Various methods have been employed to investigate the interaction of carriers with the intestinal membrane and Caco-2 cells remain a good choice of cell models [14]. When grown on semipermeable filters, Caco-2 cells differentiate into confluent monolayers that possess many of the ultrastructure and biochemical properties of mature enterocytes [15]. Several characteristics, including the exhibition of well developed microvilli and a polarized exhibition of brush border enzymes [16], have made it an ideal in vitro cell model for the study of drug absorption and transport in intestinal epithelium and hence have attracted a lot of research interests.

To enhance the cellular uptake and transport of orally administrated drugs, receptor-mediated endocytosis is supposed to be beneficial [17]. However, there were limited reports on how targeting moieties could affect the cellular uptake and transport of micelles across the intestinal epithelium [18], [19]. The transferrin receptor (TfR) is a transmembrane glycoprotein composed of two identical subunit monomers [20]. TfR-directed targeting has achieved efficient delivery of therapeutic agents to malignant tissues after iv administration based on the overexpression of TfR on the surface of tumor cells [21], [22], [23]. Besides, it is worth noticing that the TfR also exists in appreciable amounts in small intestine epithelium [24]. Due to polarization of epithelial cells, TfR is primarily distributed on the basolateral surface in differentiated cells, which presents an obstacle for receptor mediated endocytosis. Up to now, only transferrin conjugates were proved to be efficient in transcytosis across Caco-2 cell monolayer [25], [26], but related mechanisms were not clearly elaborated. 7peptide (7pep, Histidine- Alanine-Isoleucine-Tyrosine-Proline-Arginine-Histidine, HAIYPRH) is a peptide obtained through phage display and has exhibited high affinity to TfR [27]. However, its potential as a ligand of targeted drug delivery system has been barely reported.

Based on above background, it is supposed that the conjugation of 7pep on the surface of polymeric micelles may lead to their favorable uptake and transport in GI endothelial cells, providing a potential delivery system for orally administrated drugs. For the proof-of-concept, a functional material with TfR targeting ability was synthesized through the conjugation of 7pep to PEG-b-PCL copolymer for the first time, and then the functional nanocarriers were constructed by a solvent evaporation method using 7pep-PEG-b-PCL as one of the carrier materials. The endocytosis, intracellular trafficking and transcytosis of the functional nanocarriers loaded with coumarin 6 (7pep-M-C6) in a human colon carcinoma cell line (Caco-2) were conducted using unmodified micelles (M-C6) as the control, followed by the in vivo intestine distribution study. Especially, the mechanisms involved were explored as well.

Section snippets

Materials

N-hydroxysuccinimidyl-PEG4000-b-PCL2500 (NHS-PEG-b-PCL, Mw/Mn = 1.30 as determined by GPC) and mPEG3000-b-PCL2500 (Mw/Mn = 1.09 as determined by GPC) were purchased from Advanced Polymer Materials Inc. (Montreal, QC, Canada). Histidine- Alanine-Isoleucine-Tyrosine-Proline-Arginine-Histidine (HAIYPRH; 7pepside) was obtained from GL Biochem Peptide Ltd. (Shanghai, China). Chlorpromazine, sucrose, methyl-beta-cyclodextrin (MβCD), filipin and EIPA were purchased from Sigma–Aldrich (St. Louis, MO,

Synthesis of 7peptide conjugated PEG-b-PCL polymer

7peptide was conjugated to the distal end of PEG through a reaction between the NHS group and amino group (Fig. 1A). The conjugation reaction was monitored by HPLC. As shown in Fig. 1B, the retention time for 7peptide was around 30 min and after 120 h reaction the peak of 7peptide almost disappeared, indicating that 7peptide had been successfully conjugated to the polymer. The UV–Vis spectrum of the products is shown in Supplemental Fig. S1. There was an absorption peak of 7peptide at around

Conclusion

This study demonstrated that a kind of TfR-targeted functional nanocarriers increased the intracellular uptake, altered their intracellular trafficking and enhanced their tanscytosis in polarized Caco-2 cells. Flow cytometry and confocal microscope study showed a higher uptake of 7pep-M-C6 over M-C6 via the specific binding to TfR. Real time confocal imaging analysis of cellular uptake further validated this conclusion. Endocytosis pathway study indicated that both micelle systems were

Acknowledgments

This work was supported by the National Research Fund for Fundamental Key Project (2009CB930300) and the National Natural Science Foundation of China (81130059).

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