Intestinal permeability of oxytetracycline from chitosan-montmorillonite nanocomposites

Highlights

• Montmorillonite-chitosan nanocomposites were loaded with oxytetracycline.

• The loaded nanocomposites were internalized by Caco-2 cells.

• The loaded nanocomposites avoided P-glycoprotein effect on drug absorption.

Abstract

A nanocomposite based on chitosan and montmorillonite was developed as carrier to improve oral bioavailability of oxytetracycline. The nanocomposite was prepared by simple solid–liquid interaction and loaded with the drug. The loaded nanocomposite was characterized by X-ray powder diffraction, thermal analysis, FTIR spectroscopy and zeta potential. Caco-2 cell cultures were used to evaluate in vitro cytotoxicity and drug permeation. Confocal laser scanning microscopy was also performed to evaluate the eventual entrapment of drug into the Caco-2 cells. Results showed that the nanocomposite was internalized into the cells and effectively enhanced drug permeation, being also biocompatible towards Caco-2 cells.

Introduction

Oxytetracycline is a commonly used broad-spectrum antimicrobial agent with bacteriostatic activity. Oral absorption of OXT is incomplete and very variable among species [1], [2], [3], [4]. As a result, high oral dosages are needed to obtain effective plasma concentrations for the treatment of systemic infections. It has been also demonstrated that oxytetracycline is a substrate for the efflux transporter P-glycoprotein, with a detrimental effect on oral drug bioavailability [5].

Transport of drugs across the intestinal epithelium occurs mainly by one or more of the following mechanisms: passive transcellular diffusion, paracellular diffusion, active carrier mediated transport and endocytosis–transcytosis. Intestinal transcytosis is a kind of endocytosis in which the material internalized by cellular epithelium membranes is transported through the cell and secreted on the opposite side [6]. The normal absorptive epithelium has the possibility to transcytose particles by one of these mechanisms: pinocytosis, phagocytosis, macropinocytosis and protein-mediated endocytosis, allowing cellular internalization of particulate matters ranging from few nanometre up to several micrometre [7].

Polymeric materials have been extensively used to obtain nanoparticles with surface properties able to interact with epithelium components, promoting bioavailability of poorly absorbable drugs [8]. It is known that chitosan, a biocompatible polycationic polysaccharide, can act as absorption enhancer, affecting both paracelular and transcellular transport. In particular, chitosan solutions improve paracellular transport of drugs through interactions with cell membranes [9] as well as with Ca²⁺ and proteins involved in the structure of tight junctions [10]. The polymer may also induce reorganization of the actin cytoskeleton in Caco-2 cells [11]. Transcellular absorption is increased by activation of a chloride–bicarbonate exchanger involved in intracellular pH regulation [12]. Nevertheless, the effect of chitosan on drug permeability is pH-dependent, with a limited effectiveness at neutral and alkaline pH values [13]. To overcome this limitation, soluble chitosan derivatives have been developed [14], [15]. Chitosan nanoparticles have been also proposed to improve oral drug absorption via endocytosis and transcytosis transport [16], [17]. The addition of clay particles into a polymer matrix can alternatively provide high-performance novel nanoscale materials (clay mineral polymer nanocomposites) that find many applications in pharmaceutical field [18], [19]. In particular, chitosan has been intercalated via ionic exchange into negatively charged montmorillonite interlayers [20]. Montmorillonite/chitosan nanocomposites have been successfully used to reduce toxicity and improve bioavailability of 5-fluorouracil [21].

Caco-2 cell line is an established model to investigate the mechanisms involved in drug absorption, as the cells grown as confluent monolayers, form tight junctions, and thus resemble the intestinal barrier [22]. Caco-2 cells express morphological and biochemical characteristics similar to those of healthy human enterocytic cells, even if they are smaller than enterocytes [23]. Depending on the chemical characteristics of the compound under consideration, passive diffusion and active transport, contributing to absorption and secretion through a Caco-2 cell monolayer, can be measured [24], [25]. Consequently, Caco-2 cell cultures are extensively used in studies aimed to evaluate both the permeability of drugs through the gastrointestinal wall and to evaluate cytotoxicity of the different components of a given formulation [26].

With these premises, aim of this paper was to assess the suitability of a chitosan-montmorillonite nanocomposite to improve oxytetracycline bioavailability via oral route. The nanocomposite should enhance drug permeation by overcoming the interaction between oxytetracycline and P-glycoprotein, thus because the absorption of the drug mediated by nanocarriers should be mainly due to the transcytosis transport. The polymer clay nanocomposite was prepared by simple solid–liquid interaction, resulting in a biocompatible nanoscale material able to effectively stimulate cell proliferation, as it was previously demonstrated [27]. The obtained nanocomposite was loaded with oxytetracycline and Caco-2 cell model was used to evaluate the penetration enhancement properties of the nanocarrier towards the drug.