Elsevier

Chemical Engineering Journal

Volume 340, 15 May 2018, Pages 181-191
Chemical Engineering Journal

New active formulations against M. tuberculosis: Bedaquiline encapsulation in lipid nanoparticles and chitosan nanocapsules

https://doi.org/10.1016/j.cej.2017.12.110Get rights and content

Highlights

  • Bedaquiline was successfully encapsulated in different nanoemulsion-based nanocarriers.

  • High encapsulation efficiency and in vitro activity comparable to the free drug were observed.

  • No cytotoxicity was observed at nanocarrier concentrations needed to kill the bacteria.

  • Preliminary microscopy results showed a close interaction of carriers with bacteria surface.

  • Results open the way for a future safer employment of bedaquiline against M. tuberculosis.

Abstract

In the last years, the increase in antimicrobial resistance, together with a lack of new drugs for the treatment of bacterial infections resistant to classical antibiotics are of growing concern. Moreover, some of current therapies induce severe side effects and are often difficult to administer. In 2012 the FDA approved the use of bedaquiline, as the first new very effective drug against TB in the last 40 years. Despite its effectiveness, unfortunately bedaquiline side effects can be so dangerous that at present it is to be prescribed only when no other treatment options are available. The development of effective and safe nanotechnology-based methods can be particularly relevant to increase antimicrobial concentration at the site of infection, to reduce doses in the general circulation, which in turn reduces adverse effects. In this work bedaquiline was encapsulated in two types of nanocarriers, lipid nanoparticles and chitosan-based nanocapsules with high encapsulation efficiency and drug loading values. The efficacy of the drug-encapsulating nanocarriers has been demonstrated in vitro against Mycobacterium tuberculosis, together with the excellent compatibility of both carriers with animal cells. The obtained results open the way for further studies on multi-drug resistant strains of M. tuberculosis and for in vivo studies of the optimized nanocarriers. The promising behaviour of drug-loaded nanocarriers will hopefully lead to a reduction of the administered doses of a quite dangerous drug as bedaquiline, tuning its biodistribution and so decreasing its adverse effects, finally allowing its use in a higher number of patients.

Introduction

Novel drug delivery systems based on nanocarriers are a promising strategy to overcome current therapeutic limitations thanks to nanomaterials unique physicochemical properties. These include their small size, which allows them to reach the cellular level, their high surface to volume ratio, which increases interactions with target cells and their ability to be structurally and functionally modified to control their biodistribution. In addition, nanocarriers allow the improvement of aqueous solubility of poorly soluble drugs, the drug protection in order to avoid its degradation before reaching its target, its selective transport to the sites of infection and the controlled release of the medication to decrease the frequency of administration [1].

Many studies have been carried out for different anti-TB drugs, showing the success and usefulness of this technology to improve the treatment of tuberculosis [2].

The increase in antimicrobial resistance observed in last years is of growing concern worldwide and only thirteen new antibacterial agents were approved by the Food and Drug Administration (FDA) between 1999 and 2011 [3]. Mycobacterium tuberculosis (TB) have developed a high level of drug resistance to antimicrobial drugs. Multidrug resistant TB (MDR-TB) and extensively drug-resistant TB strains (XDR-TB, a form of MDR-TB with additional resistance to any of the fluoroquinolones and to at least one of three injectable second-line) have been identified in 105 countries worldwide, and they are making increasingly difficult to successfully treat tuberculosis. Drugs available for the treatment of bacterial infections resistant to antimicrobial drugs are very limited, induce severe side effects and/or are difficult to administer, so they could require parenteral injection [4]. Moreover the high costs of these treatments and their low success rates should be taken into account [5].

Bedaquiline, a diarylquinoline antimycobacterial drug, the first FDA approved anti-TB drug in four decades, is a very effective drug, nonetheless it shows serious side effects including induction of life-threatening cardiac arrhythmias [5]. Therefore, this drug is to be prescribed only when no other treatment options are available [4], [6].

The development of effective and safe nanotechnology-based methods would be particularly relevant to improve the safety of this drug for the treatment of drug-resistant infections. In fact, the use of a carrier can increase the local concentration of the drug at the site of infection, enabling lower doses in the general circulation, which in turn reduces the adverse effects.

Nanoemulsion-based nanocapsules present a lipidic core highly suitable for the encapsulation of a very hydrophobic molecule such as bedaquiline. For this reason, two bedaquiline-loaded nanocarriers have been developed in this work, consisting in a polymeric shell that surrounds a liquid or solid lipidic core where the drug is confined [7]. They are nanoemulsion-based chitosan nanocapsules (CS-NC), consisting in an oily core with a nanogel polysaccharidic shell grafted or not with polyethylene glycol molecules following a method previously described in literature by the same authors [8], and lipid nanoparticles (LNPs), made of a lipid core of long chains triglycerides and surrounded by a surfactant shell made of a polyethylene glycol based surfactant, phospholipids [9] and eventually cationic lipids in order to confer them a cationic character [10]. In both cases the polymeric coating has been used for nanocapsule stabilization [11].

In view of a possible finding of the optimal delivery system for bedaquiline, the two carriers have been designed for future, different administration route, being chitosan-based nanomaterials more suitable for nebulization and lipid nanoparticles very desirable for intravenous injection.

In the case of chitosan nanocapsules, bedaquiline has been encapsulated in the lipidic core and nanoemulsion was stabilized through chitosan coating by ionotropic gelation process. The same chitosan-based nanocapsules have been further grafted with a polyethylene glycol (PEG) layer to improve their colloidal stability in biologically interesting media. Among the high number of biopolymers used in drug delivery applications (alginate, hyaluronic acid, pectine, albumin, fibroin, etc.) [12], [13], chitosan is widely used in nanomedicine because of the excellent biocompatibility and safety of its degradation products [14]. Chitosan has been chosen as nanoemulsion stabilizer since it is very rich in amino groups, which makes it useful to favour interaction of nanocarrier and respiratory tract mucose. Mucoadhesive properties of chitosan are especially important for aerosol administration for pulmonary infections [15], [16].

LNPs have been chosen as promising nanocarriers for this work because they gather several advantages for intravenous administration. They are composed of molecules generally regarded as safe, display no cellular toxicity and present long term circulation time due to their high colloidal stability and to the furtivity provided by the PEG shell [17], [18]. LNPs with two different surface charges were studied, particles displaying a slightly negative surface charge (hereafter referred as LNPs(−)), due to the presence of zwitterionic lecithin molecules, and cationic ones (LNPs(+)), containing DOTAP, a molecule with a quaternary ammonium polar head group. Both types of LNPs exhibited a PEG-rich outer shell, which is known to be a good strategy for drug delivery in the field of nanomedical therapy [19].

The encapsulation of bedaquiline has been optimized in the case of both carriers, upon quantification of drug loading and encapsulation efficiency. The resulting bedaquiline-loaded nanocapsules have been also characterized in terms of their physicochemical properties, since the knowledge of such characteristics is fundamental for the prediction and control of the interaction of nanomaterials with biological media and their toxicity [20]. Finally, chitosan nanocapsules and lipid nanoparticles have been compared in terms of their behaviour in biological systems. In particular, their in vitro antibacterial efficacy towards M. tuberculosis and their cytotoxicity in human cells have been established, prior to more in depth studies on multi-drug resistant TB strains and in vivo infected models.

Section snippets

Materials

Tween® 20 and absolute EtOH, were purchased from Panreac Química S.L.U (Barcelona, Spain). Span® 85 (sorbitanetrioleate), oleic acid and chitosan (medium molecular weight) were purchased from Sigma-Aldrich Pte. Ltd. (Singapore). Bedaquiline was obtained from AURUM Pharmatech LLC (Franklin Park, NJ, USA). Bis(sulfosuccinimidyl) suberate (BS3) was purchased from Pierce Biotechnology Inc. (Rockford, IL, USA) and α-methoxy-ω-amino poly(ethylene glycol) (PEG-MW 5000 Dalton) from IRIS Biotech GmbH

Nanocarriers synthesis

Due to the lipophilic character of bedaquiline, the lipidic core of both LNPs, made of vegetable oil and wax, and of CS-NC, based on non-ionic surfactant/oleic acid micelles, have been found to be highly suitable for the encapsulation of this drug.

In this study the synthesis and characterization of the two kinds of nanocarriers is reported, from both a physicochemical and biological point of view, with the final aim of proposing them as candidates for future in vivo administration in the

Conclusions

Nanocarriers developed in this work were able to encapsulate bedaquiline, a very effective, although dangerous anti-TB drug, with excellent efficiency and very good drug loading values and the work represents the first bedaquiline encapsulation, at the best of our knowledge. The aim of the work was to obtain the development and fundamental physicochemical characterization of this promising nanocarriers that would open the way in the future for more in depth studies to verify the activity of

Acknowledgements

Authors would like to acknowledge the public funding from Fondo Social de la DGA (grupos DGA), Ministerio de la Economía y Competitividad del Gobierno de España for the public founding of ProyectosI + D + i – ProgramaEstatal de Investigación, Desarrollo e InnovaciónOrientada a los Retos de la Sociedad (project n. SAF2014-54763-C2-2-R), the European Seventh Framework Program (NAREB Project 604237) and Ministerio de Educación Cultura y Deporte for SGE fellowship. We also want to acknowledge Dr.

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