Reverse micelle-loaded lipid nanocarriers: A novel drug delivery system for the sustained release of doxorubicin hydrochloride

Abstract

In this study, we are pioneering new nanotechnology for the encapsulation of anticancer drugs (doxorubicin (DOX) and/or docetaxel (DOCE)), whatever their solubility and water affinity. The purpose of this study is to highlight the potential of this recently patented technology, by carrying out a thorough physicochemical characterisation of these multiscaled nanocarriers, followed by the study of an encapsulation and release model of hydrophilic anticancer drug. The formulation process is based on a low-energy nano-emulsification method and allows the generation of a structure composed of oil-based nanocarriers loaded with reverse micelles. Thanks to this, hydrophilic contents can be solubilised in the oily core of this kind of nano-emulsion along with lipophilic content. The results emphasise some original structure particularities due to the multistep formulation process, and the diffusion-based behaviour revealed for the DOX release profile that is shown to be intimately linked to the morphology of the particles.

Introduction

Doxorubicin hydrochloride (DOX) is commonly used in the treatment of many solid tumours, such as breast, lung, stomach or ovarian cancer and sarcoma. It inhibits cell growth by DNA intercalation, the generation of free radicals, interaction with cellular membranes and the inhibition of topoisomerase II [1]. Unfortunately, this anthracyclin has appeared to be highly toxic, by biodistribution to non-targeted tissues (heart mainly), causing severe side effects, limiting DOX dosage and harming to the patients comfort [2]. Therefore, in order to get round this critical point, research has focused on the development of new formulation strategies via DOX-encapsulated particulate drug delivery systems. CAELYX® provides an example of a commercial product, which is a DOX-containing liposomal PEGylated form available for the treatment of Aids-related Kaposi’s sarcoma, advanced ovarian cancer and advanced breast cancer. More generally, drug encapsulation in colloidal systems offers many advantages, such as: (i) the protection of the drug against in vivo degradation, (ii) the reduction in potential toxic side effects occurring with the direct administration of solution-solubilising anticancer drugs, (iii) the increase in patient comfort by avoiding repetitive bolus injection or the use of perfusion pumps, and (iv) the achievement of more favourable drug pharmacokinetics [3]. In this way, many studies have been carried out with doxorubicin, giving rise to various DOX-containing nanoparticles, such as polymeric nanocarriers [4], [5], [6], [7], [8], inorganic magnetic nanoparticles where DOX is adsorbed onto their surface [9], [10] or even solid lipid nanoparticles containing DOX as an ion-pair complex [11], [12].

Eventually, since DOX is a hydrophilic and hydrosoluble molecule, the main challenge of these formulations remains that of entrapping DOX with a significant encapsulation rate and yield into such nanoparticulate systems that are themselves in suspension in aqueous solutions. More generally, this scope is included in the general field of encapsulating hydrophilic contents in nanoparticulate systems and has hence been the subject of much research. The main difficulties arise in controlling the hydrophilic, encapsulated molecule release, while also finding a good compromise between encapsulation efficiency, drug leakage induced by diffusion and/or by material degradation, the biocompatibility and biodegradation of particle components and the formulation processes (in the case of encapsulating fragile contents, adopting soft processes for limiting degradation is necessary).

In this context, the original purpose of this study concerns the formulation of lipid nanoparticulate systems, which combine all these latter points. These formulations are based on lipid nano-emulsions [13], [14], [15], [16]. These nanoparticles, only formulated using biocompatible ingredients, are generated through a low-energy and solvent-free method (the phase inversion temperature method or PIT method) and present highly adjustable sizes [16] with a wide range of monodispersal size distribution. Accordingly, such a system is readily suitable for pharmaceutical applications. in vitro, and in vivo studies have disclosed for instance [17] an active role on the inhibition of the global multidrug resistance mechanism (MDR), hence increasing the inhibition of one ATP-efflux pump (P-glycoprotein) present in humans. This effect has been attributed to the PEG moiety of the non-ionic surfactant surrounding the nano-droplet surface [17]. In the case of anticancer drug administration, and a fortiori with DOX, this point is fundamental and reinforces the important role of PEGylated lipid nanocarriers.

The incorporation of hydrophilic or hydrosoluble contents in such lipid nano-objects is achieved by applying a simple idea that consists of incorporating a reverse micellar system to the nanocarrier lipid core since it solubilises hydrophilic molecule to be encapsulated [15], [18]. Reverse micelles (RM) are multimolecular entities of surfactant in a non-polar medium. Reverse micellar system has been widely studied and reviewed [19], [20], [21], [22], [23], [24] as it is a very simple and powerful system allowing the easy solubilisation of hydrophilic guests in oily phases. RM is a thermodynamically stable system; it forms spontaneously and is fully transparent. The most common application of RM is based on its use as a nano-reactor for nanoparticle formation or particular chemical syntheses [25]. A further potential of RM lies in their use as hydrophilic nano-reservoirs dispersed in a hydrophobic phase. Nevertheless, to date, except for a few examples [26], [23], this option has not been extensively developed, mainly due to the continuous oily phase not being well adapted to the specifications inherent in the field of drug delivery and targeting. We have got round this problem in our study by subdividing this continuous oil phase for creating a stable nano-suspension, encapsulating the RM and producing different sizes. This was actually performed by taking advantage of the PIT process, which induces the spontaneous formation of nanocarriers without drastic treatment, and thus preventing the destruction of reverse micelles during emulsification.

In addition to these new features (i.e. the encapsulation of hydrophilic contents), these lipid nanocarriers still keep their ability to encapsulate hydrophobic molecules in the full, oily core of the nano-droplets; hence hydrophilic and hydrophobic molecules are encapsulated and stable in the same core. We recently pioneered this concept on another kind of nanoparticulate system based on aqueous-core nanocapsules [27], and we propose here a new and simple alternative with different technology. The great advantage of this technology, when two different drugs are entrapped in the same nanoparticle, lies in the fact that their simultaneous location is assured in the targeted site, and this can be highly interesting when their concomitant action is needed or is necessary for the treatment. As a result, efficiency should be increased and adverse effects reduced. This specific, synergic anticancer effect in multidrug resistant human breast cancer cells has been evidenced with the simultaneous co-encapsulation of DOX and mitomycin C in solid lipid/polymeric hybrid nanocapsules [28]. We chose to associate a lipophilic antineoplastic agent belonging to the taxoid family (which is docetaxel (DOCE)) to the DOX. This anticancer drug is used in association with DOX for the treatment of certain forms of breast cancer [29].

The different aspects related to the formulation and characterisation of these reverse micelle-loaded nanocarriers will be dealt with first. Then, the study will focus on the evaluation of encapsulation and release properties, of DOX alone and DOX associated with DOCE. In this work, our aim is not only to present this new technology, but also to show that it allows the encapsulation of two major anticancer drug contents with biocompatible formulations.