Layer-by-layer surface modification of lipid nanocapsules

Abstract

Lipid nanocapsules (LNCs) were modified by adsorbing sequentially dextran sulfate (DS) and chitosan (CS) on their surface by the layer-by-layer (LBL) approach. Tangential flow filtration (TFF) was used in intermediate purifications of the LNC dispersion during the LBL process. The surface modification was based on electrostatic interactions between the coating polyelectrolytes (PEs) and the LNCs. Therefore, a cationic surfactant, lipochitosan (LC), was synthesised by coupling stearic anhydride on chitosan, and the surface of LNCs was first modified by this LC by the post-insertion technique. The PEs could be successfully adsorbed on the LNC surface as verified by alternating zeta potential and increase in size. To present a therapeutic application, fondaparinux sodium (FP), a heparin-like synthetic pentasaccharide, was introduced on the LNC surface instead of DS.

Introduction

Since the introduction of layer-by-layer (LBL) surface modification technique [1] to colloidal objects [2], coating of nanosized drug carriers by polyelectrolytes (PEs) has become a strategy of increasing interest during the latest years. The first LBL studies applied synthetic PEs such as poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) [2], [3], but later the selection of materials has been expanded toward biocompatible and thus more convenient PEs for pharmaceutical purposes. These macromolecules include chitosan, chitosan sulfate, alginate, dextran sulfate and heparin [4], [5], [6], [7], [8]. The goal of an LBL process can be improvement for the stability of the core particles [8], [9] or control of the release of an encapsulated substance [6], [7], [10]. Also, macromolecules such as DNA and RNA, with therapeutic interest, have been used as coating agents [8].

The principle of the LBL coating is to sequentially expose the substrate to be coated to solutions containing either negatively or positively charged PEs. Adsorption of the PEs is based on electrostatic interactions originating from the charged nature of the substrate and the PEs. Thickness and properties of the coating film can be controlled by the number and composition of the layers and the process conditions. After each coating layer, removal of the excess PE in the dispersing medium is crucial to avoid aggregation due to the formation of unwanted PE complexes. In most of the studies, this is done by intermediate ultracentrifugations followed by the replacement of supernatant [2], [4], [6], [7]. To avoid the force of ultracentrifugation leading to particle aggregation in some cases, another used purification approach is ultrafiltration [3], [5], [11], [12]. In this study, tangential flow filtration (TFF) [13] was applied as an intermediate purification method.

Lipid nanocapsules (LNCs) are synthetic lipoproteins, with tuneable size between 20 and 100 nm, and offer a versatile approach to drug delivery [14], [15]. LNCs are prepared by a solvent-free, low-energy phase inversion temperature process. Such a method enables a straightforward scale-up of the process with pilot scale equipment, at least up to 50-fold the volume (unpublished data). Their structure can be characterized as a hybrid between polymeric nanocapsules and liposomes (an oily core with a shell consisting of a mixture of lecithin and a PEGylated surfactant) with good dispersion stability (up to 18 months). Because of their structure which includes a semi-rigid shell, LNCs can be further modified by insertion of amphiphilic molecules. The motivation for this kind of post-insertion can be improvement of biodistribution [16], [17], [18], targeting profile [19] or creation of a template for further attachment of active targeting or other moieties [20], [21], [22].

Chitosan is a natural material possessing great potential to be used in pharmaceutical applications (reviewed e.g. in [23]). If hydrophobic chains are introduced to chitosan using a method such as acylation [24], [25], the modified chitosan can be used as a hydrophilic cationic surfactant. This study presents synthesis of an amphiphilic chitosan derivative, lipochitosan (LC), and its introduction to the LNC surface by the post-insertion technique. The positive charge provided by LC enabled further modification of the surface by the LBL technique using dextran sulfate (DS) and chitosan oligosaccharide (CS) as model PEs (Fig. 1). Being biocompatible and highly charged macromolecules, DS and CS are suitable choices for pharmaceutical (self-assembling) systems: these two PEs have been previously used e.g. as PE complexes [26] and in LBL coating of nanoparticles [4] and liposomes [12]. Due to the semi-solid structure of LNCs, their purification by a process involving a high stress toward the capsules (ultracentrifugation) might lead to destabilisation of LNCs. Therefore, TFF was introduced in the process to purify the LNC dispersion after each layer. To our knowledge, TFF has not been used as a tool in the LBL coating before. To present a possible therapeutic application, fondaparinux sodium (FP), a synthetic pentasaccharide (Fig. 1) used in the treatment and prophylaxis of venous thromboembolism, was introduced on the LNC surface instead of DS. Treatment by a drug like FP, normally administered subcutaneously, would benefit from oral administration. As LNCs are known to enhance the oral absorption of drug molecules [27], they could serve in avoiding degradation and enhancing absorption of FP in the gastrointestinal tract.