Production of solid lipid nanoparticle suspensions using supercritical fluid extraction of emulsions (SFEE) for pulmonary delivery using the AERx system

Abstract

The aims of the current work included: development of a new production method for nanoparticles of water-insoluble drugs in combination with lipids, characterization of the nanoparticles and development of lipid nanosuspension formulations, and investigation of the feasibility of delivering the nanosuspensions as aerosols for inhalation using Aradigm's AERx® Single Dose Platform (SDP) with micron-sized nozzles and the all mechanical AERx Essence™ with sub-micron-sized nozzles. The continuous SFEE method was used for particle precipitation of solid lipid nanoparticles (SLN). The method allowed for production of stable particulate aqueous suspensions of a narrow size distribution, with a volume mean diameter below 30 nm (D99% cumulative volume below 100 nm). Thus the particle size obtained was significantly smaller than previously has been achieved by other techniques. The residual solvent content in the final suspension was consistently below 20 ppm. Drug loading values between 10–20% w/w drug were obtained for model compounds ketoprofen and indomethacin in formulation with lipids such as tripalmitin, tristearin and Gelucire 50/13. It was observed that the loading capacity achieved was higher than the thermodynamic limit of the solubility of the drugs in molten lipids. Lipid nanosuspension formulations were successfully aerosolized using both of the AERx systems. As measured by both cascade impactor and laser diffraction, the aerosol fine particle fraction (FPF) was comparable to drug solution formulations typically used in these devices; i.e., greater than 90% of the aerosol mass resided in particles less than 3.5 μm aerodynamic diameter.

Introduction

For both topical and systemic administration of drug through the pulmonary route, nanoparticulate formulations (where the dispersed phase is typically between 100–700 nm) offer several important advantages when compared to more traditional microparticles (1–5 μm) suspensions. If a water-insoluble drug is intended for immediate action, nanoparticles will result in a bioavailability enhancement, attributable to either better drug delivery, more rapid dissolution or increased residence time (increased bioadhesivity) in the lung [1]. The statistical homogeneity of nanosuspensions is also greatly superior to that of microparticulate suspensions. The mean number of particles in an average 4 μm droplet of a nanosuspension increases by three orders of magnitude when compared to a microsuspension, leading to significantly higher delivered dose and better dose uniformity [2]. For example, as shown in the case of budesonide nanosuspensions, both the delivered dose and respirable dose could nearly double with the use of nanosuspensions [3]. The small size of solid particles also gives the nanosuspension a solution-like rheology that facilitates the dispersion of sprays into smaller and more uniform droplets, particularly in the case of jet-nebulizers and perforated membrane inhalers. For successful inhaler or nebulizer operation it is essential that the size of the dispersed drug is significantly smaller than that of the micro-jet or the vibrating membrane size, in order to avoid the drug retention and to improve the drug delivery efficiency [4]. Another significant benefit of using nanosuspensions is the possibility of direct sterile filtration, which can eliminate the more expensive and other problematic sterilization techniques such as thermal treatment or gamma-irradiation [5].

In addition to the delivery of drugs for immediate release, another rapidly developing area of interest is the composite drug-carrier systems for respiratory delivery. Such particles are not just intended for sustained or controlled drug release but also for improved drug bioavailability, protection of sensitive drugs against degradation, and/or for prevention of fast removal of particles by the respiratory clearance system before sufficient drug absorption occurs [5]. The use of polymeric colloidal drug carriers in drug delivery is often limited by the unknown toxicity of the carrier in the lungs, e.g., many bioerodable polymers have not undergone toxicity testing for delivery via the pulmonary route [6]. This challenge has stimulated current research into pursuing alternative formulations for drug delivery, which utilize carriers of lower expected toxicity, or eliminate the carrier altogether. Examples include lipid carriers such as liposomes, lipid emulsions, lipid complexes and solid lipid nanoparticles (SLN) [5], [6], [7], [8]. Lipids are water insoluble molecules, typically saturated and unsaturated hydrocarbons that form the essential structure of all cell membranes. Most lipids are well tolerated by the human body and rapidly metabolize into non-toxic products that do not show side effects upon delivery through injections [6]. This biocompatibility makes lipid systems ideal for parenteral drug administration. Although their role for the respiratory delivery has yet to be assessed, because of the lack of parenteral side effects, their toxicological profile is expected to be better than for polymeric systems. It is feasible that aqueous suspensions and perhaps dry powder formulations of SLN can be used for pulmonary administration of drugs using liquid and dry powder inhalers. It has been suggested that excipients or carriers in these formulations have a faster biodegradation rate and higher tolerability in the lungs compared to particles made from some polymeric materials [6].

The functionality of drug-lipid particles is derived from their structure. A distinction is usually made between a carrier-based colloid systems such as SLN [9] and nanosuspensions that consist of predominantly pure crystalline drug, stabilized by surfactants. Although many lipids, notably phosphatidylcholines, can also be used as surfactants or stabilizing agents, the difference between drug nanosuspensions and lipid-drug nanoformulations is reflected in the amount of the carrier used. Typically less than 30% of the surfactants per drug w/w is required to stabilize the nanosuspensions with mean size between 120–300 nm [9–10]. It should be noted, however, that this amount is directly proportional to the specific surface area, i.e. should increase inversely proportional to the particles surface-equivalent (Sauter) diameter. It is presumed that nanosuspensions consist of small drug crystals with a relatively thin layer of surfactant coated on the crystal surfaces. Amorphous solids of small molecules are usually unstable and convert into their crystalline state upon contact with water, resulting in particle growth. In contrast, SLN may consist of the drug molecularly dispersed in the lipid carrier matrix [11]. However, many synthetic drugs, being insoluble in aqueous vehicles, are also practically insoluble in lipids with the saturation limit well below 1% w/w. This fact, based on equilibrium thermodynamics, may determine the loading capacity of the solid lipid matrixes. Another important criterion is the crystallinity of the lipid phase. Naturally, crystalline or semi-crystalline lipids have a lesser ability to form solid solutions with drugs than lipids that are amorphous. In principle, a structure can be obtained where the drug core is encapsulated into a lipid shell [12]. The drug loading in such systems may not be limited by the solubility of the drug in the lipid phase, but rather by the physical stability of such particles in a nanosuspension.

The particle formation mechanism and precipitation kinetics play a key role in defining the structure and resulting physical properties of the SLN. The structure of particles obtained from a drug dissolved in a molten lipid or from a solution containing both the drug and the lipid are not necessarily the same, even if the resulting phase composition and thermodynamic conditions are similar in both cases. This is because several powerful kinetic factors are involved in the particle formation: nucleation and precipitation mechanisms, rate of solvent extraction or solidification, influence of the surface-active agents, supersaturation profile in the drug-lipid-solvent system as well as the mechanism of drug separation between the inner core and outer particle shell. Forming particles from the solution phase should allow one, at least in principle but not always in practice, to achieve the maximum theoretical drug loading provided that the resulting particles are stable. At the least, there should be no difference between micronized nanosuspension stabilized by a lipid and nanocapsules produced by precipitation. However, if the drug precipitates predominately outside the lipid matrix or shell, the suspension may become physically unstable, typically leading to formation of large crystals [13].

Current methods of making SLN include high-pressure homogenization [5], [8], microemulsion technique [14], [15] and oil-in-water (o/w) emulsion precipitation [16]. The high-pressure homogenization technique primarily includes two processes of the hot and cold homogenization [5], [8]. The fundamental limitation of this method is the drug solubility in the molten lipid and in the solidified lipid phase. Particles are obtained usually within the 200–500 nm size range. Clear thermodynamically stable microemulsions are obtained by dispersing in a cold aqueous phase the molten lipid and the drug [15]. Although the particles produced using this microemulsion process could be in the lower nanometer range (< 100 nm), the final dispersions obtained are extremely dilute and require a very complex balancing between the drug, lipid and surfactant/co-surfactant concentrations, which are practically very difficult to maintain. As with the high-pressure homogenization technique, the loading capacity of particles produced by the microemulsion technique is limited by the solubility of drug in lipid. The alternative method of SLN production includes direct precipitation of oil-in-water (o/w) emulsions, consisting of organic phase comprising of the lipid and the drug [16]. Precipitation of the lipid nanoparticles using this technique is carried out by solvent evaporation, liquid-solvent extraction or dilution. Although this technique is suitable for production of fine particles including nanospheres and nanocapsules, long processing times, residual solvents and scale-up issues limit its wide application.

The production method discussed in this work, supercritical fluid extraction of emulsions (SFEE), is based on a simple principle whereby the lipid nanosuspensions are produced by supercritical fluid extraction of the organic solvent from o/w emulsions. In this way, the flexibility of particle engineering using emulsion systems is combined with the high efficiency and scalability of continuous supercritical fluid extraction [17], [18]. The short processing time required for particle precipitation facilitates a more controllable consistent operation and uniform particle size distribution. The interaction between the supercritical fluid (CO₂) and the lipid during extraction may lead to plasticization (decrease of the glass transition temperature, T_g) or suppression of the melting point, and thus provide an opportunity to create solid particles with different structures and physical properties. In what follows below, we describe production of model drug-lipid systems using SFEE, physical characterization of the lipid nanosuspensions obtained and, finally, testing these formulations using both the AERx SDP and AERx Essence systems.

The AERx System consists of the AERx Strip™, a single-use disposable dosage form, and the AERx device, which has two hand-held configurations: an electromechanical version (SDP) and an all-mechanical version. The AERx SDP system was first described in 1997 [19] and although refinements have been made to the system, the general principle of aerosolization remains unchanged. The AERx System uses liquid formulations, which are relatively simple and economical to manufacture. The formulation is packaged under aseptic conditions into the AERx Strip, to create a sterile dosage form. The AERx Strip is a three-layer laminate consisting of a blister container, a lid layer and a nozzle layer. The nozzle layer contains a micro-machined (photo-ablated) array of holes (the nozzle array). The reservoir in the blister layer is loaded with formulation (50 μl) and dosage forms are prepared by heat-sealing the blister layer to the lid and nozzle layers. Aerosol generation is completed in one or two seconds via mechanical pressurization of the aqueous formulation. This pressurization causes the seal in the AERx Strip between the drug reservoir and the nozzle array to peel open, which leads to the formulation being expelled through the nozzle array as a fine-droplet aerosol. By varying the size of the nozzle holes, the size of the aerosol can be modified to optimize regional lung deposition.