Preparation and Characterization of a Lecithin Nanoemulsion as a Topical Delivery System
© to the authors 2009
Received: 16 July 2009
Accepted: 14 October 2009
Published: 29 October 2009
Purpose of this study was to establish a lecithin nanoemulsion (LNE) without any synthetic surfactant as a topical delivery vehicle and to evaluate its topical delivery potential by the following factors: particle size, morphology, viscosity, stability, skin hydration and skin penetration. Experimental results demonstrated that an increasing concentration of soybean lecithin and glycerol resulted in a smaller size LNE droplet and increasing viscosity, respectively. The droplet size of optimized LNE, with the glycerol concentration above 75% (w/w), changed from 92 (F10) to 58 nm (F14). Additionally, LNE, incorporated into o/w cream, improved the skin hydration capacity of the cream significantly with about 2.5-fold increase when the concentration of LNE reached 10%. LNE was also demonstrated to improve the penetrability of Nile red (NR) dye into the dermis layer, when an o/w cream, incorporated with NR-loaded LNE, applied on the abdominal skin of rat in vivo. Specifically, the arbitrary unit (ABU) of fluorescence in the dermis layer that had received the cream with a NR-loaded LNE was about 9.9-fold higher than the cream with a NR-loaded general emulsion (GE). These observations suggest that LNE could be used as a promising topical delivery vehicle for lipophilic compounds.
Topical delivery systems (TDS) have received increased attention during the past few years. TDS could avoid a variety of disadvantages compared with the oral administration including drastic pH changes, deleterious presence of food and enzymes and first-pass effect of the liver. TDS also eschewed injection inconvenience and needle phobia. In addition, TDS were noninvasive and can be self-administered with the minimization of side-effects in the topical drug therapy. However, the paucity of candidates for TDS was presented in the market because few molecules yielded skin permeability coefficients sufficiently high to meet the clinical therapeutic needs, and TDS was even less applicable for large hydrophilic molecules because of their very low skin permeation rates. The continuous stratum corneum (the outer layer of skin) provided a major barricade for drug penetration to the deeper skin layers and was therefore the usual target for attempts to strength topical drug permeation ability [1–5].
Various kinds of vesicular carriers had been suggested as topical delivery vehicles, including liposomes, microemulsions and lipid nanoparticles [6–10]. However, liposome formulations had certain limitations such as drug loading and stability. Microemulsions, which consisted of synthetic surfactant, oil, water and co-surfactant, exhibited a lower drug-loading capacity relative to the high concentration of surfactant, which had been generally acknowledged to induce skin irritation [11, 12]. Recently, due to advantages such as a controlled droplet size, lower concentration of surfactant and the ability to efficiently solubilize lipophilic drugs, nanoemulsions, which were composed of nanoscale droplets of one immiscible liquid dispersed within another, had been widely designed to deliver drugs by various routes of administration (e.g., intravenous, oral or ocular delivery) for therapeutic needs [13–17]. Few studies reported nanoemulsions were used as a topical carrier with following significant advantages including powerful penetrability and a high drug-loading capacity [18–21]. Most of nanoemulsions consisted of synthetic surfactant, and nanoemulsions without any synthetic surfactant were rarely reported.
In the present study, we report a LNE system without any synthetic surfactant composed of snake oil , soybean lecithin, glycerol and water. The effects of the glycerol to water ratio and soybean lecithin concentration on the emulsion’s physical properties (droplet size, viscosity, morphology and stability) were studied. Furthermore, the skin penetration ability and skin hydration levels of this LNE system were determined to evaluate its topical delivery potential.
Materials and Methods
Soybean lecithin was purchased from Cargill Texturizing Solutions Deutschland GmbH & Co. KG., USA. Refined snake oil and o/w cream were obtained from Jiangsu Longliqi Bioscience Co., Ltd., China. Nile red (NR) was obtained from Sigma–Aldrich, USA. 2-Propanol and optimal cutting temperature compound (OCT) were purchased from Leica microsystem, German. Glycerol, ethane and hexane were reagent grade.
Preparation of LNE
Composition of LNE formulation
Ingredients concentration (%,w/w)
Droplet Size and Polydispersity Index (PI)
Droplet size and the PI of LNE were measured by photon correlation spectroscopy (PCS; Zetasize 2000, Malvern Instruments, UK). The LNE was diluted with deionized water by 50-fold. Droplet size and PI value were obtained as the average of three measurements at 25 °C.
The viscosity of the LNE was measured using the small sample adapter of a Brookfield rheometer (Model DV-III, Brookfield Engineering Labs., Inc., Stoughton, MA, USA) at 25 °C. An average of three data points was obtained to determine the viscosity at a shear rate of 7.34 s−1.
Morphology of the LNE was characterized by freeze-fracture transmission electron microscopy (FF-TEM). Samples were immersed rapidly into liquid ethane cooled by liquid nitrogen. They were then transferred into liquid nitrogen after about 5 s. The samples, after being transferred into the chamber of the freeze-etching apparatus (BALZERS BAF-400D), were fractured at −120 °C and 3 × 10−7 mbar. After being etched for 1 min, Pt–C was sprayed onto the fracture face at 45°, and then C was sprayed at 90°. The replicas were taken out of the chamber and placed on a copper grid mesh after washing with hexane. After processing, morphology data from the samples were detected under a transmission electron microscope (PHILIPS-FEI TECNAI20).
Skin Hydration Test
The skin hydration effect of LNE was investigated in a blind, placebo-controlled in vivo study. LNE was stirred into o/w cream at 200 rpm evenly at 40 °C. Sample 1 was o/w cream without LNE and samples 2, 3, 4 and 5 were o/w cream enriched with 0.5, 2, 5 and 10% LNE (F12, shown in Table 1), respectively. Fifteen volunteers applied the samples on their healthy volar forearm skin. The skin hydration test was conducted at 20 2 °C room temperature and 50 5% relative humidity. Samples were spread on a 3.14 cm2 area of volar forearm skin with an amount equaling 1.5 μL/cm2. After application, skin hydration was measured at fixed time intervals of 30, 60, 90, 120 and 150 min with a Corneometer CM 825(CK Electronic GmH, Germany). The percent change in skin hydration was calculated with the following equation: ∆% = (Q t − Q 0)/Q 0 × 100.Q t was the hydration value after application time t, and Q 0 was the hydration value before application.
Skin Penetration Test
Experiments were conducted on female Wistar SD rats (180–200 g body weight). The rats were 10 weeks old and were of a similar development stage. They were anesthetized by a suitable dose of sodium barbital. The fur on the abdominal area of the rats was carefully removed by an electrical shaver to avoid damage to their stratum corneum. The furless abdominal area was used for in vivo permeation studies.
In this study, NR, a fluorescent dye, was dissolved into the oil phase during the LNE preparation procedure. The following two samples were used in a blind, placebo-controlled in vivo study: one sample was the o/w cream incorporated with NR-loaded LNE (F12, shown in Table 1); the other was the o/w cream incorporated with NR-loaded GE (F12 without HPH). The NR concentration in the o/w cream was 2.5 μg/mL, and the NR-loaded GE or NR-loaded LNE were stirred into the o/w cream at 200 rpm evenly at 40 °C. For each set of experiments, 50 mg of sample was applied to the hairless abdominal skin area of ~3.14 cm2. At fixed times of 0.5, 2 and 4 h after application, the surplus sample was removed from the skin surface, the skin surface was washed three times with PBS and dried gently under cold air with an electric hairdrier. A 0.5 cm × 0.5-cm skin piece, which was cut out from the treated area, was embedded in optimal cutting temperature compound and frozen rapidly by liquid nitrogen. The specimen was removed from liquid nitrogen and was frozen on a metal block. The metal block was then transferred to a cryostat microtome (LE ICACM 1850, Germany) for slicing through vertical cross-sections of skin. Twelve vertical skin sections with a thickness of 25 μm were obtained and stored at 4 °C until analyzed microscopically.
Skin sections were subjected to fluorescent microscopy using an Olympus CK40 microscope (Olympus, Japan) equipped with a UV source and filter for fluorescent measurements. Image capture and analysis were carried out by the Image-pro Plus program (Media Cybernetics, USA). The excitation and emission wavelengths were 543 nm and 604 nm for NR, respectively. Images were recorded with a camera integration time of 1/1.8 s, and the same parameters were used for the imaging of all samples. The fluorescence intensity value was quantified using Image-pro Plus program.
All the data tests were repeated three times and expressed as the mean SD. The statistical data were analyzed by a t-test analysis via Origin 7.0.
Results and Discussion
Particle Size and Viscosity of LNE
Droplet size, polydispersity index (PI) and viscosity of LNE (mean SD,n = 3)
Droplet size (nm)
F12 (without HPH)
Interestingly, apart from the size and viscosity, the effect of glycerol on the emulsion appearance was also observed. When the formulation was composed of 0 and 25% (w/w) glycerol in the aqueous phase (F1-F6), the nanoemulsion appeared as a milky solution. When the formulation was composed of 50% glycerol in the aqueous phase (F7-F9), the nanoemulsion appeared as a semitransparent solution. Furthermore, when the glycerol concentration was increased to 75 and 100% in the aqueous phase (F10-F15), the nanoemulsion appeared as a transparent solution. Possibly, glycerol behaved as a continuous phase solvent, whereby it not only affected the appearance of the nanoemulsion but also increased the viscosity of the continuous phase so as to decrease the droplet collision frequency [24, 25].
Morphology of LNE
Stability of LNE
Skin Hydration of LNE
Skin Penetration of LNE
The LNE, composed of snake oil, soybean lecithin, glycerol and water, was prepared by a HPH method successfully. Results revealed that LNE not only improved the skin hydration of formulation significantly, but also greatly strengthened skin penetration of NR for topical application. LNE may be a promising topical delivery vehicle for lipophilic compounds.
The authors are grateful for FF-TEM support by Shufeng Sun from the Center for Electron Microscope, Institute of Biophysics, Chinese Academy of Sciences, China.
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