Coenzyme Q10-loaded NLCs: preparation, occlusive properties and penetration enhancement

July 1, 2007
Rainer H. Müller

,
Jana Pardeike

Pharmaceutical Technology Europe

Pharmaceutical Technology Europe, Pharmaceutical Technology Europe-07-01-2007, Volume 19, Issue 7

The free radical theory of ageing, first postulated by Harman, is the most popular and widely tested.1 This theory is based on the chemical nature and ubiquitous presence of free radicals.2,3 In the cosmetic industry, the antioxidant coenzyme Q10 is widely used in anti-ageing products.4 Coenzyme Q10 is a lipid-soluble compound composed of a redox-active quinoid nucleus and a hydrophobic side chain containing 10 monounsaturated trans-isoprenoid units (Figure 1). The main biological function of coenzyme Q10 is to act as a redox component of transmembrane electron transport systems, such as the respiratory chain of mitochondria.5

For the topical application of coenzyme Q10 a system that can deliver the antioxidant most efficiently into the skin is required. Therefore, investigations with different topical applied formulations, composed of NLCs, emulsion and liquid paraffin were performed for this study.

NLCs are used as carrier systems for cosmetic ingredients and pharmaceutical drugs.6–8 They were developed following the success of solid lipid nanoparticles (SLNs). In contrast to emulsions, the solid particle matrix is composed of a blend of solid lipid and liquid lipid. The particle matrix is solid at body temperature, which protects the incorporated active ingredients against chemical degradation and allows the release of incorporated molecules to be modified.9,10 NLCs were developed in 1999/2000 and the first two cosmetic dermal products were introduced to the market at the end of 2005 (Nanorepair Q10 Cream and Nanorepair Q10 Serum [Dr Rimpler GmbH, Germany]). In 2006, more products were introduced to the market, for example, the day cream NanoVital (Dr Rimpler GmbH) and in the pipeline, IOPE the SuperVital products (cream, serum, eye cream [Amore Parcific/South Korea]).

Figure 1

In this study, NLCs were investigated to compare possible advantages with a conventional oil/water (o/w) emulsion or liquid paraffin. The occlusion properties, as well as the penetration behaviour of incorporated coenzyme Q10 into the stratum corneum (SC), were analysed.

Experimental methods

NLCs were produced from the solid lipid cetylpalmitate (Henkel KGaA, Germany) and liquid lipid Miglyol 812 (Cognis Deutschland GmbH & Co. KG, Germany). The lipid blend was heated 5 °C above the melting point of the solid lipid and dispersed in an aqueous Tego Care 450 (Goldschmidt GmbH, Germany) solution at identical temperature (65 °C) by high-speed stirring. The pre-emulsion obtained was homogenized using a Micron LAB 40 (APV Homogeniser Systems, Germany). Cooling of the hot o/w nanoemulsion obtained led to the solidification of the lipid and the formation of NLCs. The reference emulsion was produced by substituting the cetylpalmitate with Miglyol 812 using the same high pressure homogenizer. Both formulations contained 25% (mass/mass [m/m]) coenzyme Q10 (BKI Internationaler Handel GmbH, Germany) calculated on the basis of the particle mass (i.e., 5% m/m in the total formulation). For the investigations, the formulations were diluted 1:10 with water to obtain a final coenzyme Q10 concentration of 0.5% m/m. As a second reference, a solution of 0.5% m/m coenzyme Q10 in liquid paraffin was used.

The determination of the particle size of NLCs and the emulsion was performed by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instruments, UK). Further investigations on particle size were performed by laser diffractometry (LD) using a Coulter LS 230 (Beckman-Coulter, Germany). PCS yields the mean particle size and the polydispersity index (PI) as a measure of the width of the particle size distribution. The LD data were evaluated using the diameter 50% (LD 50) and 95% (LD 95), which means that either 50% or 95% (volume distribution) of the measured particles are below this size.

The particle charge was quantified as zeta potential using the Zetasizer Nano ZS. Measurements were performed in distilled water adjusted with sodium chloride to a conductivity of 50 μS/cm.11

The occlusive effect of NLCs, emulsion and liquid paraffin was investigated using an in vitro occlusion test.12 Water evaporation through a cellulose acetate membrane was measured and the occlusion factor (F) was calculated using the following equation:

F = (A–B)/A)100 [Equation 1]

Where A is the amount of water evaporated through the cellulose acetate membrane without applying a formulation and B is the amount of water evaporated through the cellulose acetate membrane after applying a sample. For the evaluation of the occlusion factor, each sample was applied five times with a final amount of 10.2 mg/cm2 . The samples were stored at 32±1 °C and 60±5% relative humidity (RH). Water evaporation was investigated by weighing after 6, 24 and 48 h. If the applied formulation had no occlusive properties an occlusion factor of 0% would be obtained and for maximum occlusion an occlusion factor of 100% would be obtained.

The penetration of coenzyme Q10 into the SC was studied using a tape-stripping test. At the beginning all volunteers were asked to wash their arms and dry them afterwards. After an incubation time of 30 min at 20±1 °C and 45±5% RH, 150 mg of NLCs, emulsion and liquid paraffin containing 0.5% m/m coenzyme Q10 were applied to a 10.4 cm2 testing area on the volar forearm of five female volunteers with healthy skin in the testing areas. Thirty minutes after application the samples were washed off and the SC was removed with nine strips of Scotch Tape No. 90 (3M, MN, USA). Each tape was taken in a controlled way; that is, a 1 kg rubber weight was rolled over it 10 times. The coenzyme Q10 was extracted from the tape strips with 2 mL of acetone. The concentration of coenzyme Q10 was determined using HPLC. Strip 4 and 5, 6 and 7, 8 and 9 were analysed together.

Figure 2

Results and discussion

The NLCs and the emulsion had the same lipid content, were produced under the same conditions using the high pressure homogenization and possessed identical particle sizes (Figures 2 and 3). Figure 2 shows the PSC mean diameter and the polydispersity index of NLCs, and emulsion measured at six different time points during 1 month. The LD mean diameters (LD 50 and LD 95) for the formulations measured at the same time points as the PCS values are displayed in Figure 2. Both figures show that the particle size of NLCs and the emulsion are identical and remain constant during 1 month. Additionally, zeta potential measurements were performed. The zeta potential right after production was –54 mV for the NLCs and –57 mV for the emulsion. Zeta potentials in this range are related to no aggregation or to only little aggregation.

Figure 3

Figure 4 shows the F values for NLCs, emulsion and liquid paraffin. The occlusion factor of the NLCs was much higher than the occlusion factor of the emulsion, although they had the same lipid content and particle size. This effect can be explained by the film formation of NLCs. NLCs showed a tendency to fuse, forming a dense film after application.8,13 NLC used for these investigations had a melting point of 47.5 °C, therefore, they are still solid at both room and skin temperature. After the water is evaporated from the NLC dispersion, only the solid NLC particles stay on the skin. The capillary force of the nanometer pores between the NLC particles are contractive, which promotes fusion and dense film formation.14,15 Thereby, the NLCs show the same occlusion properties as the highly occlusive liquid paraffin. It should be noted that the lipid content of the NLC dispersion was only 2%. The emulsion had relatively low occlusive properties although it had the same lipid content as the NLCs.

Figure 4

With the tape stripping test it can be shown that the penetration of coenzyme Q10 from the NLCs and emulsion was much higher than from liquid paraffin (Figure 5). The NLCs showed the highest penetration of coenzyme Q10 into the SC.

Figure 5

y = n log x + m [Equation 2]

The shape of the curves obtained indicates a logarithmic relationship between the cumulative values of coenzyme Q10 and the tape strip numbers. Functions of the type can fit the data if a regression analysis is performed, where y is the cumulative percentage of the applied coenzyme Q10 found inside the SC, x is the corresponding tape number and n and m the estimate equilibration coefficients (Table 1).

Table 1 Parameters describing the logarithmic relation between the cumulative amount of coenzyme Q10 and the tape number for NLC, emulsion and liquid paraffin (p,