Passive Enhancement of Retinol Skin Penetration by Jojoba Oil Measured Using the Skin Parallel Artificial Membrane Permeation Assay (Skin-PAMPA): A Pilot Study

Introduction

Jojoba (Simmondsia chinensis) seed oil is unique among the many naturally occurring plant oils. Most typical seed oils present themselves as fatty acid triglycerides which are formed in the seeds in small, individually packaged droplets called lipid bodies or oleosomes.¹ The stored oil is used to help provide nutrition during germination of the seeds. Jojoba oil, however, is comprised of liquid waxes, not triglyceride oils but is typically referred to as an oil due to the liquid nature of the waxy esters.² The oil is typically isolated on commercial scales by cold pressing to produce virgin oil often followed by a second pressing to produce non-virgin grades.³ Jojoba oil is also unique among vegetable oils because of its stability against oxidation, a trait not shared by most triglyceride oils. The jojoba plant is particularly interesting as it grows under quite arid conditions which means the plant grows abundantly without the need for large quantities of water.

Jojoba oil has been popularly used for many years in skin and hair care applications and has been shown more recently to have skin benefits including wound healing, emollient, hydrating, anti-inflammatory, and anti-microbial benefits, among others.^4–7 The linear wax esters that comprise jojoba oil share a very profound similarity to wax esters known to exist in human sebum.⁸ This compatibility with human sebaceous lipids is likely one reason jojoba oil has been so popularly used in topical skin preparations. In addition, the oil lends itself to various natural chemical modifications to provide fatty acids and fatty alcohols as well as the hydrogenated esters, acids and alcohols which tend to be higher melting solid waxes.³ Jojoba waxes and the various derivatives have been recognized as safe for use in cosmetics by the Cosmetic Ingredient Review Board since 2008.⁹

Within the pantheon of skin care actives that have been proven efficacious, retinoids form one of the most popular ingredients used among consumers. Retinoic acid is a very powerful exfoliant with demonstrated antiaging, antimicrobial, and other homeostatic effects.¹⁰ However, it is also a known teratogen, so its use is tightly controlled. The retinoic acid derivatives like retinaldehyde, retinol and various retinol esters are popular and safer, but less effective substitutes for retinoic acid, that have also been shown to improve skin. However, the use of retinol has been demonstrated to have a powerful skin dehydrating effect which can lead to skin dryness and potentially to skin irritation.¹¹ Recent work has demonstrated that jojoba oil, formulated as microemulsions, has the potential to enhance the penetration of various drugs through the skin.¹² In addition, previous work demonstrated that retinoids formulated in a soft colloidal nanocarrier system composed of jojoba oil could enhance the benefits of the retinoid in psoriatic patients.¹³ In this application, skin penetration is not as difficult as the drug being studied, tazarotene, is being delivered to skin which has a disrupted barrier.

The study of skin penetration has evolved with the development of new models to examine the ability of various ingredients to accelerate or improve passive skin permeation of active molecules of interest.¹⁴ Ideally, examining skin penetration is done clinically using skin biopsies or extensive tape stripping methods which allow examination of a particular skin active directly from the skin in which it has come into contact. These methods are expensive and quite time consuming and do not readily lend themselves to more rapid examination of multiple formulations or ingredients. A very popular method for investigating skin permeation is the use of Franz Cells which typically incorporate either a synthetic skin equivalent or actual cadaver skin maintained in a medium that keeps the skin moist. Franz Cell studies are considered the benchmark of in vitro test methods for non-clinical work. In 1998, Kansy et al reported on a new method to examine skin penetration called the parallel artificial membrane permeability assay or PAMPA for short.¹⁵ The method employs the use of a synthetic polymer membrane surface treated with a hydrophobic coating that closely mimics the skin lipid barrier.^16–23 More importantly, unlike the single chamber Franz Cell, the PAMPA method allows for studies to be conducted in 96-well plates, like those used to examine living cells and skin mimics. The method can be adapted depending on the selection of the synthetic membrane and the hydrophobic coating.²⁰ Most current commercially available assays employ polyvinylidenefluoride (PVDF) coated with phospholipids.²¹ The method is designed to specifically measure passive transdermal delivery of molecules. Studies have examined closely how the PAMPA assay provides comparable data to the skin penetration data demonstrated from Franz Cell studies.^18,22 In the following studies, the Skin-PAMPA was used to examine the influence of jojoba oil to enhance the skin permeation of retinol from various moisturizing emulsion formulations. To the best of our knowledge, this is the first time that the Skin-PAMPA has been used to examine transdermal retinol penetration.

Materials and Methods

Retinol Formulations

A summary of the formulas examined in the study is shown below in Table 1. Formulation 1 is a control formulation that comprises 10% jojoba oil without added retinol. Formulation 2 is a retinol test formulation that contains retinol without added jojoba oil. The formulation also contains 9% soybean oil as well that was added with the retinol. Formulation 3 is the second retinol test formulation containing 10% jojoba oil and 9% soybean oil.

Table 1 Summary of Formulations

Skin-PAMPA

Transdermal penetration was approximated with the Parallel Artificial Membrane Permeation Assay (PAMPA) using MultiScreen Filter/Receiver Plates from Millipore-Sigma (cat. MAIPNTR10 and MATRNPS50). This assay is designed to measure the permeability of a substance from a donor compartment through a phospholipid-coated PVDF filter into an acceptor compartment, Figure 1. Because the membrane has no transporters or efflux systems, only passive permeability is observed.

Figure 1 Schematic image of a single PAMPA permeation compartment showing the donor and acceptor sites and the lipophilic membrane designed to mimic the human stratum corneum.

The method is based on the application note “Evaluation of the Reproducibility of Parallel Artificial Membrane Permeation Assays (PAMPA)” by Millipore-Sigma Corporation.²⁴ Briefly, it consists of applying 150μL of a test material on a filter immediately after preconditioning it with 5μL of 1% (w:v) L-α-phosphatidylcholine (a natural phospholipid, Sigma, cat.# 3644). The incubation with test materials is pursued in the tissue culture incubator at 37°C/5%CO₂ for 16h on top of a receiver chamber (acceptor compartment) filled with 300µL of 5% DMSO in PBS. Each sample was run in duplicate to establish significance.

Retinol HPLC Assay

At the end of the experiment, 50μL from each acceptor compartment are extracted in cyclohexane at a 1:5 ratio and one µL is injected to the HPLC system. The chromatographic profile of the injected sample is then compared to one of a 10µL retinol control formulation, Formulation 2, containing just retinol dissolved in 300µL of the receiver chamber solution and extracted with cyclohexane. All samples were analyzed using Agilent HPLC series 1100 (Agilent Technologies, Palo Alto, CA) station equipped with the series 1100 degasser, autosampler G1313A, Zorbax SB-C8 reverse phase column protected by Javelin direct-connection column filter, diode array UV/Vis detector and ChemStation software, at 325nm (A325nm). Ethanol:Methanol:Ethyl Acetate (2:2:1) was used as mobile phase.

Calculations

The amount of retinol that elutes into the acceptor compartment was determined by the area under the curve (AUC) calculated automatically by the Agilent’s HPLC software (ChemStation) expressed in units. Statistical significance was calculated using double tail t-test and the threshold was established at p ≤ 0.05.

Results

Chromatography Results: Non-Eluted Control Sample Formulation 3 with Retinol

The chromatographic spectrum for donor chamber control sample Formulation 2 containing retinol is shown below in Figure 2. The retinol elutes at approximately 9.6 minutes confirming that retinol can be examined under the chromatographic conditions after extraction with cyclohexane.

Figure 2 HPLC elution profile of sample, Formulation 2, (10µL) mixed with 300µL of 5% DMSO/PBS and extracted 1:5 with cyclohexane, measured at 325nm.

Chromatographic Results: Eluted Control Formulation 1 Samples Without Retinol

The chromatographic spectrum for control sample Formulation 1 which does not contain retinol after 16h permeation is shown below in Figure 3. The spectrum confirms that samples without retinol do not show the retinol peak at 9.6 minutes.

Figure 3 HPLC elution profile of Formulation 1 without retinol sample from the acceptor chamber extracted 1:5 with cyclohexane, measured at 325nm.

Chromatographic Results: Eluted Sample Formulation 2 with Retinol Without Jojoba Oil

Figure 4 demonstrates that the moisturizing formulation with 1.0% retinol, Formulation 2, without added jojoba oil after 16h permeation demonstrates approximately a 0.5% enrichment in retinol in the acceptor reservoir after cyclohexane extraction.

Figure 4 HPLC elution profile of the sample Formulation 2 from the acceptor chamber extracted 1:5 with cyclohexane, measured at 325nm.

Chromatographic Results: Eluted Formulation 3 Sample with Retinol and Jojoba Oil

Figure 5 demonstrates the chromatographic spectrum of Formulation 3 after 16h permeation containing 1.0% retinol and jojoba oil at 10%.

Figure 5 HPLC profile of Formulation 3 with 1.0% Retinol and 10% jojoba oil sample from the acceptor chamber extracted 1:5 with cyclohexane, measured at 325nm.

Comparison of All Samples Eluted with the PAMPA

Figure 6 shows the Area Under the Curve (AUC) data for the three samples that permeated through the PAMPA membrane were eluted in the PAMPA. The data, based on the chromatographic spectra (Figures 2–4), indicate that the addition of jojoba oil to the sample formulation containing retinol increased the AUC from 7 units (no jojoba oil, Figure 3) to 285 units (jojoba oil, Figure 4), a nearly 40-fold increase in permeation of the retinol because of inclusion of the jojoba oil at 10%. In addition, Formulation 2 contained added soybean oil which also was present in Formulation 3 at comparable levels. However, it was found that Formulation 2 did not provide the enhanced benefits in skin permeation noted for Formulation 3 that contained both jojoba oil and soybean oil. This suggests that the sebaceous lipid mimicking nature of jojoba oil may be a principal reason the jojoba oil works superior to triglyceride oils to enhance penetration of lipophilic active ingredients like retinol.

Figure 6 Graph of Area Under the Curve (AUC) units for Formulations 1–3 eluted through the PAMPA. Formulation 1 contains 10% jojoba oil without retinol and serves as a background control. Formulation 2 contains retinol without jojoba oil. Formulation 3 contains retinol and 10% jojoba oil. The data demonstrates that the addition of 10% jojoba oil to the formulation increases retinol permeation via the PAMPA by approximately 40-fold.

Discussion

Retinol is one of the most well-known topical skin care active ingredients. However, as was recently reported, retinol can have a dehydrating effect on the skin which, over time, may lead to increased skin erythema and flaking.¹¹ Technologies that enhance the skin permeability of retinol may allow formulators who are employing retinol to lower the active levels of the molecule which can help to mitigate the skin dehydrating effects. Methods to help improve the overall efficacy and safety of retinoid use can include, for example, encapsulation or incorporation of the retinoids into unique emulsion structures such as micro- or nanoemulsions.^25,26 In addition, retinol and retinol derivative permeation has been shown to improve when the molecule is delivered to the skin using occlusive barriers.²⁷

There are various mechanisms to increase the passive diffusion of skin care actives through the skin.²⁸ The most effective is to disrupt the skin’s lipid barrier to allow molecules to move through the stratum corneum more efficiently. One molecule that does this very well is dimethylsulfoxide (DMSO). DMSO can penetrate the skin’s lipid barrier very quickly as DMSO itself has been promoted as a topical analgesic.²⁹ DMSO will carry anything dissolved into it essentially systemically to the blood stream when applied topically. Such aggressive skin penetration is not particularly desirable when ingredients are intended to only influence the stratum corneum and upper epidermal layers of the skin.

Jojoba oil wax esters share very common similarity to the skin’s sebaceous lipids as was noted earlier.⁸ Jojoba oil enhances the passive penetration of lipophilic active ingredients allowing for improved penetration of solubilized actives.⁷ This likely occurs because the oil can increase the fluidity of the lipid barrier of the skin which can create small channels that allow lipophilic actives dissolved in the oil, like retinol, to passively pass through the skin’s lipid barrier. The use of the Parallel Artificial Membrane Permeation Assay (PAMPA) to examine retinol penetration has not been reported previously. The PAMPA is a particularly rapid and effective testing methodology for examining skin penetration enhancers that work by lipid barrier disruption. The PVDF membrane of the assay is coated with phosphatidylcholine prior to commencement of the assay. This creates a barrier that can closely mimic the functional properties of the skin’s lipid barrier.²⁴ The testing method focuses on the influence of permeation enhancers to function through passive lipid barrier disruption mechanisms and may limit ingredients that do not function through such lipophilic disruption mechanisms. PAMPA would not be effective for skin permeation that might use active transport systems such as transmembrane transporters, channels, and pumps.

Conclusions

In the present study it was found that addition of 10% jojoba oil to a moisturizing formulation containing retinol was able to effectively increase the permeability of the retinol when examined using the skin-PAMPA. The increase in retinol permeation was nearly 40-fold compared to a similar formulation without jojoba oil. The findings demonstrate for retinol previously reported benefits of jojoba oil in transdermal delivery.^12,13 The demonstrated use of the PAMPA to examine retinol skin permeation suggests the test method may be useful for examining other lipophilic topical skin care actives to improve passive skin permeation rapidly and accurately.