(M1030-04-23) Formulation of a Triple-Action Nanoemulgel for Mild to Moderate Atopic Dermatitis: Impact on Skin's Mechanical and Antibacterial Properties

Purpose: The aim of this study was to develop a jojoba oil-based nanoemulsion (NE) as a nanosized dispersion for transporting insoluble bioactive compounds, including polyphenols and phenolic compounds such as t-resveratrol and curcuminoids. Various formulations were created by blending water, jojoba oil, isopropyl myristate, and a Tween 80/Span 20 mixture (Surfmix). A polyphenol-enriched extract (PPE - highly purified), serving as a model for poorly soluble drugs, was previously dissolved in jojoba oil at a fixed concentration. Formulations with varying oil-to-Surfmix ratios were then dispersed in water using ultrasonic homogenization to achieve nanosized droplets, leveraging the cavitation principle. These formulations were characterized by dynamic light scattering (DLS) and evaluated for growth inhibition in Staphylococcus aureus and their cytotoxicity. Placebo formulations containing 10% of the oily phase (jojoba oil and isopropyl myristate) were also assessed for elasticity to determine the effect of the vehicle on the skin characteristics of healthy volunteers.

Methods: The methodology was summarized in Figure 1. The NE formulations were evaluated in sequence starting with the formulations with 2, 6 and 10% of the oily phase. The minimum inhibitory concentration (MIC) was evaluated against Staphylococcus aureus, in the presence and absence of nanoemulsion loaded with active agents (Loaded NE) and nanoemulsion without active agents (Empty NE). For this, 1x10⁷ bacteria/well were inoculated in Müller Hinton Broth medium in a 96-well plate, using a range of NE concentrations from 1% v/v to 0.2% v/v. The microorganisms were incubated with shaking at 120 rpm and a temperature of 37°C. Growth inhibition was evaluated using an EPOCH spectrophotometer at 600 nm at 24 hours. Kanamycin was used as a positive control for the antimicrobial agent. The cytotoxic effect of the loaded NE was evaluated on an immortalized adult human dermal fibroblast cell line (HDFa). For this, 3x10³ cells were seeded in 96-well plates and incubated with the NEs for 24 hours in fibroblast culture medium, at 37°C and 5% CO₂. After incubation, cell viability was determined using the MTS cell viability assay, measured by spectrophotometry at 490 nm. If the compounds have a toxic effect on the cells, a decrease in the percentage of viable cells (live cells) will occur. The skin moisture was determined at the forearm using a MoistureMeterSC (Delfin Technologies) a device for the measurements of skin surface hydration in the stratum corneum in ten healthy volunteers

Results: Figure 2 summarizes the data obtained from the DLS and the hydration of the skin from the jojoba oil.  The higher hydrodynamic (389 nm) size was obtained in NE formulated at 10% F.O2. Stable and less disperse NEs were observed at formulations made with Oil Phase 2. Therefore, NE Formulation F.O2 10% was selected for following experiments. The effect of the jojoba oil in the surface hydration of the skin demonstrated a trend to increase after one hour when applied in healthy volunteers and its effect remains for four hours. The data at figure 2 supports this findings however the results should be consider as a trend due to the high variability of the results, which is common in in vivo studies. Figure 3 represents the results for the cytotoxicity and the MIC studies. NE F.O2 showed a considerable cytotoxicity effect probably due to using artificial chemical surfactants (3). The following steps will explore the possibility of reducing the concentration of surfactants without affecting the particle size of the final formulation. Empty NE F.O2 formulation showed antimicrobial activity which was promoted by a synergic effect due to the presence of PEE. Together, these results validate the carrier properties of the selected NE formulation.

Conclusion: The proposed strategy for obtaining nanoemulsions with bioactive molecules was validated through DLS characterization. Nanoemulsion encapsulation is an effective approach as a drug delivery vehicle for poorly soluble molecules intended for therapeutic purposes.Nanoemulsions loaded with a polyphenol-enriched extract showed antimicrobial activity against Staphylococcus aureus, which is a promising characteristic for using the nanoemulsion to treat atopic dermatitis, a skin disease often triggered by this opportunistic bacterium.

References: McClements, D.J. (2012) ‘Nanoemulsions versus microemulsions: Terminology, differences, and similarities’, Soft Matter, 8(6), pp. 1719–1729. doi:10.1039/c2sm06903b.
Rachmawati, H., Budiputra, D.K. and Mauludin, R. (2014) ‘Curcumin nanoemulsion for Transdermal Application: Formulation and evaluation’, Drug Development and Industrial Pharmacy, 41(4), pp. 560–566. doi:10.3109/03639045.2014.884127.
Vater, C. et al. (2020) ‘Effects of lecithin-based nanoemulsions on skin: Short-time cytotoxicity MTT and brdu studies, skin penetration of surfactants and additives and the delivery of curcumin’, International Journal of Pharmaceutics, 580, p. 119209. doi:10.1016/j.ijpharm.2020.119209.

Acknowledgements: This work was founded by Agencia Nacional de Innovación y Desarrollo, ANID. Proyecto FONDEF ID23I10359, and supported by the pharmaceutics division at the Magíster en Gestión Farmacéutica y Farmacia Asistencial.

Figure 1: Summary of the methods use in the study of the nanoemulsions for mild to moderate dermatitis

Figure 2: A) Hydrodynamic diameter comparison between empty NEs formulations (left) and Polyphenol-enriched extract “vehicularized” NEs (right). (B) Zeta potential and polydispersity index characterization in vehicularized NEs. F.O1 (Oil Phase 1); F.O2 (Oil Phase 2). C) Moisture content over time after exposing healthy volunteers to 100uL of jojoba Oil (n=10) C) Analysis of the hydration in the stratum corneum of ten healthy volunteers in the forearm after applying jojoba oil (100uL)


Biological activity validation of selected NE Formulation.
(A) Cytotoxicity evaluation of selected empty and loaded NE formulation in HFA cells. (+) Loaded NE (-) Empty NE . (B) Activity of empty and loaded NE formulation against S. aureus growth in a planktonic model (FD = 10). * ambiguous result considered as non-conclusive.