*Article* **Niosomal Nanocarriers for Enhanced Dermal Delivery of Epigallocatechin Gallate for Protection against Oxidative Stress of the Skin**

**Danhui Li <sup>1</sup> , Nataly Martini <sup>1</sup> , Zimei Wu <sup>1</sup> , Shuo Chen <sup>1</sup> , James Robert Falconer <sup>2</sup> , Michelle Locke <sup>3</sup> , Zhiwen Zhang <sup>4</sup> and Jingyuan Wen 1,\***


**Abstract:** Among green tea catechins, epigallocatechin gallate (EGCG) is the most abundant and has the highest biological activities. This study aims to develop and statistically optimise an EGCGloaded niosomal system to overcome the cutaneous barriers and provide an antioxidant effect. EGCG-niosomes were prepared by thin film hydration method and statistically optimised. The niosomes were characterised for size, zeta potential, morphology and entrapment efficiency. Ex vivo permeation and deposition studies were conducted using full-thickness human skin. Cell viability, lipid peroxidation, antioxidant enzyme activities after UVA-irradiation and cellular uptake were determined. The optimised niosomes were spherical and had a relatively uniform size of 235.4 ± 15.64 nm, with a zeta potential of −45.2 ± 0.03 mV and an EE of 53.05 ± 4.46%. The niosomes effectively prolonged drug release and demonstrated much greater skin penetration and deposition than free EGCG. They also increased cell survival after UVA-irradiation, reduced lipid peroxidation, and increased the antioxidant enzymes' activities in human dermal fibroblasts (Fbs) compared to free EGCG. Finally, the uptake of niosomes was via energy-dependent endocytosis. The optimised niosomes have the potential to be used as a dermal carrier for antioxidants and other therapeutic compounds in the pharmaceutical and cosmetic industries.

**Keywords:** niosomes; catechin; dermal delivery; antioxidant activity; oxidative stress; skin barrier; penetration; cellular uptake

## **1. Introduction**

The skin is the largest organ of the human body, which makes it the direct target of oxidative stress due to the exposure to reactive oxygen species (ROS) from the surrounding environment. The most important function of human skin is protection by providing a barrier from pathogens, and physical and chemical damages. It also plays a crucial role in thermoregulation and endocrine function such as vitamin D synthesis [1,2]. The skin comprises three layers: the epidermis, which consists of keratinocytes; the dermis consisting of connective tissue, and the subcutaneous layer [3]. The epidermis can be divided into four layers, including the stratum corneum (SC), stratum granulosum, stratum spinosum and stratum basale. The dermis is composed of connective tissues, which are also rich in glands, white blood cells and blood vessels [4,5]. SC is the highly hydrophobic surface layer that contains 18 to 21 cell layers and is composed of corneocytes that are

**Citation:** Li, D.; Martini, N.; Wu, Z.; Chen, S.; Falconer, J.R.; Locke, M.; Zhang, Z.; Wen, J. Niosomal Nanocarriers for Enhanced Dermal Delivery of Epigallocatechin Gallate for Protection against Oxidative Stress of the Skin. *Pharmaceutics* **2022**, *14*, 726. https://doi.org/10.3390/ pharmaceutics14040726

Academic Editors: Alyssa Panitch and Montse Mitjans Arnal

Received: 30 January 2022 Accepted: 23 March 2022 Published: 28 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

terminally differentiated keratinocytes anchored in a lipophilic matrix [3,4]. The 'bricks and mortar' model is often employed to describe the structure of SC, in which intercellular lipid accounts for 10% of the dry weight of this layer, and the rest is an intracellular protein (mainly keratin). Keratins are a family of alpha-helical polypeptides with a molecular weight ranging from 40,000 to 70,000 Daltons, making the corneocyte layers dense and relatively impervious to external compounds [6].

The skin is continuously exposed to environmental threats such as UV radiation, pollution, micro-organisms, and viruses, which lead to ROS production. ROS are also formed during the normal cellular metabolism and immune reactions. More than 80% of environmental ROS that damage the skin is produced by UV [7]. Antioxidants such as glutathione, ubiquinol and thiols inhibit oxidation reactions by donating electrons to free radicals [7]. Our bodies also produce enzymatic antioxidants, such as superoxide dismutase and glutathione peroxidases. Other non-enzymatic antioxidants such as vitamin E (alpha-tocopherol) and vitamin C (ascorbic acid) are obtained from the diet [8]. However, the antioxidants produced by our bodies are inadequate to protect against oxidative stress, and antioxidants are often used as dietary supplements to replenish the level of endogenous antioxidants and hence help to delay the onset of aging or diseases [8].

Catechins are a group of powerful antioxidants with health-promoting effects, and epigallocatechin gallate (EGCG) is one of the catechins found in green tea. EGCG has several beneficial effects on the skin, including anti-aging, anti-inflammatory, and anticancer properties. According to a study, treating normal human epidermal keratinocytes with EGCG prevented UVB-induced intracellular release of hydrogen peroxide while also inhibiting UVB-induced oxidative stress-mediated skin damage [9]. EGCG has been shown to inhibit UV-induced collagen production and collagenase transcription in human dermal fibroblasts [10]. In addition, on the human model, catechins were shown to have anti-aging functions [9]. A double-blind, placebo-controlled experiment of adult women found that catechins can reduce total sun damage when given as oral catechins supplements [11]. The oral administration route is generally the most accepted for drug administration, particularly for long-term prevention purposes. However, when administered orally, catechins readily undergo several metabolic transformations by intestinal microflora and enzymes; therefore, they are poorly bioavailable [12]. The application of EGCG is also limited by its unstable physiochemical properties, which can be degraded quickly. Many studies have reported that green tea catechins were vulnerable to degradation caused by the elevation of temperature, pH, and metal ions of incubation media [13]. The instability is part of the reason for the poor bioavailability and also presents as an issue in the manufacturing process. Therefore, the oral bioavailability of catechin represents a big challenge. Topical application of these bioactive compounds may be able to overcome the problem, as this route bypasses metabolism by the liver and gastrointestinal track with relatively low enzymatic degradation. However, the skin barrier, which is due to the SC layer, impedes the transport of exogenous compounds into the skin and restricts diffusion of external substances into the deeper dermis layer. Therefore, we hypothesise that loading EGCG into a drug carrier would help overcome the skin barrier, improve penetration into the deeper skin layers and improve their stability. The compound can therefore exert its beneficial effect at the site of interest.

Niosomes are versatile drug carrier systems that have been administered via various routes; they are surfactant-based nanocarriers that are mainly composed of non-ionic surfactant and cholesterol [14–17]. Niosomes have been extensively studied in topical drug delivery due to their ability to significantly improve penetration across the skin barrier and deposition in the dermis layer [18–24]. Drugs with a variety of physicochemical properties have been investigated for topical and transdermal delivery using niosomes, for example, diacerein [25], itraconazole [26], tretinoin [27], salidroside [28] and finasteride [29], demonstrating their advantages in topical delivery. In addition, a range of bioactive compounds has also been loaded into niosomes, such as curcumin, rutin, and Ginkgo

biloba extract [30–32]. Silymarin-loaded niosomes demonstrated superior antioxidant activity over silymarin suspension in vitro [33].

In formulation development, various factors might impact the final product's performance. Design of Experiment (DOE) is a planned set-up of experiments to acquire information efficiently and precisely. It applies to any process that has quantifiable inputs and outputs. DOE was first designed for agricultural applications, but it has since become a frequently used technique in process sectors, including the chemical, food and pharmaceutical industries [34]. It may be used to explore the effect of multiple variables on responses by altering them all at once in a small number of tests. By this strategy, the costs and time involved with the research and production of medicine may be significantly decreased [34,35]. Furthermore, it aids in the creation of the "best possible" formulation composition and gives a comprehensive knowledge of the process and product behaviors [36]. This study aimed to develop an optimal niosome formulation using the DOE methodology and evaluate the formulation for topical administration of EGCG for protecting the skin from external oxidative stress. An ex vivo investigation on human skin was carried out to evaluate drug deposition and the antioxidant activity of the EGCG-niosomes. The uptake of niosomes by human skin fibroblasts was also investigated.

#### **2. Materials and Methods**

#### *2.1. Materials*

Span® 60, EGCG <sup>≥</sup> 98% (HPLC), cholesterol (CH), Triton™ X-100, dihexadecyl phosphate (DCP), Fluorescein 5(6)-isothiocyanate (FITC), Sulforhodamine B (SRB), trichloroacetic acid (TCA), dimethyl sulphoxide (DMSO), methanol and acetonitrile (ACN) were purchased from Merck (Merck, Kenilworth, NJ, USA). Dulbecco's Modified Eagle Medium (DMEM) with high glucose and L-glutamine, Phosphate-Buffered Saline (PBS), Hank's Balanced Salt Solution (HBSS), penicillin-streptomycin, fetal bovine serum of New Zealand origin (FBS), trypsin-EDTA, DAPI and CellTracker were purchased from Thermo Fisher Scientific (Auckland, New Zealand). Malondialdehyde (MDA), glutathione peroxidase (GSH-px) and superoxide dismutase (SOD) kits were purchased from Biovision (Biovision Inc., Milpitas, CA, USA). Trifluoroacetic acid (TFA) was purchased from Fluka (Fluka, Darmstadt, Germany). Distilled, deionised water was used throughout and was obtained from a Millipore water purifier.

#### *2.2. High-Pressure Liquid Chromatography Method for Quantification of EGCG*

An Angilent Technologies 1100 series high pressure liquid chromatography (HPLC) system equipped with a vacuum degasser, autosampler, thermostatted column compartment and photodiode-array detector (PDA) was used. A C18 column (Jupiter, 250 × 4.6 mm, 5 mm, Phenomenex, Torrance, CA, USA) was used for HPLC method development for EGCG. The mobile phase consisted of Milli Q water (0.1% TFA) and methanol at 75:25 ratio. EGCG was analysed at flow rate of 0.8 mL/min, with an injection volume of 20 µL and wavelength of 280 nm at 25 ◦C.

#### *2.3. Preparation of EGCG Loaded Niosomes*

A total of 150 mmol of surfactant, cholesterol and DCP (2 µmol) was dissolved in organic solvents (methanol/chloroform, 4:1, *v*/*v*) and then the mixture rotatory evaporated to form a thin lipid film on the wall at 45 ◦C. The the lipid film was purged with nitrogen to remove any organic solvents. The thin film was then hydrated with PBS (pH 7.4, 10% ethanol) containing 2 mg of EGCG at 58 ◦C to form EGCG-niosomes. The niosome suspension was then extruded through a 400 nm polyester membrane with an ER-1 extruder (Eastern Scientific, Rockville, MD, USA) for 10 cycles and then stored at 20–25 ◦C for the niosome membrane to anneal.

#### *2.4. Optimisation of Formulation with Design of Experiment*
