Liposomes

Liposomes are colloidal and vesicular delivery systems, composed of at least one bilayer amphiphilic lipid membrane and a hydrophilic core [25]. These systems have many advantages, including the capability of encapsulating hydrophilic, hydrophobic, or amphiphilic components, target potential, slow-release properties, and biocompatibility due to the similarity with cell membranes, as well as biodegradability, low toxicity, ease of preparation and the ability to extend product shelf-life [25,26]. The use of liposomes as a drug delivery system allows partially overcoming the problems related to the poor stability, water-solubility, and bioavailability of AST [27] (Figure 2). For skin treatment purposes, liposomes are an excellent option for drug delivery because they can be absorbed by endocytosis, particularly by cells of the reticuloendothelial system, and release entrapped drugs. They are also able to fuse with cells or exchange lipids with the cell membrane [28]. However, in aqueous solutions, liposomes have reduced vesicle flexibility, which is a problem in the preparation of efficient liposomal formulations. In addition, absorption from topical application to the skin requires that it reaches the stratum corneum, which poses a challenge for the development of new alternatives to increase efficiency. The loaded-AST liposomes studies found are summarized in Table 1**.**

**Figure 2.** Disadvantages of the topical administration of AST and the use of delivery systems to improve the properties of the molecule, favoring skin absorption. Below: a schematic representation of vesicular systems (liposomes and nanoliposomes) acting as delivery systems for AST. Created with BioRender.com (accessed on 17 August 2021).

Dopierała et al. [29] have recently investigated the interactions between AST and the lipid membrane, in terms of the thermodynamic, morphological, and viscoelastic behavior and surface charge density, to understand how this molecule can affect liposomal formation and characteristics. The study showed that AST can be incorporated into the lipid monolayer and form a stable film, which regulates membrane fluidity and enhances the stability of the liposomal delivery system. Through steady-state fluorescence measurements, these authors observed that the interaction between AST and the lipid membrane reduced membrane fluidity but increased its micropolarity within a certain range of AST concentration, giving more stability to the saturated lipid bilayer. This system appears to be a promising way to improve lipid-based drug formulations, designing a dosage form that may help overcome the problem of unstable bioactive compounds. In addition to AST's antioxidant and biological activities, it is possible to enhance the stability of other formulations that are acting as adjuvants. Previously, Goto et al. [30] suggested that the two terminal rings of AST are likely to interact with the hydrophilic polar region of membrane phospholipids, and intermolecular hydrogen bonds could be formed between polar ends of the hydroxyl-ketocarotene and polar groups of phospholipids, thus regulating the fluidity of lipid membranes.

As an alternative liposomal delivery system, Lee et al. [31] developed liposomes with solid-supported lipid bilayers to overcome the flexibility issues of vesicles in an aqueous solution. Silicified liposomes were synthesized via silicification with tetraethyl orthosilicate on the hydrophilic regions of lecithin vesicles and assembled with boron nitride to enhance their stability. The resulting AST-loaded silicified-phospholipids boron nitride complex showed a release pattern and an antioxidant activity according to the 2,2- diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging method that is compatible with the literature. In addition, it presents a simple and low-cost method for AST applications in cosmetics.

**Table 1.** Summary of AST vesicular delivery systems, describing the preparation technique, liposome type, characterization and stability data, and assays (in vitro/in vivo) that were performed for each system.


However, regular liposomes have some disadvantages, due to their large particle size (around 1–100 μm). As an alternative way to enhance the use of this delivery system, Pan et al. [27] developed nanoliposomes with a lower particle size that exhibited better penetration and new targeting properties. Using a film-dispersion ultrasonic technique and soybean phosphatidylcholine as the lipid, AST-incorporating nanoliposomes (AST-LN) were prepared, characterized, and evaluated for their in vitro release, compared with the pure molecule. Nanoliposomes ensured higher encapsulation efficiency (97.5 ± 0.3%) than in previous reports, and an average particle size of 80 ± 2 nm, which was confirmed by transmission electron microscopy (TEM). AST-LN was 17 times more soluble than pure AST and had good water dispersibility, without insoluble particles or precipitation. X-ray diffraction (XRD) analyses suggested that this improvement of water solubility could be due to the change of AST's crystalline natural form into nanoliposomes. To evaluate the release of AST-LN, an in vitro assay was carried out with pure AST solution and nanoliposomes, using a dialysis bag technique, which stopped after given intervals. The AST release from nanoliposomes after 24 h was much slower (28.74%) than from the pure solution (95.27%), which supports the potential of continuous release from nanoliposomes.

Hama et al. [18] developed liposomal topical AST formulations to assess their action against skin damage that has been induced by UV radiation. The lipid hydration method was used to prepare either neutral liposome formulations (Asx-EPC-lipo) using egg phosphatidylcholine (EPC) liposomes or 1,2-dioleoyl-3-trimethylammonium-propane-based cationic liposomal formulations (Asx-DOTAP-lipo), intended to reach the skin stratum corneum by exploiting iontophoretic delivery. The diameter of the neutral vesicles varied from 170 μm to over 300 μm, and that of the cationic ones was around 170 ± 40 μm. The in vitro test of antioxidant activity with singlet oxygen generation and chemiluminescence detection showed that liposomal formulations did not interfere with AST antioxidant activity compared with the free solution, which increased in a dose-dependent manner. The results of in vivo tests performed to protect the skin from UV radiation showed that pretreatment of the skin surface with Asx-EPC-lipo prevented morphological skin changes, which suggests that liposomal AST formulation effectively prevented stratum corneum thickening by scavenging reactive oxygen species (ROS). The effect of iontophoretic transdermal delivery with cationic Asx-DOTAP-lipo pretreatment resulted in the significant inhibition of UV-induced melanin production in the basal laminae region. This result suggests that this formulation prevented melanocyte activation by efficiently scavenging ROS in deep regions of skin exposed to UV irradiation, acting as a potential whitening agent.

In another article, Hama et al. [32] compared the hydroxyl radical scavenging activity in an aqueous solution of Asx-EPC-lipo, prepared by the method of lipid hydration using EPC as the lipid base with those of β-carotene and α-tocopherol liposomes. The average diameter of EPC-lipo increased from 150 μm to over 300 μm, alongside increasing the amount of entrapped AST. Chemiluminescence intensity, which was assessed by the Fenton reaction to check the scavenging potential of AST and its encapsulated formulations, decreased in a dose-dependent manner with respect to AST in EPC-lipo. In addition, ASTcontaining liposomes were more powerful than either EPC-lipo-encapsulated β-carotene or α-tocopherol. An in vitro cytotoxicity test was performed to evaluate if Asx-EPC-lipo could protect a cultured mouse skin fibroblast cell line (NIH3T3 cells) against hydroxyl radicals damage. Results showed that the formulation prevented hydroxyl radical cytotoxicity in a dose-dependent manner since liposomes could suffer from cell endocytosis, and suggested that this effect was probably due to the action on the cell surface of AST molecules distributed in the plasma membranes.
