Carnosine (β-alanyl- l-histidine) is a water-soluble dipeptide characterized by three ionizable groups [1,2]. The molecule is endogenously predominant in tissues such as skeletal muscles, the nervous system and the cardiac muscle. The main dietary sources of carnosine for humans are meat and fish, but it can also be synthesized endogenously in glial cells and myocytes [3,4]. It exhibits significant antioxidant properties, as demonstrated in several studies. The antioxidant properties of carnosine are mediated by different mechanisms involving metal ion chelation and scavenging reactive oxygen species (ROS) and peroxyl radicals [1].

Accumulation of advanced lipid peroxidation end products (AGEs) in the skin may be associated with the aging process because it has been found to affect extracellular matrix proteins such as collagen, vimentin and elastin [5]. Carnosine has been shown to protect against the formation of AGEs in the skin, particularly carboxymethyl lysine (CML) and pentosidine, while the carnosine carrier plays a significant role in its bioavailability in the skin which in turn, affects its effectiveness in reducing AGEs [6]. Α combination of injected carnosine and locally applied carnosine in mice with diabetes mellitus type 2 has shown a remarkable enhancement of wound healing. In the same study, an in vitro experiment with human dermal fibroblasts and microvascular endothelial cells showed that carnosine increased cell viability in the presence of high glucose. This is especially important because in diabetes numerous factors delay the wound healing [7]. In a study regarding a night cream formula containing melatonin, carnosine and Helichrysum italicum extract, it was found that the application of this product significantly improved all markers regarding oxidative stress, hydration and clinical signs of aging [8]. Carnosine has been studied several times for its effect on skin and ageing, however, most of the studies utilized a combination of molecules, making it difficult to attribute the benefits to carnosine [[9], [10], [11], [12], [13]]. In addition, bioavailability plays a key role in the effect that carnosine can facilitate on the skin.

To enhance active ingredients permeability through the skin, liposomes are utilized as delivery systems because they possess unique abilities. Due to their vesicular structure, liposomes resemble biological membranes. They are composed of amphiphilic molecules, usually cholesterol and nontoxic phospholipids in a bilayer conformation. Liposomes are defined as pseudo-spherical vesicles with particle sizes ranging from 30 nm to several micrometers [14,15]. Besides improving the biodistribution of active ingredients, liposomes are known to make therapeutic compounds more stable, can be used with hydrophilic and hydrophobic molecules, are biocompatible and biodegradable [16]. Several types of lipid nanoparticles (LNPs) have been developed for topical dermatological use including conventional liposomes, composed of phospholipids and cholesterol, ultradeformable liposomes or transferosomes, composed of phospholipids and surfactant molecules, niosomes, ethosomes etc. All these classes exhibit several advantages for specific uses [14]. Evidence in the literature suggests that there is not one unifying theory regarding the way liposomes improve active substance topical delivery through the skin, rather than different types of liposomes loaded with different drugs exhibit different behavior due to different interactions [17].

The physicochemical characteristics of liposomes affect their ability to deliver substances to the skin. Specifically, the transition temperature (Tm) of the phospholipids forming the liposomes has an impact on the fluidity of the liposomal membranes as well as the passive permeability of water and small molecules through the membrane of the vesicles. The size of the vesicles also plays a significant role in liposome ability to deliver drugs and active substances into the skin with vesicles of size greater than 600 nm being unable to reach deeper layers of the skin, vesicles of 300 nm being able to reach deeper layers of the tissue and vesicles of 70 nm size showing more promise for topical skin delivery. The surface charge of liposomes can modify their permeation capacity. Whether anionic or cationic liposomes perform better is rather controversial, as studies suggest that both types show promising results, because surface charge plays a role in the final formulation and depends on the drug encapsulated in the liposome [14].

Since elasticity of the bilayer is a key factor for effective skin delivery, ultradeformable liposomes (transferosomes) exhibit great potential as a drug delivery system for skin medications and active ingredients in general. Transferosomes were introduced by Cevc et al. and are composed of phospholipids and an edge activator, typically a surfactant. The combination of these two types of molecules in a single vesicle destabilizes the lipid bilayer and increases its deformability by lowering the interfacial tension, thus creating elastic liposomes. Surfactants used as edge activators are typically sodium cholate, Span 60, Span 65, Span 80, Tween 20, Tween 60 and Tween 80 [14,18]. Ceramides are a complex group of sphingolipids, containing derivatives of sphingosine bases in amide linkage with fatty acids and are one of the components of the lipid matrix of the stratum corneum (SC) that regulates skin barrier function, cell adhesion and epidermal differentiation. In the SC, at least 8 ceramides have been identified that differ from each other in terms of headgroup architecture and fatty acid chain length. The less dense lipid organization is associated with a reduction of lipid chain length in the SC, in patients with atopic dermatitis and patients with impaired skin barrier function [19,20]. Numerous skin products utilize ceramide combinations to improve skin conditions and liposome formulations containing ceramides have gained some attention in recent years [[20], [21], [22], [23], [24]].

In this study, three different liposomes containing 2.5 % w/w carnosine were developed by the modified heating method (MHM) [25,26], namely a conventional liposome, a liposome containing Tween 80 as edge activator (transferosome/elastosome) and a ceramide-containing liposome (ceramidosome). Several carnosine-containing liposomal formulations have been previously developed for various purposes, while a recent review discusses the therapeutic potential of nanoparticulate carnosine [[27], [28], [29], [30]]. Concerning the MHM, it is based on the classic heating method, which includes hydrating and heating phospholipids until liposomes are formed and by not utilizing any volatile organic solvents, but by employing higher concentrations of polyols, avoids overheating the formula and leads quicker and one-step to improved physicochemical characteristics [25]. A carnosine concentration of 2.5 % w/w was formulated and the encapsulation efficiency of the molecule inside the nanoparticles was evaluated. Since topical formulations in the literature contain 0.2 % – 1 % w/w carnosine, the rationale in this work was to develop a formula that could be further formulated in other dosage forms (e.g. a gel or a cream) for topical use and retain its therapeutic properties [6,[8], [9], [10]]. The stability of the formulations was evaluated for 60 days under room temperature and 4 °C storage conditions, to decide which kind of liposome shows more potential for topical use. An in vitro skin permeation study (IVPT) was also performed to assess the bioavailability of the herein prepared carnosine liposomal formulas [31,32]. The formulas were used without purification, in order to have the same concentration of nanoparticles and carnosine in all cases, whether the latter would be encapsulated or not.