# Physicochemical Characterization of Hyaluronic Acid and Chitosan Liposome Coatings
## 1 Introduction  
Liposomes are self-assembled phospholipid structures showing biocompatibility properties. These systems have been applied to improve the bioavailability of hydrophobic bioactive compounds. Liposomes and nanoparticles are widely used for stabilizing drugs and improving their pharmacological properties by acting as carriers in drug delivery processes and promoting the controlled release of drugs at selected target sites [1]. By modifying the surface of these carriers with polymers, such as polyethylene glycol, they become able to prevent mechanisms of plasma-protein adsorption, interfering with therecognition by the reticuloendothelial system [2; 3]. These vesicles are known as stealth liposomes, and their ability to prolong blood circulation, improving the pharmacological effects of drugs, is well documented [4; 5; 6]. On the other hand, these systems provide new opportunities in the field of pharmaceutical research and the design of innovative liposomes. To inhibit/reduce chemical degradation of a liposome and undesirable loss of the encapsulated drug, polymers can be used to modify the physicochemical properties of vesicles [7; 8]. In particular, the surface properties can be modified to reduce toxicity and to control drug release [9; 10]. Coated liposomes have demonstrated that drug activity was synergically increased, along with many other desirable biological effects [11]. The surface modification of liposomes can be obtained by following different processes, such as polymer adsorption [12]. Furthermore, surface coating with a polymer is able to preserve liposome stability [13], and this behavior depends on the ability of the polymer to adhere to the lipid bilayers [14]. Two different strategies can be considered to modify the liposome surface: (i) the classical adsorption of a polymer, promoting an excess of polymer in solution and inducing a random distribution at the surface and (ii) the innovative hydration of phospholipids from a polymeric solution [15]. Moreover, liposomes are systems that are thermodynamically unstable and the coating could interfere, decreasing the colloidal stability. The study of chemical modification of vesicles becomes a very challenging aspect and highlights potential new target sites and applications [16]. Two different polymers were used in the present study to obtain the surface modification of liposomes: chitosan (CH; molecular weight: 50-190 kDa; Figure 1a) and hyaluronic acid (HA; molecular weight: 8.7 kDa; Figure 1b), both at low molecular weights. Chitosan is a linear copolymer composed of D-glucosamine and N-acetyl-D-glucosamine units, linked via \(\beta\)-(1 \(\rightarrow\) 4) bonds. It is a biodegradable polymer obtained from the alkaline deacetylation of chitin [17]. Chitosan is one of the most widely used biopolymers in biomedical applications (textiles, cosmetics and food treatment) due to its ability to bind to a variety of natural or synthetic species such as nanoparticles, polymers, metal ions and cell surfaces. Moreover, owing to its high biocompatibility, biodegradability, absorption capacity and non-antigenicity, chitosan gels and supports are often adopted in the biomedical field and for the synthesis of organic-inorganic nanocomposites with bioactive substrates. Chitosan-coated liposomes have been used as a mucoadhesive delivery system. Their positively charged surface favors adhesion to cell membranes, which are normally negatively charged [14]. Hyaluronic acid is an anionic glycosaminoglycan and is one of the fundamental components of the connective tissue of humans and mammals. HA is a polymer constituted of disaccharides, D-glucuron acid and N-acetyl-D-glucosamine, connected by alternating \(\beta\)-(1 \(\rightarrow\) 4) and \(\beta\)-(1 \(\rightarrow\) 3) glycosidic bonds. HA has a polymeric structure, having different  
Figure 1: Numbering and chemical structure of (**a**) chitosan (CH) and (**b**) hyaluronic acid (HA). molecular weights (5 and 20,000 kDa).