1. INTRODUCTION
The human body has different ways for protecting itself against potential injuries and infections, by means of the skin characteristics acting as an effective barrier. The physiological process of tissue repair at an injured site is the wound healing, which represents a complex integrated system of biological and molecular processes, such as cell migration, proliferation, extracellular matrix deposition and remodelling.[1] Under normal conditions, the healing process cascade is initiated by an inflammatory phase yielding to granulation tissue accumulation and scar remodelling after epithelial wound closure has been accomplished.[2] A normal wound healing process is constituted by various phases: coagulation (hemostasis), inflammation, cellular migration and proliferation, protein synthesis and wound contraction (fibroplasia), and scar formation (remodelling). Among these phases, three are the most important for a successful wound healing: fibroplasia, angiogenesis and re-epithelialisation. A delay in the development of these mechanisms leads to failure or prolong wound healing process with a crucial part attributable to the wound neovascularisation.[3,4] However, any change or pathological problem (for example ischemia, diabetes mellitus and so on) may result in the formation of chronic wounds difficult to heal, due to the failure of the normal wound healing process.[3,5] Another very important objective of the wound treatment is the inhibition of all the pathogenic organisms able to cause serious infection, by accelerating the healing process with reducing cicatrices and pain to the patients of all ages.[5] In fact, any delay during the tissue repair process could be defined as chronic injury and can have a highly detrimental impact on human health being more subjected to pathogens. Rapid closure of dermatological wounds is of vital importance in preventing infection and reducing post-treatment side-effects.[6] So, shortening the chronic wound healing process is a prime challenge in modern dermatology.[7]
Several different topical and systemic agents, together with the antimicrobial ones have been developed for improving the cure of surgical and traumatic wounds. However, these functional topical biomaterials might delay re-epithelialisation and might result a risk for oversensitivity and resistance.[4] Hence, some alternative wound treatment methods have been searched for and, in this regard, the use of nanotechnology could be useful as a prominent scientific discipline in the technological revolution of this millennium.[8] Nanobiotechnology-based therapies in the field of wound healing are considered to be a promising area with new benefits based on the utilization of bionanomaterials in promoting wound healing, including antibacterial, antiinflammatory, regulating extracellular matrix production, promoting stem cell proliferation and differentiation, and enhancing growth factors.[9] Researches using gold nanoparticles (AuNPs) of different sizes and morphologies have been proposed in biomedical applications for skin injury therapy with great potential.[6] AuNPs have received great attention due to their low toxicity to animal and microorganism cells compared to other metal nanoparticles,[10] promoting their use as drug delivery systems, markers, and photothermal agents, as well as radiotherapy enhancers as well.[6] Recent studies have established new prospects for developing properly functionalized AuNPs for wound healing, acting as triggers to activate proliferation and migration of wound cells.[7,11,12] The AuNPs use should exhibit benefits including enhanced mechanical stability and resistance against enzymatic degradation when incorporated into tissue scaffolds, in presence of antibodies, growth factors, and peptides, rendering them promising candidates for skin tissue engineering applications.[8]
Chemical methods, in which reduction or co-precipitation processes take place for the generation of nanoparticles, have been investigated since ancient times. However, the main drawbacks of these synthetic routes are the use of harsh chemical conditions of work, organic solvents and the production of toxic by-products during the synthesis and/or functionalization of the nanoparticles. For this purpose, many studies are focused on finding alternative therapeutic approaches by adopting biocompatible, safer and greener methods employing plant extracts for the fast and economical synthesis of biogenic nanogold.[13-15] Green synthesized AuNPs can also exhibit anticancer and antimicrobial activity,[16] antioxidant moisture retention, skin lightening properties,[17] and sunscreen ability, enlarging their applicability.[18,19] As reported by Gonnelli et al. ,[20] synthetic routes relying on green chemistry show other advantages for large scale production adopting mild conditions of work.[20] For green synthesized AuNPs, additional assembling steps are not necessary since the AuNPs properties are ascribed to main components present on their surface and used to induce their formation. Thus, considering these factors, the snail secretion (SS) from Helix Aspersa , currently revolutionizing the world of cosmetics and human skin care, has been used in this work for synthesizing AuNPs (AuNPs-SS), conferring them interesting properties. Indeed, the SS protein content plays a key role in cell regeneration and growth, preventing the effect of the inflammatory disease.[21,22] The SS main compounds are used as reducing agents enabling the AuNPs synthesis via reduction of Au(III), from a HAuCl4 solution, to Au(0), while forming an organic layer around the AuNPs. AuNPs-SS are thus synthetized by using a single step reaction, avoiding the use of additional and toxic agents. The nanoconfined main components of the SS on the AuNPs surface could be an interesting way for improving the whole process, combining the properties of gold and SS. For example, Conte et al .[23] reported that the pharmacokinetics of snail components do not permit their optimal absorption, so the combined use of AuNPs, well-known antibacterial agents, and SS could be a functional ingredient in the treatment of acne and in biotechnology, or used as a preservative in cosmetic products formulation. Considering the overall literature published,[21,22,24-27] although interesting information can be found on the SS use, the presence of fragmentary and not detailed investigations suggests that this research field has not been extensively explored yet. In a time when cosmetic dermatology and beauty care are increasingly influential,[28,29] we intend to discuss the biogenic formation of AuNPs-SS by characterizing them and by applying as accelerating wound healing and anti-inflammatory agents in biomedicine. More specifically, we tested the effect of AuNPs-SS on improving wound healing in human keratinocytes. Furthermore, considering that gold has been used historically in the treatment of painful inflammatory conditions, such as rheumatoid arthritis, and snail slime has been proved to clear up skin inflammation, we evaluated the potential anti-inflammatory properties of AuNPs-SS in Murine Macrophages.
2. EXPERIMENTAL SECTION