**1. Introduction**

Gelatin is a polymer obtained from the thermal denaturation or chemical degradation of collagen. These processes involve the loss of the collagen triple-helix structure and the formation of random coil structure typical of gelatin. Gelatin macromolecules can rearrange, under certain conditions, thus forming again sequences of the triple helix, even if the fibrillar collagen structure cannot be recovered and the material becomes highly soluble in an aqueous environment. Gelatin is employed in different fields, such as food industry, tissue engineering, medical applications [1–4], thanks to its biocompatibility, biodegradability, and low cost. Moreover, gelatin shows binding sites for cell adhesion, signaling, and differentiation, which make this polymer suitable in tissue engineering, wound dressing, and drug delivery [5–9]. In these sectors, electrospun nanofibrous mats are highly demanded, since they mimic the extracellular matrix and promote cell adhesion and proliferation due to their high porosity and surface area.

However, due to water solubility, gelatin nanofibrous mats do not maintain their morphology when they come in contact with water. In order to improve water resistance, physical [10] and chemical [11–13] crosslinking methods have been proposed. Focusing on chemical crosslinking approaches, agents such as genipin, diisocyanate, glutaraldehyde, and carbodiimide, have been

successfully employed, being included in the gelatin solution prior to spinning [8,11,14–16]. Chemical methods also include post-treatment strategies based on the introduction of crosslinking agents after the spinning of the solution [13,16–20]. However, this approach demands a further step and, in some cases, has been reported to induce the flattening of the fibers, fusing them together [17,20]. Furthermore, some of the above-mentioned agents cause cytotoxicity [13,17] and are not suitable for applications in the medical field.

Citric acid is a natural acid and it has been already demonstrated to be suitable for the crosslinking of proteins [21–24]. The carboxylic groups of citric acid can undergo nucleophilic acyl substitution with the ε-amines of lysine, leading to the formation of stable amide bonds [21]. Saito et al. reported the use of a citric acid derivative, obtained through the modification of citric acid carboxyl groups with N-hydroxysuccinimide in presence of 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride, for the preparation of crosslinked type A porcine skin gelatin gels [25]. More recently, Shafagh et al. reported the use of citric acid to crosslink porcine skin gelatin in presence of Ag nanoparticles to produce, through a green approach in which water was used as a solvent, gelatin/Ag nanocomposite hydrogels with swelling and a drug release behavior both depending on pH [26]. Uranga et al. developed citric acid-crosslinked fish gelatin films, starting from an aqueous solution containing also glycerol and chitosan, with potentials for packaging applications since the presence of citric acid in the films did not produce any change of color, but improved both the light barrier and mechanical properties of the obtained films [27].

Among the different techniques for the production of polymeric substrates, electrospinning enables to develop micro- and nano-materials, and has been successfully used also to process fish-derived proteins [28], suitable for biomedical applications [29]. Citric acid has been used to crosslink collagen electrospun nanofibers. Cumming et al. successfully produced intra-fibrillar crosslinked marine collagen nanofibers by using the optimized citric acid: collagen molar ratio of 260:1 at pH 3.5 and performing a thermal treatment at 160 ◦C for 16 h after the mat fabrication [21]. Jiang et al. employed citric acid to produce crosslinked electrospun gelatin fibers using acetic acid as solvent and sodium hypophosphite as a catalyst of the citric acid, followed by a thermal treatment at 150 ◦C for 4 h to induce crosslinking [30].

To the best of our knowledge, no works have dealt so far with the production of crosslinked fish gelatin electrospun fibers by using an aqueous solution of citric acid, avoiding acetic acid or other chemicals. Indeed, only one previous work is present in the literature on the investigation of fish gelatin electrospinning in water/citric acid solution [31], but in this work the authors demonstrated that it was possible to obtain electrospun fibers only in the presence of additional acetic acid in the solution. Moreover, no studies or considerations are reported in this paper on the strategies to crosslink the as-produced mats.

The aim of the present work is to develop a protocol to make feasible, for the first time, the electrospinning process of fish gelatin by using only a natural and non-toxic crosslinker, such as citric acid, in aqueous solution to obtain crosslinked fibers, as illustrated in Scheme 1. The effect of the solution's pH on electrospinnability and morphological and chemical properties of the resulting fibers was investigated. Furthermore, the effect of a subsequent thermal treatment of the mat on its morphological and chemical properties was also assessed.

**Scheme 1.** Scheme of the proposed approach to produce electrospun crosslinked gelatin fish fibrous mats.
