**3. Results and Discussion**

## *3.1. Electrospinning of Fish Gelatin*

It has been widely reported that the addition of an acid to water solutions, or the use of solvents such as 1,1,3,3,3-hexafluoro-2-propanol, is required for gelatin electrospinning to prevent gelation that hinders and even blocks the solution flow through the syringe needle and capillary during the spinning process [32,33]. Due to the possible toxicity of the most employed acids or organic solvents, efforts have been devoted to the fabrication of gelatin electrospun fibers using solutions of benign acids in water. Acetic acid, malic acid, and citric acid water solutions, as binary, ternary, and quaternary solvents, were recently used to manufacture FG based electrospun mats [31,32]. Although positive results on the feasibility of the process were achieved for water/acetic acid solvents, citric acid based binary solvent was reported to be non-suitable for developing fibers [31].

Considering both the absence of toxicity of citric acid and its potential crosslinking action, in this work efforts have been carried out to identify a procedure for the production of crosslinked FG electrospun mats using a spinning solution containing only citric acid in an aqueous solution. In particular, the effect of solution's pH on the electrospinning process, mat properties, and crosslinking degree was investigated by adding NaOH to the FG citric acid/water solution in order to increase the pH of the FG solution from 1.8 to 3.7, on the basis of a previous study on collagen electrospun fibers crosslinked with citric acid [21]. In the mentioned paper the highest crosslinking extent was achieved from a solution containing collagen and citric acid with a pH 3.5, a result that was attributed by the authors to the more effective formation of citric anhydride at this pH [21,34].

Given the importance of flow properties and viscoelastic behavior of the solution in the electrospinning process, FG solution composition was optimized on the basis of rheological measurements, taking as a reference a PG solution whose electrospinnability was previously demonstrated by some of the Authors [8]. Figure 1 reports the storage modulus (G ) and the loss modulus (G") over 1 h 40 min period for the analyzed solutions. FG shows a rheological behavior similar to that of PG. Indeed, although FG moduli are slightly lower than those of PG, both solutions behave as viscoelastic liquids (G < G") at initial times (up to 12 min for PG and up to 23 min for FG), then they switch to a solid-phase dominated behavior (G > G"), as demonstrated by the achievement of the cross-over, referred to as the "gel point" attributed to the increased molecular association. Finally, the moduli reach a fairly constant value. The presence of NaOH affects the viscoelastic properties of the solutions. Indeed, FG+NaOH presents higher values of G and G" than FG, even higher than PG, and it shows no cross-over and a solid-phase dominated behavior since the beginning of the measurement. This result might indicate that crosslinking reactions due to citric acid are more likely to happen in the solution at pH 3.7 rather than in that at pH 1.8.

**Figure 1.** Time dependence of storage modulus G' (square) and loss modulus G" (circle) of PG (green curves), FG (red curves), and FG+NaOH (black curves) solutions.

For all the solutions, even if a gel point was observed and the solid-like behavior dominated over time, the small differences between G and G" indicate the formation of a weak gel that did not hinder electrospinnability of the solutions.
