**2. Materials and Methods**

#### *2.1. Materials*

Hyt (>98% purity), chitosan medium molecular weight (50,000–190,000 Da, 75–85% deacetylated, viscosity < 200 mPa.s, 1% in acetic acid), lactic acid (DL-Lactic acid, powder), sodium tripolyphosphate (TPP, technical grade), Pluronic F-127, and Fluorescein isothiocyanate (FITC), 3,3 ,5,5 -Tetramethylbenzidine (TMB), Thiobarbitoric acid (TBA), Dichloro-dihydro-fluorescein diacetate (DCFH-DA) and Ultrapure HA in the form of sodium hyaluronate medium molecular weight were purchased from Sigma Aldrich (Milan, Italy) and used as received. All other reagents used in the experiment were of analytical grade and when not indicated were purchased from Sigma Aldrich (Milan, Italy).

#### *2.2. Preparation and Physico-Chemical Characterization of Hyt-Loading Nanoparticles (Hyt-NPs)*

A series of three Hyt-loading nanoparticles (Hyt-NPs) with varying chitosan concentration (0.1%, 0.5%, and 1% *w*/*w*) were obtained, with slight modifications, according to the well-known ionotropic gelation method [34]. Briefly, chitosan solution in 1% (*v*/*v*) lactic acid was prepared and stirred overnight at room temperature. TPP (5 mg/mL) and Hyt (10 mg) were dissolved in double distilled water to achieve different CS:TPP mass ratios. All solutions were filtered using 0.45 μm pore size membrane filters. TPP/Hyt solution was added dropwise into the chitosan solution under magnetic stirring (750 rpm) until a translucent Hyt-NPs suspension was formed. The suspension was stirred for 1 h at 1000 rpm at 25 ◦C to allow complete interaction. Then, the solution containing nanoparticles was ultrasonicated for 5 min at 40 kHz. Finally, Hyt-NPs were collected by cooling centrifugation (Frontiers 5718R, OHAUS, Milan, Italy) at 15,000 rpm for 45 min at 4 ◦C and washed with deionized water. FITC-loaded NPs were obtained by adding hydrophilic fluorescent probe into the TPP aqueous phase instead of Hyt, and the NPs prepared as described previously. Blank NPs were produced as negative control.

Particle Size (hydrodynamic diameter), polydispersity index (PDI), and zeta potential measurements were carried out on freshly prepared samples as reported in Conte et al. [35]. All samples were diluted in deionized water and measured at 25 ◦C using a Malvern Zetasizer (Malvern Instruments Ltd., Malvern, UK). The reported data are an average value of three measurements of the same sample. The particle size was confirmed by NanoSight NS300 Nanoparticles Tracking Analysis (NTA, Malvern Instruments, Amesbury, United Kingdom, UK). The fresh nanoparticle dispersions were centrifuged at 14,000 rpm for 30 min (5718R, OHAUS, Nänikon, Switzerland). The amount of drug entrapped in NPs was determined in triplicate indirectly by analyzing the amount of free Hyt in supernatant. The free Hyt in supernatant was quantified as described in Section 2.4.4 paragraph.

The encapsulation efficiencies of a series of Hyt-loaded nanoparticles were determined based on the following equation:

Encapsulation Efficiency (EE %) <sup>=</sup> Total amount of Hyt loaded <sup>−</sup> Free Hyt in supernatant Total amount of Hyt loaded <sup>×</sup> <sup>100</sup>
