*2.2. DSPE–HA Conjugate*

The DSPE–HA conjugate was produced with the protocol described by Yao et al. with a few modifications [32]. Briefly, 50 mg of HA sodium salt (8–15 kDa) was added to 20 mL of purified water. The solution was dialyzed (2 kDa cut-off) against 1 L of 0.01 M hydrochloric acid (HCl) for 24 h to acidify the HA, and then against 1 L of purified water for 24 h. After the dialysis, the solution pH was increased to 9 with a few drops of 20% tetrabutylammonium hydroxide (TBA-OH). The mixture was then stirred for 2 h at room temperature and thereafter dialyzed against an excess of purified water. Water was removed from the final dialysis product by lyophilization with SCANVAC CoolSafe 110 (LaboGene ApS, Allerød, Denmark).

The freeze-dried HA–TBA powder was dissolved in 3.5 mL of anhydrous dimethyl sulfoxide (DMSO) under an argon atmosphere, and the solution was stirred at 60 ◦C until the powder dissolved completely. Then, 16.3 mg of DSPE and 6.1 µL of dry triethylamine were mixed with 0.87 mL of anhydrous chloroform, and the solution was sonicated for a short period until all the particles visibly dissolved. The HA and DSPE solutions were combined and stirred for 2 h at 60 ◦C. Thereafter, a solution containing 9.2 mg of sodium triacetoxyborohydride (NaBH(OAc)3) and 0.87 mL of anhydrous DMSO was added dropwise to the DSPE–HA–TBA solution. This mixture was allowed to react for 72 h at 60 ◦C and then cooled to room temperature.

Chloroform was evaporated from the suspension under a constant nitrogen gas flow. Then, 30 mL of purified water was added to the suspension, which was then centrifuged at 10,000× *g* for 30 min. The supernatant was dialyzed against 0.01 M HCl for 24 h and under purified water for an additional 24 h to remove the acidic form of the TBA salt. Finally, the product was freeze-dried with the SCANVAC CoolSafe 110 (LaboGene ApS) over the duration of two days.
