*2.1. Materials and Methods*

Silver nitrate (AgNO3, 99.5%, Sigma-Aldrich, St. Louis, MO, USA) and sodium borohydride (NaBH4, 98%, Sigma-Aldrich, St. Louis, MO, USA) were used for the synthesis of the nanoparticles. 3-mercapto-1propanesulfonic acid sodium salt (C3H7S2O3Na, 3MPS, 98%, Sigma Aldrich, St. Louis, MO, USA) was used as a capping agent. For all of the solutions, we used deionized water (electrical conductivity less than 1 μΩ/cm at room temperature) obtained from a Millipore Milli-Q water purification system. HEPES salt (sodium 2-(4-(2-hydroxyethyl) piperazin-1-yl) ethanesulfonate), cholesterol, Sephadex G75, Pyrene, DPH (1,6-diphenyl-1,3,5-exatriene), Span 20 (sorbitan monolaurate), Tween 20 (polyoxyethylene sorbitan monolaurate), human/bovine serum, calcein, and Nile Red were purchased from Sigma-Aldrich (Milan, Italy). All the other products and reagents were of analytical grade. All of the reagents were purchased from Sigma Aldrich and were used without further purification.

### *2.2. Preparation of AgNPs Loaded Niosomes*

The AgNPs–3MPS synthesis (see Figure S1) consisted in a wet reduction of silver nitrate to metallic silver by means of sodium borohydride in the presence of 3MPS [17,18]. Briefly, a solution of AgNO3 in deionized water (0.200 g in 10 mL) was added in a flask with 3MPS water solution (0.830 g in 10 mL), and the mixture was maintained under stirring in argon atmosphere at room temperature for 10 min. Then, an NaBH4 water solution (0.220 g in 10 mL) was added dropwise, under vigorous stirring. The reaction mixture was allowed to react for 2 h. The obtained black product was centrifuged with deionized water three times (20 min, 5000 rpm), and the solid obtained was characterized.

Several niosomal formulations by Tween 20 (NioTw20) or Span 20 (NioSp20) were prepared using AgNPs-3 MPS at di fferent concentrations. Only the results obtained by selected samples in terms of the size, ζ-potential, entrapment e fficiency, and stability features were reported and discussed.

AgNPs–3MPS were loaded into Span 20 and Tween 20 niosomes through a protocol already used to internalize chemicals [49]. Niosomes were prepared using the thin film hydration method [50]. Span 20 or Tween 20 (15 mM) and cholesterol (15 mM) were dissolved in organic solvent mixture (chloroform/methanol 3:1 *v*/*v*). The solvent was evaporated using a rotary evaporator (VV2000, Heidolph, Schwabach, Germany) to form a thin "film". The film was hydrated using 5 mL of AgNPs solution, which was then vortexed and sonicated at 60 ◦C and 18%/16% amplitude for 5 min using an ultrasonic microprobe (Vibra-Cell VCX-400, Sonics & Materials, Newtown, CT, USA). The unilamellar vesicular suspension was purified by gel filtration chromatography using Sephadex G75 (glass column of 50 × 1.2 cm) with HEPES bu ffer as the eluent. The purified vesicles were filtrated by using cellulose filters (pore size 1.2 μm).
