*2.3. Characterizations*

The AgNPs water suspension has been characterized by means of UV-Vis collected using a Perkin-Elmer Lambda 19 and a Cary 100 spectrophotometer using quartz cuvettes. The dynamic light scattering (DLS) measurements on the AgNPs colloidal suspensions (0.200 mg/mL) at T = 25.0 ± 0.2 ◦C were performed by the Malvern Zetasizer Nano ZS90 instrument (Malvern, UK), as reported in previous studies [49,51]. The ζ-potential was calculated from the measured electrophoretic mobility by means of the Smolukovsky equation [52]. UV-Vis: λmax (nm) 415; DLS: <2R H> 5 ± 2 nm; ζ-potential: −35 ± 2 mV.

The mean size, size distribution, and ζ-potential of empty and AgNPs-loaded niosomes were characterized by using DLS. UV-Visible spectroscopy was employed to evaluate the amount of AgNPs entrapped in niosomal formulations. The AgNPs' entrapment e fficiency was expressed as encapsulation yield, i.e., the percentage of nanoparticles loaded with respect to the total amount added for the preparation. Bilayer characterization has been carried out on empty Span 20 or Tween 20 niosomes and on AgNPs-loaded ones, employing DPH and pyrene (lipophilic probes) that provided di fferent bilayer information (fluidity, microviscosity, and polarity).

Span 20/Tween 20 (15 mM), cholesterol (15 mM), and DPH solution (2 × 10−<sup>4</sup> M) were codissolved in chloroform/methanol, which was removed using a rotatory evaporator (VV2000, Heidolph, Schwabach, Germany), and then hydrated with HEPES bu ffer or AgNPs solution (0.5 mg/mL), with the same preparation methods as those mentioned above. A cellulose filter with a 450-nm cut-o ff (Spectrum, New Jersey, New Brunswick USA) was used to purify the DPH–niosomal formulations. Fluorescent measurements were performed (λ = 350–425 nm) using a luminescence spectrometer (LS5013, PerkinElmer) in order to obtain fluorescence anisotropy. The florescence anisotropy (r) was determined by using Equation (1) [53–55].

$$A = \frac{I\_{\rm vv} - I\_{\rm vlb} \times G}{I\_{\rm vv} + 2I\_{\rm vlb} \times G} \tag{1}$$

where *Ivv, Ivh, Ihv*, and *Ihh* are fluorescent intensities, and subscript *v* (vertical) and *h* (horizontal) represent the orientation of polarized light. The G factor is ratio of the sensitivity of the detection system for vertically and horizontally polarized light.

Pyrene-loaded niosomes were prepared by adding the probe (4 mM) to niosomes (Nio) components in order to obtain empty and Nio-AgNPs following the same preparation method as above. Pyrene is a florescence probe, whose monomer exhibited a spectrum characterized with five emission peaks (from I1 to I5) and excimer has only one peak (IE). The monomer and the excimer have di fferent fluorescence signals, and the ratio between the several fluorescence intensities is directly related to the probe distribution in the bilayer. In particular, the ratio I1/I3, corresponding to the first and third vibration bands in the pyrene spectrum, is related to the polarity of the probe environment. Pyrene can form an intramolecular excimer based on the viscosity of the probe microenvironment [56], and it is estimated with the ratio IE/I3, where IE is the excimer intensity. The fluorescence signals emitted by pyrene-loaded niosome suspension were scanned (λ = 350–550 nm) using a luminescence spectrometer (LS5013, PerkinElmer) and the intensities of the excimer florescence (IE), first (I1), and third (I3) peak were recorded [57].

An atomic force microscopy (AFM) study of the morphology of the niosomes has been performed using a standard AFM setup (Dimension Icon, Bruker Inc., Milan, Italy) equipped with Si cantilevers suitable for tapping mode imaging. Samples have been prepared by depositing a droplet of solution on a clean monocrystalline Si surface and waiting until partial dehydration. AFM imaging has been performed in air and at room conditions.

Small-angle X-ray scattering (SAXS) experiments were performed at a ID02 high-brilliance beamline at the ESRF (Grenoble, France). The measured SAXS profiles report the scattered radiation intensity as a function of the momentum transfer, q = (4 π/λ) sin (θ/2), where θ is the scattering angle and λ is the X-ray wavelength (0.1 nm). Analysis was carried out to obtain information on the dimension, homogeneity, and shape of the particles in solution. The form factors of niosomes have been reconstructed as unilamellar or multilamellar closed bilayers. Details are reported in the Supporting Information.

Biological studies were also carried out in the presence of bovine or human serum to evaluate the in vitro stability. NioTw20/AgNPs and NioSp20/AgNPs, as well as empty niosomes, were diluted in fetal bovine or human serum to obtain a final serum concentration of 45% at 37 ◦C. The average size, polydispersity index, and ζ-potential were evaluated by means of DLS at di fferent time points (15 min, 30 min, 60 min, and 180 min) [58].

Stability studies up to 90 days at di fferent temperatures—room temperature (RT) and 4 ◦C—were carried out by DLS to asses the dimension and ζ-potential of empty niosomes and Nio-AgNPs [58].

Release studies were carried out following the release calcein from empty niosomes and Nio-AgNPs using a Fluorimetric apparatus.

In order to obtain information about the ability of Nio to release lipophilic and hydrophilic probes, also in presence of AgNPs, Nile Red (lipophilic fluorescent probe) and calcein (hydrophilic fluorescent probe) release studies were carried out.

The hydrophilic probe (calcein 10−<sup>2</sup> M) was added to the film during hydration, and the excess of calcein was purified by gel filtration chromatography, as already mentioned [58].

On the contrary, due to its apolar nature, the fluorescent probe Nile Red was added to the vesicle components at a final concentration of 10−<sup>3</sup> M, and it will be located in the hydrophobic bilayer.

The in vitro release experiments were carried out using dialysis tubes (molecular weight cut-o ff 8000 and 5.5 cm<sup>2</sup> di ffusing area) at 37 ◦C in HEPES bu ffer (10 mM, pH 7.4). The Nile Red/calcein concentration was measured using the UV spectrophotometer/luminescence spectrometer at di fferent time points over 1–24 h.

In vitro release experiments were performed at 37 ◦C, and the defined volume of vesicle dispersions was included in dialysis sacs (cut-o ff 8.000) with a fixed di ffusing area (5.5 cm2) adding to a niosomal formulation of 45% human serum or HEPES (in order to maintain the same probe concentration). The probe concentration was detected in the outer solution at fixed time intervals (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, and 24 h) by means of the Fluorometric apparatus, taking into account the dilution factor.
