*2.5. Characterization of HSD Nanocrystals*

The obtained HSD was pre-milled and further nanonized on a homogenizer or ultrasound of high potency. The e ffectiveness of pre-milling was clearly revealed by the reduction in particle size distribution observed after applying di fferent analytical methodologies for HSD nanonization. The average size, zeta potential, and polydispersity index of the HSD nanoparticles are shown in Table 2.



The results were obtained after 6 months of preparation of the emulsions. The sizes of the HSD nanoparticles, after passing through the homogenizer and ultrasound, reduced from 600 to 150–350 nm, depending on the type, concentration of the polymer, and technique used for preparation. Zeta potentials were very variable, and it was not possible to obtain the desired stability for all nanoemulsions (for example, III, Table 2) with the best zeta potential being around −30 ± 6 mV. Part of the particles had sizes up to 400 nm as measured using the Dynamic Light Scattering (DLS) and will not a ffect their permeation via *stratum corneum*.

Scanning and transmission electron micrographs obtained for the formulations I, II, and IV–VIII are presented in Figure 4 and show particles whose sizes are in accordance with the sizes measured by (DLS) and NanoTracking Analysis (NTA) techniques. Nanoparticles of formulation I were around 200 nm in size, as shown in Table 2 and Figure 4. The formulation II contained bigger particles, around 1 μm, probably because of sodium-carboxyl methyl cellulose as surfactant (image not shown). Formulation IV (Figure 4) had particles with sizes of 250 nm (DLS and NTA), probably due to the aggregation of poloxamer around the hesperidin particles, whereas NTA experiments showed the presence of smaller particles of 50–100 nm.

Formulations V and VI were prepared utilizing the technique NANOEDGElike, where hesperidin was dissolved in small amounts of propylene glycol and glycerol, respectively. SEM experiments were not adequate to characterize the VI formulation and it was necessary to apply atomic force microscopy (AFM) to perform such characterization. The particles were in the size range of 50–200 nm, and in the image of formulation VI, nanofibers of nanocellulose could be seen. Formulations V and VI showed above average stability for 6 months without any precipitation of the nanoparticles. Their stability was achieved mostly because of the significant amount of nanocellulose used for their production, while good dispersibility of hesperidin was achieved because of propylene glycol and glycerol used in their formulation. As the formulations were prepared in aqueous solutions, there was no observed HSD binding with CNF, which was also especially important from the point of view of skin permeation. The stability of the prepared nanoemulsions (I–VIII) was very good (up to 6 months), which despite showing a small percentage of agglomerates, returned to the initial state by a process involving the use of an ultrasound bath (5 min).

**Figure 4.** Scanning electron micrographs (**left**; scale bar = 500 nm) of formulation **I** and transmission electron micrographs (**right**; scale bar = 200 nm) of formulation **I**. Scanning electron micrographs of formulation **III** (**left**; scale bar = 1 μm) and formulation **IV** (**right**; scale bar = 500 nm), respectively. Scanning electron micrographs of formulation **V** (**left**; scale bar = 1 μm) and atomic force micrographs of formulation **VI** (**right**; scale bar = 4 μm). Scanning electron micrographs of formulations **VII** and **VIII** (**left**; scale bar = 2 μm).
