*2.6. Stability of HSD Cream Formulations*

The stability tests for HSD incorporated in silky cream formulations, kept in plastic boxes made of polypropylene at 4 ◦C and at room temperature, were carried out 72 h, 30 days, and 12 months after manufacture. Organoleptic tests included appearance, color, brightness, consistency, and homogeneity of the creams, evaluated by visual observation. Applied and sensory characteristics of samples, such as spreadability, absorption, stickiness, and fat film on the skin were assessed after applying the cream on the skin. Shortly after preparation, creams were homogenous, bright, and easily spreadable on the skin. During the following 12 months, their consistency did not change, and the cream formulations maintained odor and color as well. Measurement of the pH value was carried out potentiometrically, by direct immersion of the glass electrode into the tested samples of creams at room temperature. Values of the pH were in the range from 5 to 6, as is recommended by pharmacopoeia.

#### *2.7. Evaluation of Toxicity of HSD Nanoemulsions and Cream*

Full thickness skin (FTS) models for the toxicity (corrosion) test of hesperidin cream formulations were synthesized in the Laboratory of Biology of the Skin, Faculty of Pharmaceutical Sciences, University of Sao Paulo. As the experiment is quite complicated and expensive, just some of the HSD cream formulations were tested.

Figure 5 presents photomicrographs of histological analysis—eosin stain (samples in para ffin) of the FTS skin model after the cream application assay (24 h). This model was synthesized from the fibroblast and keratinocytes isolated from normal human skin cells-donated foreskin samples. The model was composed from two main skin layers-epidermis and dermis-and used for the evaluation of the toxicity of cosmetic and other preparations. Epidermis was composed from five layers: stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. The last one was formed from the undi fferentiated cells, with a full metabolic activity, which was lost in later proliferation of the cells and formation of the corneocytes. It is important to mention that macrophage and Langerhans cells were not present, which are the first defensive line of the skin [36].

**Figure 5.** Photomicrographs of Histological Analysis—Eosin stain (sample in para ffin) of the full thickness skin (FTS) equivalent skin model after the cream application assay (**a**) negative control: sodium chloride 0.9%; (**b**) positive control: HSD-DMSO, (**c**) A1 cream, and (**d**) A2 cream. The skin models were generated using primary human keratinocytes and fibroblasts—the FTS model; (Scale bar = 500 μm).

In the negative control sample (Figure 5a) photomicrograph, epidermis and dermis layers were preserved and there was no presence of vacuolization in the stratum basale. After application of the A1 cream (without HSD-nanoemulsion), the basal layer was preserved, with remaining proliferation despite some vacuoles' presence (Figure 5b). The stratum corneum of this sample was preserved and thicker, and the cream demonstrated good moisturizing e ffects. HSD in 10% DMSO showed corrosive and cytotoxic e ffects on the FTS skin model, and led to complete degradation of the basal layer (Figure 5b). Further, the formulation with even a small percentage of DMSO was avoided. Application of nanoemulsion VI showed an irregular basal layer and elevated stratum corneum; therefore, a slight corrosive effect for this formulation was observed (Figure 5d). Nanoemulsion VII demonstrated less cytotoxic effects, inclusively in the basal membrane, compared to the A2 cream. The corrosion test with the A2 and A3 creams (data not shown) showed moderate hydration effects on the FTS skin model, probably due to the hygroscopic effect of some of their ingredients. As this skin model is a living organ, some of the ingredients of the nanoemulsion or the surfactant alone could have led to lower vitality of the cells and prevented their further proliferation. The A2 cream contained only a Pluronic-F127 as a hygroscopic compound, and therefore, the viability of the cells was much higher than in the case of the A3 cream. Nanoemulsions VI and VII demonstrated corrosive effects on the skin model (data not shown) due to the dilution of the buffer solution as well as the inadequate environment for the development and proliferation of cells. It is important to mention that the generated FTS skin model does not contain macrophages or Langerhans cells, which are the first defense of the skin tissue's immunity, which makes it much more sensitive than normal skin tissue.
