*4.8. Preparation of Hesperidin Nanocrystals*

HSD was utilized to formulate nanoemulsions. Polymers utilized in preparations were poloxamer (Pluronic F127, Sigma Aldrich, St. Louis, MO, USA) and nanocellulose, which served as stabilizers and showed different capacities regarding the preservation of hesperidin nanostructure. These were dissolved in water and mixed with an Ultra Turrax mixer for 15 min at a speed of 14,000 rpm min−1. Pre-milling with the mixer avoided blocking the homogenizing gap of the homogenizer present in the prepared formulation. Hereupon, the formulations I, II, and VII were processed through the homogenizer (Gea Niro Soavi, Model NS 1001L—Panda 2k, Dusseldorf, Germany) at high pressure (600 bar) for 5 cycles. During the homogenization process, a pressure decrease was observed, especially after the third cycle. The process utilized for HSD nanonization for formulations III, IV, V, and VI was based on the technology NANOEDGE™, where HSD was solubilized in a minimum quantity of solvent (or homogenous mixture of complex composition). The resulting solution was added to a mixture of purified water and stabilizers and it underwent a homogenization process with high-potency ultrasound (Ultronique, Model: DESRUPTOR; Freq. US: 20 kHz; Potency US: 750 W, Indaiatuba SP, Brazil), 70% potency for 30 min. Formulations V and VI were prepared through a NANOEDGE-like technique, where initially HSD was dissolved in the mixture of essential oil (orange peels) and glycerol and added into the solution of purified water and nanocellulose; afterwards, it was sonicated. Formulations prepared using two nano-techniques and their ingredients were as follows:

I—H69, combination of pre-milling with an Ultra Turrax and high-pressure homogenization, ingredients used: hesperidin and water;

II—H69, combination of pre-milling in an Ultra Turrax and high-pressure homogenization, ingredients used: hesperidin, DMSO, glycerol, sodium-carboxyl methyl cellulose, and water;

III—NANOEDGE-like, combination of microprecipitation and ultrasound, used ingredients: hesperidin, DMSO, glycerol, poloxamer (Pluronic F127), and water;

IV—NANOEDGE-like, combination of microprecipitation and ultrasound, used ingredients: hesperidin, DMSO, orange oil, poloxamer (Pluronic F127), and water;

V—NANOEDGE-like, combination of microprecipitation and ultrasound, used ingredients: hesperidin, propylene glycol, nanocellulose, and water;

VI—NANOEDGE-like, combination of microprecipitation and ultrasound, used ingredients: hesperidin, glycerol, nanocellulose, and water;

VII—H69, combination of pre-milling in an Ultra Turrax and high-pressure homogenization: hesperidin, glycerol, orange oil, poloxamer (Pluronic F127), and water;

VIII—Milling in an Ultra Turrax, ingredients used: hesperidin, glycerol, orange oil, poloxamer (Pluronic F127), and water.

### *4.9. Formulation of the Silky Cream*

Oil-in-water silky cream formulations were prepared following the protocols of the EVONIK ® group (Essen, Germany), for use of the surfactant ABIL ® Care 85 (Bis-PEG/PPg-16/16 PEG/PPG16/16 Dimethicone; Caprylic/Capric Triglyceride) and presented in Table 3. Muru muru (*Astrocaryum murumuru* Seed Butter), Cupuaçu butter (*Theobroma grandiflorum* Seed Butter), and Andiroba oil (*Carapa Guianensis* Seed Oil) were sampled from Amazon oil industry; Glyceryl monostearate, Mineral oil, Carbopol ULTREZ 10, Triethanolamine, and Vit E-acetate ( α-Tocopheryl acetate) were from Fagron ®. Emulsifier ABIL ® Care 85 that gives a velvety-silky skin feel was sampled from EVONIK ® and was used in exceptionally low usage concentration. Ethylhexylglycerin and Phenoxyethanol (Fagron ®) were used for formulation preserving in exceptionally low concentration, instead of the commercial paraben's components.


**Table 3.** Silky velvet cream formulations prepared for application of hesperidin nanoemulsions.

> \*gtts is drops.

Ingredients of the phases A and B were measured, transferred into two beakers, and heated to 50 ◦C. When the oil phase was homogenized and all the ingredients were molten, phase A was added into phase B. The mixture was stirred and homogenized until it cooled down to a temperature of 25 ◦C. Ingredients of the phases C and E were added at room temperature, along with stirring. A whitish cream, oil-in-water, silky, with soft consistency, and easy to apply with no greasy film leftovers after applying was obtained. Nanoemulsion VI was used for the preparation in the A2 cream and Nanoemulsion VII was used for preparation in the A3 cream formulation.

#### *4.10. Skin Model USP-FTS Skin Corrosion Test of Cream Formulations*

Full thickness skin models (FTS) for the toxicity test of hesperidin cream formulations were prepared in the Laboratory of Biology of the Skin, coordinated by Prof. Silvya Stuchi Maria-Engler, Faculty of Pharmaceutical Sciences, University of Sao Paulo. Fibroblast and keratinocytes were isolated from the normal human skin cells, from donated foreskin samples obtained from the University of Sao Paulo Hospital (Sao Paulo, Brazil). Performed assays were under the approval of the local Ethics Committee (HU CEP Case No. 943/09 and CEP FCF/USP 534). Isolated cells were seeded on top of the collagen I matrix model in a 6-well plate containing enough specific medium mixture for the FTS model as to maintain the skin at the air–liquid interface, as it is described by Catarino et al. [32]. For the corrosion tests, the following system was used: 100 μg mL−<sup>1</sup> of hesperidin in 10% DMSO, cream without the hesperidin nanoemulsion (A1), two formulations of hesperidin nanoemulsions (Nanoemulsion VI and Nanoemulsion VII), and the last two in the cream formulations as A2 and A3 creams. As a negative control, 0.9% sodium-chloride solution was used. The 100 μg mL−<sup>1</sup> of hesperidin in 10% DMSO were tested by adding 5 mL in the well plate, and the 0.5 mL of cream formulations were applied over epidermis. Samples were left over night for incubation (37 ◦C, 5% CO2). All tests were performed in duplicate. After 24 h, skins were washed with 0.9% of sodium-chloride solution and preserved in para ffin for further microscopic tests.

#### *4.11. Di*ff*raction Light Scattering and Zeta Potential*

Mean size, polydispersity, and zeta potential of nanoparticles from each formulation were determined using di ffraction light scattering technique (Nano ZS Zetasizer—Malvern, PANalytical Almelo, The Netherlands). Known also as Photon Correlation Spectroscopy—PCS, this is the technique that was used to measure the hydrodynamic diameter of particles in a range from microns up to 1 nm.
