*2.6. Permeation Assays*

### 2.6.1. Ex Vivo Permeation Assay

Porcine skin was obtained from the back of pig ears within 12 h after slaughter. The pig ears were cut into circular sections 3 cm in diameter. The excess of fat was removed with surgical scissors, and the samples were washed with saline solution. The samples were divided into three groups: drug dispersion with 0.01% *w*/*v* of curcumin (2 mL; *n* = 3), CNp dispersion with 0.01% *w*/*v* of curcumin (2 mL; *n* = 3), and CNp-M4 membranes (3 cm in diameter; *n* = 3).

The experiments were conducted in 12 independent Franz cells with a diffusion area of 7.07 cm2. The freshly excised skin was placed into Franz cells, and the stratum corneum remained in contact with the donor compartment, with the dermis facing the receptor compartment. This was filled with 30 mL of 0.1 M PBS solution (pH 7.4) with 2.5% of Tween 80 and maintained under constant stirring at 400 rpm.

Franz cells were immersed in a water bath at a constant temperature of 37.0 ± 0.5 ◦C. At predetermined times (1, 2, 3, 4, 5, 6, 7, 8, 22, 24, 26, 28, and 30 h), 1000 μL of medium was removed from the receptor compartment and replaced with 1000 μL of fresh receptor medium.

At the end of the test, the skin samples were carefully removed from the Franz cells to conduct the tape-stripping test, following the in vivo permeation assay methodology (described in Section 2.6.2). All tapes were placed in a flask with 40 mL of acetone and mechanically stirred for 15 h. Subsequently, each skin sample was fragmented into small pieces using surgical scissors. The curcumin was extracted with 25 mL of ethyl acetate:methanol at a ratio of 9:1. The extract was centrifuged at 10,174 *g* for 10 min. All samples were analyzed by UV-visible spectrophotometry at a wavelength of 420 nm.

### 2.6.2. In Vivo Permeation Assay

To evaluate the in vivo permeation of the CNp-M4 membrane through the stratum corneum, the tape-stripping technique was employed. Four healthy Mexican males aged 22–36 years were recruited as volunteers. The individuals had neither a history of skin disorders nor had used cosmetic products on their forearms 24 h prior to the test. Written informed consent was obtained from each volunteer before each study.

Four sites (3 × 3 cm) were demarcated: two on the right forearm and two on the left forearm. Before administering the treatment, the sites were cleaned with cotton impregnated with water. Three treatments were administered to each volunteer. The CNp dispersion with 0.01% of curcumin (2 mL) and the drug dispersion at the same concentration were contained within a glass cylinder on the left forearm. The CNp-M4 membrane (3 cm in diameter) was put on the right forearm, and 1.75 mL of distilled water was added onto the surface of the membrane. The last site on the right forearm was used for the blank. The treatments were in contact with the skin for 6 h [20]. Once the time had elapsed, the membrane was removed with steel nippers. The skin was subjected to 15 successive tape strips (Scotch® 3M®), previously cut into 3 × 3 cm squares. In each case, the site was pressed uniformly by sliding a spatula over the surface of the tape five times; then, it was removed by pulling it with steel nippers from the lower right to the upper left end. The blank site was subjected to the same procedure. Each tape was immersed in 15 mL of acetone in a different amber glass bottle with a lid. All of the bottles were placed under mechanical agitation for 15 h; subsequently, the samples were filtered to remove the glue particles. The quantification of curcumin was performed by UV–visible spectrophotometry at a wavelength of 420 nm. Each sample was analyzed in triplicate.

### **3. Results and Discussion**

### *3.1. Physicochemical Characterization of CNp*

#### 3.1.1. Particle Size and Zeta Potential Assessment

CNps were obtained, and their mean particle size and PDI were 148.3 ± 1.9 nm and 0.044 ± 0.020, respectively. These values were expected to improve dermal permeation [21], since nanoparticles below 500 nm have a larger surface area-to-volume ratio, which ensures direct contact with the stratum corneum and skin appendages [6,7]. Moreover, the PDI value was below 0.1, which indicates that the small size measured in the sample is reliable and monodisperse [22].

On the other hand, CNp exhibited a zeta potential value of −7.32 ± 0.03 mV. In this regard, zeta potential is commonly employed to measure the charge in the nanoparticles and/or electrostatic repulsion [1], and the literature indicates that nanoparticles are stable in suspension with a zeta potential above ±30 mV [23]. Despite the CNp zeta potential value not being in this range, it should be considered that Pluronic® F-68 was added as a stabilizer of nanoparticles, which provides them with stability by means of a repulsion effect through a steric mechanism [24]. Likewise, CNp possesses a negative charge, which is related to the carboxylic end group of PCL [25]; thus, negatively charged nanoparticles could permeate adequately in conjunction with the negative charges existing on the skin [26].

### 3.1.2. Atomic Force Microscopy (AFM)

The morphology and size of CNp were evaluated by AFM. In agreemen<sup>t</sup> with the particle-size and zeta-potential assessments, the nanoparticles demonstrated a spherical shape and a size of approximately 200 nm with no agglomeration (Figure 1) [27]. These results support the idea that the small size of CNp could improve the dermal permeation of curcumin, which would increase its anti-inflammatory, antimicrobial, and wound-healing activities [6,12].

**Figure 1.** Atomic force microscopy (AFM) topography images of the CNp. The size bar is 1 μm.

#### 3.1.3. Drug Loading and Entrapment Efficiency of Nanoparticles

In order to determine the amount of curcumin entrapped inside the CNp, the DL and EE were determined. The CNp showed DL and EE values of 4.9 ± 0.7% and 96.01 ± 0.95%, respectively. It is known that the emulsification diffusion method ensures high encapsulation efficiencies (generally >70%) [28]. On the other hand, DL depends on nanoparticle structure and methodology [29], whereas both DL and EE depend on the interactions between the drug, the matrix, and the medium [29].

### *3.2. Physicochemical and Mechanical Characterization of Nanoparticle-Coated Alginate Membranes as Wound Dressings*
