2.2.9. Statistical Analysis

GraphPad Prism was used for statistical analysis using tests stated in the figure legends. Grouped analyses were performed by *t*-test. Significance was established when *p* < 0.05.

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

### *3.1. Preparation of Gel Formulations*

Previous studies concerning the preparation of physical gels based on natural or synthetic polymers, namely carbomers, sodium alginate, and carboxymethyl cellulose, have pointed out that these materials are nontoxic and nonhazardous and easily synthesized. We aimed to prepare different topical CD-NHF-loaded gel formulations based on polymers with antitumoral activity. Herein, physical gel formulations based on CARB, AS, and CMC were synthesized according to a slightly modified version of the method reported by Hellerbach et al. [19]. This method offers some advantages compared with other methods reported, such as being simple, rapid, and consistent; performed in the absence of crosslinkers (hence, we eliminated the possibility of toxic chemical contamination commonly associated with methods which use covalent crosslinkers); and the product is a homogeneous soft material formed at room temperature.

Different concentrations of CARB, AS, or CMC were tailored and their effects were observed. First, simple gels were prepared and analyzed before loading with CD-NHF. The gel formulations with or without CD-NHF obtained were stable, odorless, transparent, and highly viscous. Of them, the best formulations presenting a suitable consistency and the best viscoelastic properties, respectively, were considered for further study and loading with CD-NHF, namely samples CARB-F2, AS-F5, and CMC-F3. The carbon-dot-loaded gel samples were denoted as CARB-F4, AS-F6, and CMC-F6.
