## 2 Materials and Methods
### Methods
#### 2.2.13 In Vitro Safety Assessment for Potential Topical Use  
\(\bullet\) Cell Viability of Human Keratinocyte Cells  
The potential toxicity of free extract and extract-loaded in PLGA and PCL NPs for skin application was evaluated by the MTT assay in the human keratinocyte cell line, HaCaT. These cells were seeded in a 96-well plate at a concentration of 5\(\times 10^{4}\) cells/mL in DMEM with high-glucose (4500 mg/L), supplemented with 10% of FBS, and 100 IU/mL of penicillin and 100 \(\upmu\)g/mL of streptomycin [(Pen/Strep, 1%, \(v\)/\(v\))] (complete medium), in a humidified chamber at 37 \({}^{\circ}\)C in a 5% CO\({}_{2}\) atmosphere, and allowed to adhere for 24 h [25]. After that time, the medium was removed and extracts at concentrations levels ranging from 2.11-33.75 \(\upmu\)g of rutin/mL in free form or loaded in NPs were added to the HaCaT cells. After 48 h of incubation, the medium was discarded and the cells were washed 2 times with PBS (USP41). Then, 50 \(\upmu\)L of MTT at 0.5 mg/mL (\(p\)/\(v\)) in incomplete medium was added to the cells and the plates were incubated for 4 h at 37 \({}^{\circ}\)C in a 5% CO\({}_{2}\) atmosphere. After the incubation time, 100 \(\upmu\)L of DMSO were added to each well for solubilizing the formazan crystals. Then, the Abs was measured, and cell viability was calculated using Equation (7):  
\[\textit{Cell Viability}\left(\%\right)=\ \frac{\textit{Abs}_{t}}{\textit{Abs}_{c}} \times 100 \tag{7}\]  
where \(\textit{Abs}_{t}\) is the absorbance of tested samples and \(\textit{Abs}_{c}\) is the absorbance of control cells (incubated with complete medium). A total of three independent experiments, with six replicates per condition, were conducted.  
* Cell Viability of Human Fibroblast Cells  
HFF cells were cultured in Glutamax(tm) IMDM and supplemented with heat-inactivated FBS (10%, _v/v_), 100 units/mL of penicillin and 100 mg/mL of streptomycin. All experiments were performed in culture medium supplemented only with 0.5% of FBS without any antibiotic [26]. The cells were seeded at \(1\times 10^{5}\) cells/mL of density in 96-well plates and allowed to grow for one week (to assure the complete confluence), with medium renewal every 24 h. The cells were cultured in a humidified atmosphere at 37 \({}^{\circ}\)C with 5% CO\({}_{2}\). After that time, the cells were incubated with different concentrations of extract in free form or loaded in PLGA and PCL NPs, diluted in Glutamax(tm) IMDM and FBS (0.5%). The tested concentrations ranged from 1.81 to 29.02 mg for rutin/mL and according to the previous study [6]. After 48 h of incubation, the cell proliferation was measured using PrestoBlue(r) Cell Viability Reagent and the fluorescence values were analyzed in a Microplate Fluorimeter FL\(\times\) 800 (Bioteck Instruments, Winooski, VT, USA) (excitation and emission wavelengths of 580 nm and 595 nm, respectively). The cell's viability was calculated in relation to control with complete medium (without samples). At least three independent experiments were made using three replicates. The fluorescence was measured, and the cell's viability was calculated using Equation (8):  
\[\textit{Cell Viability}\left(\%\right)=\ \frac{\textit{Flu}_{t}}{\textit{Flu}_{c}} \times 100 \tag{8}\]  
where \(\textit{Flu}_{t}\) is the fluorescence of tested samples and \(\textit{Flu}_{c}\) is the fluorescence of control cells (incubated with complete medium). A total of three independent experiments, with six replicates per condition, were conducted.