*2.8. Antioxidant Effect of EGCG Loaded Niosomes on Human Fibroblasts* 2.8.1. Cell Culture

The primary human fibroblasts (Fbs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were routinely maintained in complete DMEM medium in T-75 tissue culture flasks (Corning, Phoenix, AZ, USA) at 37 ◦C in an atmosphere of 5% CO<sup>2</sup> and 95% relative humidity. Complete DMEM medium was prepared by adding 10% fetal bovine serum, 1% penicillin-streptomycin-glutamine, and 1% nonessential amino acids. Culture medium was changed every 2 days until cells grew to 90% confluence.

## 2.8.2. Cellular Viability after UVA-Irradiation Using Sulforhodamine B (SRB) Assay

Optimised EGCG-loaded niosomes were prepared and centrifuged as per the abovementioned method and were resuspended in cell culture medium. Fbs were seeded in 96-well plates (5000 cells/well) 24 h before UVA-irradiation to allow cells to attach. To administer UVA-irradiation, a UVA lamp (EN-160 L/FE, Spectroline, Melville, NY, USA) with a dose of 0.72 J/cm<sup>2</sup> and wavelength of 320–400nm and wave peak at 365 nm was used. After irradiation, EGCG niosome suspension and free EGCG in culture medium solution were added to the 96-well and incubated for 24 h. The SRB assay was used to assess the cell viability. Briefly, the cells were gently washed with ice-cold PBS and fixed with 10% TCA, then 0.1 mL of 0.4% (*w*/*v*) SRB in acetic acid was added to stain the cellular proteins. The

cell-bound dye was extracted using 0.1 mL 10 mM unbuffered Tris base solution (pH 10.5), and absorbance was measured at 596 nm with a plate reader (SpectraMax® Plus, Molecular Devices, San Jose, CA, USA). Cell viability was expressed as a percentage of the control.

#### 2.8.3. Intracellular MDA Level and the Antioxidant Enzyme Activities

EGCG and EGCG niosomes were diluted with a serum-free medium and then added to the cells after being irradiated by UV light and then cultured for 24 h. After incubation, cells were removed from the 6-well plate and the amount of MDA was determined by the MDA assay kits. The antioxidant enzyme activities of SOD and GSH-px were determined using the same method described above, and the cellular enzymatic activities were determined using the respective assay kits.

#### *2.9. Cellular Uptake of Niosomes by Human Fibroblasts*

FITC was added to the hydration medium to prepare FITC-labelled niosomes, then subjected to centrifugation to remove free FITC. The niosome pellets were then resuspended in the medium and diluted to the predetermined concentrations. The control solution was prepared by dissolving FITC in DMSO and then diluted with the medium. For the uptake studies, Fbs suspension (5 <sup>×</sup>10<sup>5</sup> cell in 5 mL) was seeded onto Petri dishes (100 mm, Corning, Phoenix, AZ, USA), fed with completed DMEM every 2 days and incubated at 37 ◦C in an atmosphere of 5% CO2 and 95% relative humidity to allow cells to attach and proliferate. On reaching 90% confluence, the culture medium was replaced with 2 mL of HBSS. After incubation at 37 ◦C for 15 min, the HBSS was replaced with FTIC-labelled niosomes at concentrations from 50 to 2000 µg/mL to determine the effect of concentration on cellular uptake; to study the effected of incubation temperature and duration, FITClabelled niosomes were incubated with Fbs at 4 and 37 ◦C for 0.5–24 h and at 37 ◦C for 0.5–24 h, respectively. Then the cells were washed with ice-cold HBSS for three times and then the cells were collected into a tube containing lysis medium (Methanol with 10% Triton™ X-100 solution), followed by ultrasonication for 15 min. Finally, 25 µL of the cell lysates was subjected to BCA protein assay using a Pierce® BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) to determine the amount of protein in the cells. The remainder of the cell lysates were subjected for quantitative measurement using a fluorescein spectrophotometer (PerkinElmer Precisely, Waltham, MA, USA), at excitation wavelength of 495 nm, emission wavelength of 525 nm.

A Confocal Laser Scanning Microscope (CLSM) was used to examine whether niosomes were taken up and localised intracellularly or were simply adsorbed onto the cell surface. Fbs were transferred into 2-well chamber slides (BD Falcon, Phoenix, AZ, USA) at a density of 2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well (5 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/cm<sup>2</sup> ) and grown in complete DMEM culture medium. The cells were treated with FITC-labeled niosome (2 mL) at the given doses for 2 h at 37 ◦C. After incubation, the cells were washed with ice-cold HBSS and then incubated with serum free medium for 15 min. Then the medium was withdrawn and a cytoplasm dye (CellTracker, Invitrogen, Auckland, New Zealand) (5 µM) in serum-free medium for 30 min in a cell incubator at 37 ◦C. After incubation, the cells were washed with PBS followed by a fixation solution of 3% paraformaldehyde for 30 min at room temperature. Then nuclei staining dye, DAPI (100 nM) (Invitrogen, Auckland, New Zealand) was added to the cells for 3 min. The culture chambers were removed, and the slides were rinsed and mounted with CITI-Fluor (Electron Microscopy Sciences, Hatfield, PA, USA). Coverslips were cemented in place with application of nail polish around their edges. Then the slides were observed by a confocal microscope FV1000 (Olympus, Hamburg, Germany).

#### *2.10. Statistical Analysis*

Statistical analysis was performed using the GraphPad Prism® (GraphPad, San Diego, CA, USA) version 8.0 software via one-way ANOVA. A *p*-value of < 0.05 was considered the minimum level of significance. All data were expressed as mean ± SD.

#### **3. Results**

#### *3.1. Formulation Development and Optimisation*

Six formulation variables: surfactant type (X1), drug amount (X2), molar ratio of CH to surfactant (X3) and DCP (X4), hydration medium amount (X5) and hydration time (X6) were selected, and their effect on drug entrapment efficiency (Y) was evaluated. The experimental matrix for EGCG-niosome and responses of different batches obtained are presented in Table 3. The EE of EGCG in the niosomes had a range from 2.3 to 49%, suggesting that the factors investigated were influential on the drug encapsulation. Calculations were carried out based on the responses to determine the main effects of the factors and the interaction effects



The results calculated to determine the main effects of the factors and the interaction effect are shown in Table 4.

After the estimation of the main effects, ANOVA was performed to determine the significant factors. The ANOVA results of the EGCG-niosome are shown in Table 5. A *p* value less than 0.05 (*p* < 0.05) indicated the effect was statistically significant. In this screening, surfactant type (X1), drug amount (X2), and the ratio of CH to surfactant (X3) were significantly influential on the response. The ANOVA showed that none of the two-factor interactions had a significant effect in the EGCG-niosome screening experiment. Surfactant type (X1), drug amount (X2) and the ratio of CH to surfactant (X3) were significantly influential factors on EE. Surfactant type (X1) played an important role in determining EE, and therefore, in the optimisation step, Span 60 was used, whereas drug amount (X2) and the ratio of CH to surfactant (X3) were optimised.


**Table 4.** Main effects of single factors and two-factor interactions in EGCG-niosome variable screening.

**Table 5.** Summary of analysis of variance (ANOVA) for the 2(6−2) factorial design for EGCG-niosome variable screening.


\* statistically significant *p* < 0.05, R-Squared = 0.96, Adj R-Squared = 0.861, Pred R-Squared = 0.598. \*\* df: degree of freedom. \*\*\* Cor Total: corrected total sum of square.

#### *3.2. Optimisation of EE by CCD*

In this step, significant factors detected by the screening design were optimised using a CCD. This design provides a solid foundation for generating a response surface plot, from which it is possible to get a target response. In the current study, it was the maximum EE% that the optimisation aimed to achieve. Transformed values of all the batches along with results of EGCG-niosome are shown in Table 6.


**Table 6.** Optimisation design of EGCG-niosome showing variables in coded values and responses.

Equation (4) below represents the polynomial model for EGCG-niosome as obtained from the above experiment.

$$\text{Y(EE\%)}=47.06-3.98\text{X}\_2 + 2.61\text{X}\_3 - 4.30\text{X}\_2^2 - 2.14\text{X}\_3^2 - 0.61\text{X}\_2\text{X}\_3\tag{4}$$

The correlation coefficient (*r* 2 ) of 0.92 indicated that the model fitted the data very well and the ANOVA of the model reported a high significance (*p* < 0.001) (Table 7). The threedimensional response surface and contour plots showing the variation in the entrapment efficiency with changes in drug amount (X2) and CH to surfactant ratio (X3) are presented in Figure 1. The highest EE was predicted to be achieved when drug amount (X2) is 1.4 mg and the molar ratio of CH to surfactant (X3) is 0.9.

**Table 7.** Analysis of Variance (ANOVA) of the drug entrapment efficiency.



*Pharmaceutics* **2021**, *13*, x 11 of 23

**Table 7.** *Cont.*

\* statistically significant *p* < 0.05; \*\* df: degree of freedom; \*\*\* Cor Total: corrected total sum of square. sum of square. 412

#### *3.3. Check Point Analysis* 416 *3.3. Check Point Analysis*

Having studied the effect of independent variables on the response, EE%, the levels 417 of the factors were further determined by the optimisation process. Check points were 418 evaluated to confirm the mathematic models' predictivity by comparing the experimental 419 EE (mean value out of four experiments) with the predicted value. In EGCG-niosome, the 420 average experimental EE was 53.05 ± 4.46%, which was close to the predicted value EE of 421 53% with low percentage of bias (0.4%), suggesting that the optimised formulation pa- 422 rameters were reliable. The optimised formulation composition for EGCG-niosome is 423 shown in Table 8, and the following characterisation studies were carried out on the opti- 424 mised EGCG-niosome. 425 Having studied the effect of independent variables on the response, EE%, the levels of the factors were further determined by the optimisation process. Check points were evaluated to confirm the mathematic models' predictivity by comparing the experimental EE (mean value out of four experiments) with the predicted value. In EGCG-niosome, the average experimental EE was 53.05 ± 4.46%, which was close to the predicted value EE of 53% with low percentage of bias (0.4%), suggesting that the optimised formulation parameters were reliable. The optimised formulation composition for EGCG-niosome is shown in Table 8, and the following characterisation studies were carried out on the optimised EGCG-niosome.

11

426

413


**Table 8.** Optimised formulation composition for EGCG-niosome. *Pharmaceutics* **2021**, *13*, x 12 of 23

#### *3.4. Characterisation of EGCG-Loaded Niosomes 3.4. Characterisation of EGCG-loaded Niosomes* 428 The developed HPLC method was validated for linearity, repeatability, accuracy and 429

The developed HPLC method was validated for linearity, repeatability, accuracy and sensitivity as per International Conference on Harmonisation (ICH) Q2(R1) guidelines. The standard curve was linear in the range between 1.93 to 145 µg/mL with a correlation coefficient (*r 2* ) of 0.999. Percentage of coefficient of variation (% CV) was determined to assess instrumental precision; both instrumental precision and intra-assay precision had % CV of less than 1.5%, indicating the method for EGCG is precise. Intermediate precision of the method was determined by assessing intra-day and inter-day repeatability; the % CV values were below 2.5%, which is acceptable according to the ICH guidelines. The sensitivity of the method was determined by limit of detection (LOD) and limit of quantification (LOQ), which were 0.33 µg/mL and 0.98 µg/mL, respectively. sensitivity as per International Conference on Harmonisation (ICH) Q2(R1) guidelines. 430 The standard curve was linear in the range between 1.93 to 145 µg/ml with a correlation 431 coefficient (*r 2* ) of 0.999. Percentage of coefficient of variation (% CV) was determined to 432 assess instrumental precision; both instrumental precision and intra-assay precision had 433 % CV of less than 1.5%, indicating the method for EGCG is precise. Intermediate precision 434 of the method was determined by assessing intra-day and inter-day repeatability; the % 435 CV values were below 2.5%, which is acceptable according to the ICH guidelines. The 436 sensitivity of the method was determined by limit of detection (LOD) and limit of quanti- 437 fication (LOQ), which were 0.33 µg/ml and 0.98 µg/ml, respectively. 438

#### *3.5. Particle Size, Size Distribution, Zeta Potential Analysis and EE% 3.5. Particle Size, Size Distribution, Zeta Potential Analysis and EE%* 439

The average particle size of optimised EGCG-niosomes was 235.4 ± 15.64 nm, and the PDI value was 0.267 ± 0.053. A PDI of less than 0.5 indicates a narrow distribution of the particles [17]. EGCG-niosomes had a zeta potential of −45.2 ± 0.03 mV and EE% of 53.05 ± 4.46%. The average particle size of optimised EGCG-niosomes was 235.4 ± 15.64 nm, and the 440 PDI value was 0.267 ± 0.053. A PDI of less than 0.5 indicates a narrow distribution of the 441 particles [17]. EGCG-niosomes had a zeta potential of −45.2 ± 0.03 mV and EE% of 53.05 ± 442 4.46%. 443

#### *3.6. Morphological Study 3.6. Morphological Study* 444

As shown in Figure 2, the niosomes were 200 to 300 nm, spherical in shape with a closed vesicular structure and narrow size distribution. These findings were consistent with the size determined by the Zetasizer. As shown in Figure 2, the niosomes were 200 to 300 nm, spherical in shape with a 445 closed vesicular structure and narrow size distribution. These findings were consistent 446 with the size determined by the Zetasizer. 447

**Figure 2.** Scanning Electron Microscopy (SEM) image of the optimised EGCG niosomes. 449 **Figure 2.** Scanning Electron Microscopy (SEM) image of the optimised EGCG niosomes.

#### *3.7. DSC and FTIR* 450 *3.7. DSC and FTIR*

The DSC curves of the optimised EGCG niosomes, physical mixture of the formula- 451 tion components, cholesterol, surfactant and EGCG are shown in Figure 3a. The endother- 452 The DSC curves of the optimised EGCG niosomes, physical mixture of the formulation components, cholesterol, surfactant and EGCG are shown in Figure 3a. The endothermic

mic peaks for Span 60 and cholesterol were 53 °C and 149 °C, respectively, which corre- 453 spond to their melting points. The endothermic transition of EGCG (120 and 225 °C) are 454

12

448

peaks for Span 60 and cholesterol were 53 ◦C and 149 ◦C, respectively, which correspond to their melting points. The endothermic transition of EGCG (120 and 225 ◦C) are also reported in other studies. The physical mixture of formulation components showed similar transitions as EGCG and surfactant, where the characteristic peaks were not observed with EGCG niosomes. Additional peaks were found in EGCG niosomes between 100 to 150 ◦C, indicating there were interactions between the excipients. A large peak that appeared between 200 and 300 ◦C in EGCG-niosomes may suggest drug and excipient breakdown. FTIR spectroscopy verified the above results (Figure 3b. The FTIR graph showed the characteristic peaks for EGCG such –C–O stretching at 1200–1000 cm−<sup>1</sup> and –C=C stretching at 1600–1500 cm−<sup>1</sup> . The spectrum of EGCG niosomes was similar to the surfactant; the other characteristic peaks were not observed, which confirmed the encapsulation of EGCG. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 15 of 25

**Figure 3.** (**a**) Differential Scanning Calorimetry (DSC) thermograms and (**b**) Fourier Transform Infra-red Spectroscopy (FTIR) spectra of EGCG, Span 60 and EGCG-niosomes. **Figure 3.** (**a**) Differential Scanning Calorimetry (DSC) thermograms and (**b**) Fourier Transform Infra-red Spectroscopy (FTIR) spectra of EGCG, Span 60 and EGCG-niosomes.

The in vitro drug release of EGCG from niosomes was examined using Franz diffusion cells. Figure 4 shows the release profile for control (EGCG solution) and EGCG-nio-

the niosomes within the first 3 h, and then a sustained release was observed over 21 h, with 73% of EGCG was release at the end of the study. As shown in Table 9, the release data was fitted in several mathematical models of release kinetics. Based on the results,

*3.8. In Vitro Drug Release Profile*

#### *3.8. In Vitro Drug Release Profile Pharmaceutics* **2021**, *13*, x 14 of 23

The in vitro drug release of EGCG from niosomes was examined using Franz diffusion cells. Figure 4 shows the release profile for control (EGCG solution) and EGCG-niosomes. Within 2 h, the EGCG solution was released immediately. The EGCG-niosomes, on the other hand, displayed a biphasic phase; around 35% of EGCG were released from the niosomes within the first 3 h, and then a sustained release was observed over 21 h, with 73% of EGCG was release at the end of the study. As shown in Table 9, the release data was fitted in several mathematical models of release kinetics. Based on the results, EGCG release from niosomes followed the Korsmeyer–Peppas model (*r* <sup>2</sup> = 0.996). The release exponents were found to be 0.461, which indicates the drug release was governed by an anomalous diffusion mechanism with multiple steps. the niosomes within the first 3 h, and then a sustained release was observed over 21 h, 473 with 73% of EGCG was release at the end of the study. As shown in Table 9, the release 474 data was fitted in several mathematical models of release kinetics. Based on the results, 475 EGCG release from niosomes followed the Korsmeyer–Peppas model (*r* 2 = 0.996). The re- 476 lease exponents were found to be 0.461, which indicates the drug release was governed 477 by an anomalous diffusion mechanism with multiple steps. 478

**Figure 4.** In vitro drug release of EGCG-niosomes and EGCG solution (mean ± SD, *n* = 3). 480 **Figure 4.** In vitro drug release of EGCG-niosomes and EGCG solution (mean ± SD, *n* = 3).


**Table 9.** Drug release kinetic parameters of EGCG niosomes.

**EGCG-niosomes** 0.996 0.461 3.885 0.876 2.555 0.832 0.002 0.521 0.077 *3.9. Ex Vivo Skin Permeation and Deposition Studies*

deposition levels of EGCG-niosome were 69.0 ± 13.87 µmg/cm<sup>2</sup>

*3.9. Ex vivo Skin Permeation and Deposition Studies* 482 No drug was found in the receptor chamber at the end of the permeation study, and 483 this could be caused by hydrolysis of EGCG in the aqueous medium and the limited sen- 484 sitivity of the HPLC method. Figure 5 shows the amount of EGCG deposited in the human 485 skin from niosomes and EGCG solution at 12 and 24 h. The deposition of EGCG-solution 486 were 30.02 ± 2.45 μg/cm<sup>2</sup> , 29.00 ± 1.36 μg/cm<sup>2</sup> at 12 h and 24 h, respectively. The drug 487 No drug was found in the receptor chamber at the end of the permeation study, and this could be caused by hydrolysis of EGCG in the aqueous medium and the limited sensitivity of the HPLC method. Figure 5 shows the amount of EGCG deposited in the human skin from niosomes and EGCG solution at 12 and 24 h. The deposition of EGCG-solution were 30.02 <sup>±</sup> 2.45 <sup>µ</sup>g/cm<sup>2</sup> , 29.00 <sup>±</sup> 1.36 <sup>µ</sup>g/cm<sup>2</sup> at 12 h and 24 h, respectively. The drug deposition levels of EGCG-niosome were 69.0 <sup>±</sup> 13.87 <sup>µ</sup>mg/cm<sup>2</sup> and 54.38 <sup>±</sup> 8.86 <sup>µ</sup>mg/cm<sup>2</sup> at 12 h and 24 h, respectively. When the deposition of the EGCG-niosome and the drug solution was compared at 12 and 24 h, it was discovered that the deposition from the EGCG-niosome was about 2-fold higher than that of the drug solution.

at 12 h and 24 h, respectively. When the deposition of the EGCG-niosome and the drug 489 solution was compared at 12 and 24 h, it was discovered that the deposition from the 490 EGCG-niosome was about 2-fold higher than that of the drug solution. 491

**Figure 5.** The amount of drug deposited in the human skin layers from EGCG-niosomes and EGCG- 493 solution (mean ± SD, *n* = 3). 494

*3.10. Visualisation of Skin Penetration and Deposition* 495

14

492

479

488

and 54.38 ± 8.86 µmg/cm<sup>2</sup>

 2

**0**

**20**

**40**

**60**

**Cumulative EGCG** 

**released (%)**

**80**

**100**

were 30.02 ± 2.45 μg/cm<sup>2</sup>

**Figure 5.** The amount of drug deposited in the human skin layers from EGCG-niosomes and EGCG- 493 **Figure 5.** The amount of drug deposited in the human skin layers from EGCG-niosomes and EGCGsolution (mean ± SD, *n* = 3). *3.10. Visualisation of Skin Penetration and Deposition*

the niosomes within the first 3 h, and then a sustained release was observed over 21 h, 473

with 73% of EGCG was release at the end of the study. As shown in Table 9, the release 474

data was fitted in several mathematical models of release kinetics. Based on the results, 475

lease exponents were found to be 0.461, which indicates the drug release was governed 477

by an anomalous diffusion mechanism with multiple steps. 478

**Figure 4.** In vitro drug release of EGCG-niosomes and EGCG solution (mean ± SD, *n* = 3). 480

**Table 9.** Drug release kinetic parameters of EGCG niosomes. 481

*3.9. Ex vivo Skin Permeation and Deposition Studies* 482

this could be caused by hydrolysis of EGCG in the aqueous medium and the limited sen- 484

sitivity of the HPLC method. Figure 5 shows the amount of EGCG deposited in the human 485

skin from niosomes and EGCG solution at 12 and 24 h. The deposition of EGCG-solution 486

at 12 h and 24 h, respectively. When the deposition of the EGCG-niosome and the drug 489

solution was compared at 12 and 24 h, it was discovered that the deposition from the 490

ℎ

No drug was found in the receptor chamber at the end of the permeation study, and 483

 2 1

EGCG-niosome

EGCG-solution

at 12 h and 24 h, respectively. The drug 487

and 54.38 ± 8.86 µmg/cm<sup>2</sup>

 2 0

**Formulation Korsmeyer-Peppas model Higuchi model First-order Zero-order**

**EGCG-niosomes** 0.996 0.461 3.885 0.876 2.555 0.832 0.002 0.521 0.077

 2

**0 4 8 12 16 20 24**

**Time (hour)**

, 29.00 ± 1.36 μg/cm<sup>2</sup>

deposition levels of EGCG-niosome were 69.0 ± 13.87 µmg/cm<sup>2</sup>

2

= 0.996). The re- 476

479

488

EGCG release from niosomes followed the Korsmeyer–Peppas model (*r*

#### solution (mean ± SD, *n* = 3). 494 *3.10. Visualisation of Skin Penetration and Deposition* A small amount of fluorescence was seen in the epidermis after 12 h of ethanol solu-

*3.10. Visualisation of Skin Penetration and Deposition* 495 A small amount of fluorescence was seen in the epidermis after 12 h of ethanol solution application (Figure 6). On the other hand, the niosome carrier improved fluorescence penetration through the SC and greater fluorescence intensity can be observed in the epidermis and dermis. This result matched with the results obtained from the deposition studies and confirmed that niosome could increase drug deposition into the human skin layers. tion application (Figure 6). On the other hand, the niosome carrier improved fluorescence penetration through the SC and greater fluorescence intensity can be observed in the epidermis and dermis. This result matched with the results obtained from the deposition studies and confirmed that niosome could increase drug deposition into the human skin layers.

14

492

**Figure 6.** Sections of the full-thickness human skin after been treated with Fluorescein 5(6)-isothiocyanate (FITC) solution (**a**) and FITC-loaded niosomes (**b**) after 12 h. **Figure 6.** Sections of the full-thickness human skin after been treated with Fluorescein 5(6) isothiocyanate (FITC) solution (**a**) and FITC-loaded niosomes (**b**) after 12 h.

UVA-irradiation caused substantial reduction in cell viability of 40% when compared to control (*p* < 0.05) (Figure 7a). Fbs treated with EGCG-niosomes demonstrated higher viability, (*p* < 0.05) as compared to the UVA-irradiation group, considerably greater than

The extent of cellular lipid peroxidation can be determined by measuring intracellular MDA levels. As shown in Figure 7b, intracellular MDA levels after UVA-irradiation was 5.12 ± 0.76 µmol/L/mg protein, which was significantly higher compared to untreated cells (*p* < 0.01), showing that UVA has a strong oxidative effect on skin cells. The intracellular MDA levels of Fbs treated with EGCG-niosomes were much lower (0.80 ± 0.33 µmol/L/mg protein) compared to Fbs treated with free EGCG (2.08 ± 0.33 mol/L/mg protein). Figure 7c,d shows that the activity of the intracellular antioxidant enzymes following UVA-irradiation were reduced significantly for both SOD and GSH-px. EGCG-niosome had a greater enhancing effect on the SOD activity compared to the pure drug, but the difference was insignificant (*p* > 0.05) (Figure 7c), the level of SOD was 36.48 ± 1.98 µ/L/mg protein, the group treated with free EGCG was 31.92 ± 1.67 µ/L/mg protein. The GSH-px level in Fbs after UVA irradiated was increased by EGCG-niosomes to 12.53 ± 0.01 mU/L/mg protein, significantly higher when compared to the group treated with free

EGCG (10.88 ± 0.55 mU/L/mg protein) (*p* < 0.05) (Figure 7d).

3.11.2. Intracellular MDA Level and the Antioxidant Enzyme Activities

*3.11. The Pharmacological Effects of EGCG-Niosomes on Fbs*

3.11.1. Cell Viability after UVA-Irradiation

the group treated with EGCG (*p* < 0.05).
