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

#### 3.11.1. Cell Viability after UVA-Irradiation

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 group treated with EGCG (*p* < 0.05). *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 18 of 25

**Figure 7.** (**a**) Cellular viability after UVA-irradiation and treatment with EGCG and EGCG-niosomes. The effect of EGCG and EGCG niosomes on (**b**) intracellular malondialdehyde (MDA) level (**b**), (**c**) superoxide dismutase (SOD) and (**d**) glutathione peroxidase (GSH)-px after UVA-irradiation (mean ± SD, *n* = 3). **Figure 7.** (**a**) Cellular viability after UVA-irradiation and treatment with EGCG and EGCG-niosomes. The effect of EGCG and EGCG niosomes on (**b**) intracellular malondialdehyde (MDA) level (**b**), (**c**) superoxide dismutase (SOD) and (**d**) glutathione peroxidase (GSH)-px after UVA-irradiation (mean ± SD, *n* = 3).

*3.12. Cellular Uptake of Niosome by Fbs Cells* 3.11.2. Intracellular MDA Level and the Antioxidant Enzyme Activities

Three factors influenced cellular uptake including niosome concentration, exposure duration, and incubation temperature were studied. Figure 8a shows that increasing the concentration from 50 to 500 µg/mL enhanced cellular uptake, but a further increase from 500 µg/mL did not lead to further increase. Figure 8b shows that cellular uptake was time dependent. Maximum uptake was reached after 3 h before it was declining. At 37 °C, cellular absorption was 6.89 µg FITC/mg protein, 6-fold higher than at 4 °C (0.90 µg FITC/mg protein), which indicated that this process requires energy. No cellular uptake was observed in cells incubated free FITC and no intercellular fluorescence was detected. Confo-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 UVAirradiation were reduced significantly for both SOD and GSH-px. EGCG-niosome had a

cal microscopy was used to examine whether niosomes were taken up into the cells; it provides the observation of a three-dimensional cross-sectional images of the cells and the

lowed the cells to be visible under the microscope.

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).

#### *3.12. Cellular Uptake of Niosome by Fbs Cells*

Three factors influenced cellular uptake including niosome concentration, exposure duration, and incubation temperature were studied. Figure 8a shows that increasing the concentration from 50 to 500 µg/mL enhanced cellular uptake, but a further increase from 500 µg/mL did not lead to further increase. Figure 8b shows that cellular uptake was time dependent. Maximum uptake was reached after 3 h before it was declining. At 37 ◦C, cellular absorption was 6.89 µg FITC/mg protein, 6-fold higher than at 4 ◦C (0.90 µg FITC/mg protein), which indicated that this process requires energy. No cellular uptake was observed in cells incubated free FITC and no intercellular fluorescence was detected. Confocal microscopy was used to examine whether niosomes were taken up into the cells; it provides the observation of a three-dimensional cross-sectional images of the cells and the location of niosomes within the cells. Labelling the cells with CellTracker and DAPI allowed the cells to be visible under the microscope. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 19 of 25

**Figure 8.** (**a**) Effects of niosome concentrations and (**b**) duration of exposure on the uptake of vesicles by Fbs. **Figure 8.** (**a**) Effects of niosome concentrations and (**b**) duration of exposure on the uptake of vesicles by Fbs.

Figure 9a,b illustrates the distribution of green FITC-labelled niosomes inside Fbs after 2 h of uptake. The images indicated that the niosomes were distributed throughout the cytoplasm and perinuclear region. The planar section observation confirmed that FITC was internalised rather than just adsorbing on the cell membranes. Figure 9a,b illustrates the distribution of green FITC-labelled niosomes inside Fbs after 2 h of uptake. The images indicated that the niosomes were distributed throughout the cytoplasm and perinuclear region. The planar section observation confirmed that FITC was internalised rather than just adsorbing on the cell membranes.

**Figure 9.** Confocal laser scanning microscopy images of Fbs after incubation with FITC-labelled niosomes for 2 h at 37 °C showing perinuclear accumulation of particles. Nuclei: blue (**a**), FITClabelled niosomes: green (**b**), cytoplasm: red (**c**), merged images (**d**) confirming uptake of intake niosomes. Eight images of optical sections taken in the vertical axis at interval of 1 µm from the by Fbs.

was internalised rather than just adsorbing on the cell membranes.

**Figure 9.** Confocal laser scanning microscopy images of Fbs after incubation with FITC-labelled niosomes for 2 h at 37 °C showing perinuclear accumulation of particles. Nuclei: blue (**a**), FITClabelled niosomes: green (**b**), cytoplasm: red (**c**), merged images (**d**) confirming uptake of intake niosomes. Eight images of optical sections taken in the vertical axis at interval of 1 µm from the **Figure 9.** Confocal laser scanning microscopy images of Fbs after incubation with FITC-labelled niosomes for 2 h at 37 ◦C showing perinuclear accumulation of particles. Nuclei: blue (**a**), FITClabelled niosomes: green (**b**), cytoplasm: red (**c**), merged images (**d**) confirming uptake of intake niosomes. Eight images of optical sections taken in the vertical axis at interval of 1 µm from the apical surface (**e**–**l**) from left to right; top to bottom, depths 0, 1, 2, 3, 4, 5, 6 and 7 µm, demonstrating particle internalisation. Magnification (600×).

**Figure 8.** (**a**) Effects of niosome concentrations and (**b**) duration of exposure on the uptake of vesicles

Figure 9a,b illustrates the distribution of green FITC-labelled niosomes inside Fbs after 2 h of uptake. The images indicated that the niosomes were distributed throughout the cytoplasm and perinuclear region. The planar section observation confirmed that FITC

#### **4. Discussion**

In this study, EGCG-loaded niosomes were fabricated and optimised by using first a 2 6−2 fractional factorial design followed by a central composite design. The development of niosomes involves many factors, which may affect their properties such as size and encapsulation of the drug in niosomes. The traditional experimental approach implies altering one factor at a time while keeping the other constant. In this case, to evaluate a certain number of factors, a great effort and long period of time are required. In contrast to the traditional method, utilisation of fractional factorial design is able to provide the maximum amount of information with the least experiments [38]. From a pharmaceutical viewpoint, EE is one of the most important attributes of niosome formulation; a high EE would result in less time and effort spent removing unentrapped material and a greater therapeutic effect of the product [39].

The effect of drug content used in preparation on EE% was statistically significant. Generally, increasing drug amount led to improved EE, but in the EGCG-niosomes, further increase in the drug amount above 1.4 mg showed a decrease of EE. This might be due to saturation of drug entrapment, i.e., further addition of the drug was not able to induce more drug entrapped. The ratio of CH to surfactant was found to significantly influence the entrapment of the EGCG-niosome. CH acts as a membrane stabiliser. It increases rigidity of the bilayer and reduces leakage of drugs from the vesicles; it has been reported that as the amount of CH increases in the formulation, the entrapment efficiency of the drug also increases [40]. Nevertheless, the addition of CH above a certain level may cause

disruption of the regular vesicle structure, thus decreasing the entrapment [41]. This finding is consistent with those reported by other researchers. Incorporation of CH into Span 60 niosome loaded with flurbiprofen resulted in an increase of EE from 55% to 67%, but EE was reduced by 30% when CH was increased to 60% [42]. The EE of caffeine decreased from 80% to 50% when the molar ratio of CH to surfactant increased from 3:7 to 3:5 [43]. In the current CCD study, it was obvious that the response surface had curvature in the optimisation phase of both niosome formulations. It indicated that in both niosome preparation, as the CH amount in preparation increased, the EE increased at first, whereas after a certain level, further increase of CH caused a decrease of EE.

The optimised nano-size EGCG-niosomes had an average particle size of 235.4 ± 15.64 nm and a zeta potential of −45.2 ± 0.03 mV. SEM confirmed the findings obtained from Zetasizer, that niosomes were in the 200 to 300 nm size range with a narrow distribution. In topical drug delivery, the particle size of the carriers plays an important role in penetration across the skin barrier. Studies have shown that when the particle size of carriers is greater than 600 nm, no skin deposition was observed. Carriers with a smaller particle size, such as 300 nm promote dermal delivery, while a size lower than 300 nm may result in excessive transdermal drug transport [17]. The zeta potential is an extremely useful measure of a formulation's stability. A zeta potential of less than −30 mV indicates high stability [44]. Adding DCP in the EGC-niosomes resulted in a much lower zeta potential than −30 mV.

The EGCG-niosomes achieved a high EE of 53.05 ± 4.46%, and DSC and FTIR showed that EGCG was successfully encapsulated in the niosomes. In EGCG-niosomes, an additional peak was observed between 100 to 150 ◦C, indicating a surfactant-cholesterol interaction. This interaction is crucial, as CH acts as a membrane stabiliser in niosomes. Drug release from EGCG-niosomes showed a biphasic pattern, where an initial burst release and a subsequent slow release were observed. The release kinetics followed the Korsmeyer release model, (*r* <sup>2</sup> = 0.996), demonstrating an anomalous diffusion mechanism regulated by many processes [45,46]. When it comes to topical drug delivery, this type of release pattern is appealing because the initial fast release improves drug penetration, while the subsequent sustained release provides the drug delivery over a longer period to maintain a therapeutic level in the skin and reduces the frequency of reapplication [47–49].

According to research, the use of the niosome carrier has a considerable impact on enhancing topical drug penetration, as well as increasing drug deposition in the human skin, which are both advantageous for dermal formulations. As such, niosomes have been extensively used in topical treatments [17,21,28,50,51]. A number of theories have been put forward to explain their ability to enhance penetration, firstly the adsorption and fusion of carriers onto the skin's surface results in a significant thermodynamic activity gradient of the drug at the surface of the carriers and the skin's surface, which serves as a driving force for drug penetration into the skin [52–54]. Secondly, disruption of the tightly packed lipids that occupy the extracellular spaces of the SC increases drug permeability through structural alteration of the SC. Thirdly, the carrier may disturb the densely packed lipids of the SC to promote drug penetration by modifying the SC structure. Lastly nonionic surfactants may act as penetration enhancers, increasing membrane fluidity [26,32,52,55]. Finally, niosomes alter the SC characteristics by reducing trans-epidermal water loss, increasing SC hydration and leading to the relaxation of its tightly packed cellular structure and, hence, better penetration [52,53]. Ethanol is also known as a penetration enhancer [56]. It reduces the phase transition temperature of SC lipids, improving fluidity of SC. In addition, ethanol imparts soft properties to the carrier's membrane, facilitating vesicle skin penetration [17]. On the other hand, no drug was detected in the receptor chamber of the Franz diffusion cells, indicating that EGCG did not permeate across the skin. The entrapped EGCG and released EGCG molecules may partition into and diffuse through the SC. A drug depot may be formed in the SC, and then the remaining free drug and vesicles penetrate farther into the epidermis until they reach the interface between the SC and the epidermis. The free drug, as well as any remaining intact vesicles, are subsequently released into the skin

layers. It is possible that the drug was metabolised by the enzymes in the skin. Catechin is unstable in aqueous environments, and it has been shown that it is rapidly hydrolyzed or degraded. Based on these findings, it may be feasible to explain why no drug was detected in the receptor chamber.

The assay is based on the ability of the dye sulforhodamine B to bind electrostatically and pH-dependently on protein basic amino acid residues of TCA fixed cells. A significant reduction in cellular viability was observed after UVA-irradiation; however, the Fbs viability was significantly improved by both EGCG solution and EGCG-niosomes, with the EGCG-niosomes showing greater protective effects against UVA-irradiation. ROS may cause cell and tissue dysfunction, which is partly manifested as lipid peroxidation. Malondialdehyde (MDA) is the main product of lipid peroxidation, and it reveals the level of cell damage under oxidation [57]. In addition, antioxidant molecules in the skin interact with ROS or their by-products such as MDA to minimise the deleterious oxidation effect. After being exposed to oxidative stress, the antioxidants in the skin SOD and GSH-px are activated [58]. UV irradiation causes an accumulation of ROS in the skin, overwhelming the tissue antioxidants, and thus it causes oxidative stress-related skin problems [6]. ROS may be alleviated by SOD and GSH-px. The decrease in SOD and GSH-px levels observed after UV irradiation might be attributed to the formation of a large number of free radicals that exceeded the antioxidant enzymes' scavenging capacity [59]. Furthermore, the reduction in enzymatic activity might be related to enzyme inactivation caused by ROS damage to DNA. MDA content, which indicates the lipid peroxidation state, increases following UV irradiation, showing damage induced by oxidative stress. EGCG is a polyphenol compound with a wide range of pharmacological actions. This compound has strong antioxidant properties. It is capable of scavenging ROS or their precursors, blocking ROS synthesis and upregulating antioxidant enzymes [60]. Following UV irradiation, skin Fbs treated with EGCG-niosomes had higher SOD and GSH-px activity compared to UV-treated cells. Furthermore, the MDA levels in the EGCG solution-treated group were lower than in the UV group. EGCG-niosomes showed significantly higher antioxidant activity, which might be due to the following explanations: when in cell culture, the medication is subject to autooxidation, but within a vesicle, it is somewhat shielded from destruction [61]. Furthermore, the drug-loaded niosomes produced prolonged release, keeping the level of the drug constant, resulting in an increased antioxidant effect. Furthermore, the carrier may influence drug internalisation by cells [62]. The improved antioxidant activity of EGCG encapsulated in the niosome carrier prompted researchers to investigate niosome-cell interactions.

Since free FITC had difficulties penetrating cells, the increased FITC intake should be attributed to the niosome carriers. Many studies have indicated greater drug absorption mediated by drug carriers; tamoxifen citrate loaded niosomes showed in an in vitro study on MCF-7 breast cancer cells that the amount of cellular uptake and cytotoxicity of tamoxifen were greatly enhanced when it was loaded in niosomes. Incorporating antimicrobial agents into carrier systems, such as nanoparticles or microemulsions, might be a successful technique for increasing cellular uptake [62]. Furthermore, niosomes loaded with salidroside improved the drug's intracellular absorption by both human epidermal immortal keratinocytes and human embryonic skin fibroblasts [53].

Endocytosis is a primary mechanism through which cells internalise chemicals and macromolecules. It is essential for cell-to-cell communication and cell-to-microenvironment communication [63]. To internalise foreign particles, human cells employ multiple endocytosis processes. Phagocytosis, macropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis are all examples of endocytic processes [64–66]. Endocytosis demands energy, as opposed to passive transport, which does not involve any expenditure of energy [64]. According to a study, depending on their size, liposomes are mostly endocytosed by clathrin- or caveolae-mediated endocytosis [65]. Recent findings have revealed that endocytosis of niosomes is an energy-dependent process, follows a concentration- and time-dependent pattern and has a saturation point [66]. As a result, it is likely that cell surface proteins are involved in the process of niosome endocytosis. Niosome carriers have the potential to increase cellular absorption of encapsulated compounds, even if the medication has a low permeability into the cells. Further studies are required to fully understand the uptake process.

#### **5. Conclusions**

In this work, the niosome-carrier system was fabricated to encapsulate EGCG for cutaneous administration. Based on the findings, we can conclude that EGCG-niosomes can penetrate the skin barrier and improve drug deposition in the viable layers. Because of increased cellular absorption and based on the studies of the antioxidant enzymes, EGCGniosomes demonstrated an improved antioxidant effect on skin cells. Because antioxidants have numerous roles in skin health, this topical formulation has the potential to be used in the treatment of skin diseases. Furthermore, in both the pharmaceutical and cosmetic industries, this carrier has the potential to be used as a dermal drug carrier for a variety of bioactive compounds.

**Author Contributions:** Conceptualization, J.W.; methodology, D.L., N.M., Z.W., M.L. and J.W.; software, D.L.; formal analysis, D.L., N.M. and Z.W.; investigation, D.L. and M.L.; resources, D.L., M.L., Z.Z. and J.W.; data curation, D.L., S.C. and J.R.F.; writing—original draft preparation, D.L. and S.C.; writing—review and editing, J.W., D.L., N.M., Z.W., S.C., J.R.F. and Z.Z.; visualization, D.L and S.C.; supervision, J.W. and Z.Z.; project administration, D.L., N.M. and Z.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by New Zealand Pharmacy Education Foundation (NZPERF), grant number is 236.

**Institutional Review Board Statement:** The full-thickness skin samples were kindly donated by patients who underwent elective skin reduction surgeries at Middlemore hospital, Auckland. This project has been approved by the University of Auckland's Human Participants Ethics Committee (approval number: 010990).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

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