Cellular and Non-cellular Antioxidant Properties of Vitamin E–Loaded Metallic-Quercetin/Polycaprolactone Nanoparticles for the Treatment of Melanogenesis

Inhibition of melanogenesis by quercetin and vitamin E is extensively reported in the literature, independently, with limitations in antioxidant potential owing to less permeation, solubility, decreased bioavailability, and reduced stability. Thus, the aim of the present study was to synthesize a novel complex of metal ions (copper and zinc) with quercetin to enhance antioxidant properties which were confirmed by docking studies. Polycaprolactone-based nanoparticles of the synthesized complex (PCL-NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, Cu-Q-PCL-NPs) were made later loaded with vitamin E which made the study more interesting in enhancing antioxidant profile. Nanoparticles were characterized for zeta size, charge, and polydispersity index, while physiochemical analysis of nanoparticles was strengthened by FTIR. Cu-Q-PCL-NPs-E showed maximum in vitro release of vitamin E, i.e., 80 ± 0.54%. Non-cellular antioxidant effect by 2,2-diphenyl-1-picrylhydrazyl was observed at 93 ± 0.23% in Cu-Q-PCL-NPs-E which was twofold as compared to Zn-Q-PCL-NPs-E. Michigan Cancer Foundation-7 (MCF-7) cancer cell lines were used to investigate the anticancer and cellular antioxidant profile of loaded and unloaded nanoparticles. Results revealed reactive oxygen species activity of 90 ± 0.32% with the addition of 89 ± 0.64% of its anticancer behavior shown by Cu-Q-PCL-NPs-E after 6 and 24h. Similarly, 80 ± 0.53% inhibition of melanocyte cells and 95 ± 0.54% increase of keratinocyte cells were also shown by Cu-Q-PCL-NPs-E that confirmed the tyrosinase enzyme inhibitory effect. Conclusively, the use of zinc and copper complex in unloaded and vitamin E–loaded nanoparticles can provide enhanced antioxidant properties with inhibition of melanin, which can be used for treating diseases of melanogenesis.


Introduction
An excessive formation of melanin and abnormal hyperpigmentation carried on by excessive exposure to ultraviolet (UV) radiation. However, the development of pigmented patches can be seen as an aesthetic issue that arises from the accumulation of an abnormal amount of melanocyte cells in different parts of the skin [1]. Inflammation, age spots, melasma, acne, and freckles are just a few problems of the skin that may result from melanocytes as producers and distributors of the pigment melanin (which play the key role in pigmentation development) [2]. Skin thinning, stretch marks, dilated blood vessels, and increased hair growth were the drawbacks of steroidal creams [3]. Allergic reactions, skin atrophy, easy bruising, and stretch marks were reported when high doses of NSAIDs were used [4].
Natural remedies, i.e., carrageenan, rutin, and quercetin, were used as an alternative to allopathic drugs with the limitations of their astringent effect and dehydration of skin tissues [5]. Since it has good clinical properties and few side effects, the representative polyphenol quercetin has recently attracted attention in the cosmetics field [6]. However, due to its poor water solubility, high metabolism, rapid elimination, inactive metabolites, and limited absorption, quercetin has low bioavailability and insufficient clinical effectiveness for its anti-melanogenic properties [7].
Anti-oxidant and anti-melanogenesis attributes of quercetin are supplemented by the amalgamation of metal ions with this principal flavonoid [8]. Copper and zinccontaining substances, like copper sulfate and zinc sulfate, were utilized for the control of skin diseases involving the synthesis of melanin since both have a broad spectrum of effectiveness against degeneration of melanin [9]. Quercetin and its metallic complex can be used for the production of hygiene products in cosmetics that exhibit hypopigmentation efficacy (i.e., anti-melanogenesis activity) [10,11]. Recently, there has been increased interest in transdermal copper and zinc delivery exhilarating scientists to pull out more from metallo-polyphenol complexes. In an unremitting quest to combat melanogenesis, vitamin E alpha-tocopherol has proven to target melanin synthesis by inhibiting tyrosinase enzyme activity [12]. The ambition was to prepare Zn-Q and Cu-Q complex-loaded nanoparticles utilizing low molecular weight polycaprolactone (PCL) (MW-14,000), which would effectively regulate or prevent melanin formation by delivering vitamin E (used as an antioxidant, anti-melanogenesis)-loaded nanoparticles to the skin for a prolonged period [13].
The aim of the present study was to inhibit the melanin effect by using quercetin, Zn-Q, and Cu-Q complex and their nanoparticles. Preparation of Zn-Q and Cu-Q complex was done by simple solvent evaporation technique and their confirmation was done by FTIR which made the study more useful. The stability of a new synthesized complexes can be predicted by the 3-D structural configurations of the atoms in the complex. Before conducting the experiments, molecular docking was used to determine quercetin's increased ability to attach to the proteins of the tyrosinase enzyme and suppress the melanin effect. Novel nanoparticles of zinc-quercetin-polycaprolactone (Zn-Q-PCL-NPs), copper-quercetin-polycaprolactone (Cu-Q-PCL-NPs), and vit. E-loaded nanoparticles of copper and zinc inclusion complexes, Cu-Q-PCL-NPs-E and Zn-Q-PCL-NPs-E, respectively, were prepared by ultra-homogenization method with the subsequent investigation of antioxidant properties, cytotoxicity, and permeation study in melanogenesis disease, successfully. The transport of zinc and copper complexes through the skin was considered an essential treatment to impede melanin production.

Metallic-Quercetin Complex Preparation
Copper and zinc quercetin complexes were prepared by the previously reported, slightly modified method of SB Bukhari et al. [14]. Briefly, 20mM methanolic solutions of quercetin and 10mM aqueous solutions of CuSO 4 were used for the preparation of the Cu-Q complex at 25°C. The mixture was stirred for 30 min and the progress of the reaction was observed by TLC. It resulted from brownish yellow precipitate that was filtered, washed with ethanol, and dried at room temperature then in vacuum desiccators and stored in an airtight container for further use. A similar procedure was adopted for the preparation of Zn-Q complex using zinc sulfate. The schematic illustration is shown in the Fig. 2.

Synthesis of Vitamin E Grafted Zn-Q and Cu-Q Nanoparticles
Vitamin E-loaded and Zn-Q-and Cu-Q-containing nanoparticles were prepared by already-reported, slightly modified ultra-homogenization method of Natarajan et al. [15]. Briefly, two dispersed phase solutions were prepared in such a way that 300mM quercetin was dissolved in dimethyl sulfoxide and considered solution 1. Accurately weighed 5mM PCL was dissolved in dichloromethane (DCM) at 25°C and considered as solution 2. The dispersion phase was prepared by dissolving 0.5 and 0.25% w/v polyvinyl alcohol (PVA) and gelatin in 1000mL (1:1) and homogenized at 6000 rpm and 40mg of vitamin E as a drug was added in this dispersion phase. Both dispersed phases were poured into the dispersion phase and DCM was evaporated by vigorous stirring. Resulted nanoparticles (Q-PCL-NPs) were filtered, washed with distilled water, dried at 50°C for 8h, and stored in an airtight container for further use. A similar method was used for the preparation of Zn-Q-PCL-NPs and Cu-Q-PCL-NPs.

FTIR Analysis
For the confirmation, FTIR study was performed by taking 3mg of each sample, i.e., quercetin, Cu-Q complex, Zn-Q complex, PCL, PVA, gelatin, and their PCL-NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, Cu-Q-PCL-NPs, Zn-Q-PCL-NPs-E, and Cu-Q-PCL-NPs-E. An average of twelve spectra were reported by analyzing all reagents with drug-loaded and unloaded nanoparticles from 500 to 4000 cm −1 by using ATR-FTIR (Brucker α-I Germany) [16]. All the experiments were repeated in triplicate and an average of twelve spectra was reported with ± SD.

Scanning Electron Microscopy
The particle shape analysis of Zn-Q-PCL-NPs and Cu-Q-PCL-NPs has been observed under a scanning electron microscope (SEM, S-3000H, Hitachi, Japan). For scanning electron microscopy, the nanoparticles were mounted on an aluminum sputtered with gold palladium. An accelerating voltage of 10 kV, having a working distance of 10-25 mm, was used to scan the nanoparticles and different parameters of samples were recorded by using (ImageJ) software [17].

Atomic Force Microscopy, Zeta Sizer, PDI, and Zeta Potential
Atomic force microscope (AFM, Digital Instruments) data were obtained with a J scanner and silicon probes (TESP, 2080 N/m spring constant, Bruker). All the curves were taken in air-tapping mode with a pixel resolution of 256×256 and a scanning rate of 0.5−1.0 Hz. Mean particle size, height, width, and surface roughness (300×300) were also measured from 10×10 μm and 5×5 μm AFM images of nanoparticles as described in the International System. The "Particle size Analysis" model of the Nanoscope software was used to analyze AFM data with a height of 75 nm. By intensity, the size distribution, particle dispersion index (PDI), and zeta potential were determined by dynamic light scattering with a zeta-sizer [18].

Drug Loading and Encapsulation Efficiency
The vitamin E content from nanoparticles was determined from the formulations (Zn-Q-PCL-NPs and Cu-Q-PCL-NPs) by dissolving 5mg of vitamin E-loaded nanoparticles in 10mL of DCM and vortexed for 10 min. An equal volume of ethanol was added and vortexed further for 5 min then centrifugation is carried out at 3000 kgm/s 2 for precipitation. In the supernatant layer of nanoparticle suspension, the conc. of vitamin E was calculated by using the UV-spectrophotometer at 292 nm by already-prepared standard curve of vitamin E [19][20][21][22].
The percentage entrapment efficiency (EE) of the nanoparticles was calculated by the percentage of vitamin E loading (Vit. E L) and percentage of theoretical loading by using the following formulae.

Drug Release Studies
The centrifugation method was used for the release of vitamin E from the Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E. Briefly, 1% m/v vitamin E-containing nanoparticles were suspended in 10mL phosphate buffer pH 7.4 at 37 °C in a 15-mL glass screw-capped tube. The tubes were sonicated (Eba 20 Zentrifugenz Hettich) at 30 Hz and centrifuged at (9.81 kgm/s 2 ) for 10 min and the upper layer was collected. The same volume of fresh PBS was changed to maintain the sink conditions. The drug release in the upper layer was collected occasionally at 1, 2, 4, 6, and 8h and stored at 37 °C. The conc. of vitamin E in the release medium was measured by UV-vis spectrophotometer at 292 nm [19]. To determine the drug release kinetics of Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E nanoparticles by applying different models such as time-dependent zero-order kinetics, first-order kinetics concentration-dependent, Higuchi model and the Weibull model by using the following equations [23].

Weibull model
where in F t , F is the fraction of release of the drug in time t and K 0 , K 1 , K H , is the release constant.

Maximum Absorbable Dose from Rat Skin
Maximum absorbable dose from rat skin was observed by using the already-reported method by Zhen Yang et al. after required modifications [24]. Briefly, taking a weighed amount of Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E nanoparticles having 1% m/v of vitamin E was used for permeation studies by using Franz diffusion cell (flat ground joint, 9 mm orifice diameter, and 6 mL receptor volume). A recently dissected rat skin (2cm 2 ) was used as a permeation barrier. The amount of drug in the receiving chamber was measured after 1 mL was aliquoted and replaced with freshly made phosphate buffer solution pH 7.4 at 25°C. By using the already-prepared standard curve of vitamin E in the same medium, the maximum absorbable dose (MAD) was calculated by using the following equation.
MAD is the maximum absorbable dose, K a is the absorption rate constant, sol skin is the solubility of the drug in the rat skin medium, v skin is the volume of fluid, and Tr is the resident time.
Kα is the absorbance rate constant, Flux was calculated from the slope of cumulative drug amount over time, and C 0 (mmol/L) is the initial concentration at the beginning of the permeation study. The value of 1.78cm 2 represents the effective surface area of the using chamber.

Non-cellular Antioxidant Activity
Antioxidant activity was determined by the free radical DPPH (2,2-diphenyl-1-picrylhydrazyl radical) method by using Zn-Q and Cu-Q complex-based nanoparticles that were an already-reported method of Ramzan et al. [25]. Briefly, methanolic solution of DPPH was prepared by using the conc. 600 μg/ml and considered solution 1. Equal conc. of already-prepared Zn-Q and Cu-Q complex-based nanoparticles were used for their aqueous solutions and considered solution 2. Precisely measured 3.9 ml of both complexes and their nanoparticles (solution 2) was added with 0.9 ml DPPH solution (solution 1). DPPH reduction was observed by using an already-prepared standard curve of increasing conc. of DPPH at 370 nm. The remaining percentage of DPPH was calculated by using the following equation.
where A 0 is the initial absorbance, and [At] is the absorbance at increasing time.

Cytotoxicity and Cellular Antioxidant Activity
Cytotoxicity study of prepared Zn-Q and Cu-Q complex and their nanoparticles was determined by using an alreadyreported method by Barry Halliwell et al. [26]. Briefly, the MCF-7 cell lines supplied by Merck were cultured in 24-well plates under optimized conditions of 90% relative humidity and 5% CO 2 at 37 °C for 14 days. Every well had approximately 20,000 MCF-7 cells and fresh minimum essential medium (MEM) replaced old MEM. Ten percent of m/v fetal bovine serum for cell growth was used. Incubated cells were assayed by HEPES-buffered saline solution at 7.4 pH (25 mM). Freshly prepared 0.5% suspensions of vitamin E-loaded and unloaded nanoparticles (PCL-NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, Cu-Q-PCL-NPs Zn-Q-PCL-NPs-E, and Cu-Q-PCL-NPs-E) were replaced with alreadypresent white MEM. Nanoparticles containing wells were incubated at 6h and 24 h under optimized control conditions of relative humidity and CO 2 . We used positive control only as untreated MCF-7 cells. 2.2 mM resazurin 350μl solution was added in prewashed wells containing fresh HBS buffer at 37°C for 3h. MCF-7 cells were observed at 570 nm for fluorescence and metabolism of resazurin by using a UVmicroplate reader Shimadzu UV-1600 at 570 nm.
Antioxidants at various concentrations (PBS for the control samples) were added to already-prepared wells and oxidation was initiated by the addition of DPPH to the wells at a final concentration of 10 mM, and absorbance was read after 120min by using a UV-microplate reader (Shimadzu UV-1600) at 370 nm. The following equation was used for the calculation of the toxic activity of nanoparticles.
where Xt and Xs are the absorbances of the sample and standard respectively.

Synergistic Effect of Cellular and Non-cellular Antioxidants with Vitamin E
The synergistic effect of cellular-and non-cellular-based antioxidants with vitamin E was determined by using the already-reported method of DPPH reduction of Hait-Darshan et al. [27]. Briefly, the ratio between the obtained inhibition (IF) of the oxidation reaction and the estimated anticipated inhibition was used to determine the synergistic effect of vitamin E in combination with other antioxidants on DPPH oxidation (IE). The total amount of inhibition observed with each antioxidant alone equals the anticipated inhibition (percent of control). If the ratio of IF to IE is higher than 1, this suggests synergism.

Superficial Rat Skin Model
A superficial rat model was applied by using the previously reported and slightly modified method of Gisby et al. [28]. Briefly, 6 to 8 weeks of rats were used for all experiments after getting approval from the Ethical committee of Bahauddin Zakariya University Multan (Ref No.326/ PHP/2019). The rat's hair of a 2-3 cm 2 area was stripped with an elastic adhesive bandage. Among the 4 groups, the rats of group 1 were the control but no formulation was (11) Synergistic ef fect = IF ∕ IE applied, and group 2 was considered control and treated with hyderquin plus©; the remaining two groups, i.e., groups 3 and 4, were treated with the 3% m/v of suspension of Zn-Q-PCL-NPs-E and treated with Cu-Q-PCL-NPs-E regularly twice a day after 8h, respectively. According to the previously reported protocol, a second double dose of all nanoparticles was applied after 16 h in the morning and the evening for 5 days. All rats were killed and approximately 2 cm 2 area was separated and stored in formalin (4%) for further use [29].

Histological Examinations
To characterize the histopathology, biopsy specimens with the following treatments were taken immediately after 5 days of applying the dose. Already-stored mice epidermal layer was cut 5-mm punch for histopathological slides while identification and calculation of the number of melanocyte cells were done by applying Gram's crystal violet solution (Sigma-Aldrich). Furthermore, the following parameters and semi-quantitative scoring system were used to describe the melanogenesis response: for scoring of the melanin, 0 showed no melanocyte cell present, 1 represented little melanocyte cell, while score 2 was used for more melanocyte cell present but score 3 was considered moderate melanocyte cell and score 4 represented the presence of severe melanocyte cell. Similar trends of scoring were applied for keratinocyte cells. All the experiments were repeated thrice and mean ± SD (n=3) was reported [30].

Statistical Approach
All the experiments were repeated three times and results were presented as mean and standard deviations from the three replicates. The effect of PVA and gelatin concentrations on the properties of PCL nanoparticles was evaluated by a one-way analysis of variance (ANOVA) test using biostatistics SPSS 22 software. A significance level of p < 0.05 was considered statistically significant.

Stability Studies
The physical stability of the nanoparticle was evaluated after storage for 11 and 13 months. Fifty milligrams of nanoparticles were stored in closed amber-colored glass vials at 5 ± 2°C in the refrigerator. Ten milligrams of the nanoparticles was taken at different time intervals to measure shelf life by using Rgui software (R 4.1. 3 versions) of nanoparticles.

Density Functional Theory
All the investigated structures were optimized by using the density functional theory (DFT) calculations which used three parameters Becke, Lee-Yang-Parr (B3-LYP) functional [31] including the GD3 correction [32] for dispersion and 6-31+g(d,p) for C, N, O, and H atoms and Los Alamos National Laboratory 2-double-z (LanL2DZ) basis set for Zn and Cu [33][34][35] basis sets. The geometric characteristics, frontier molecular orbitals, and the global reactivity biological descriptors were explored as optimized parameters connected to the electronic properties. For all DFT calculations, the gaussian09 [36] suite and for the visualization Guass View 6 [37] utility were used.

Preparation of Ligand and Protein for Molecular Docking Simulation
Tyrosinase-related protein 1 (TYRP1) is one of three tyrosinase-like glycoenzymes in human melanocytes that produce melanin, which controls skin, eye, and hair pigmentation. Using the Discovery Studio 2016 client package, the human tyrosinase protein in complex with kojic acid structure was prepared by removing water molecules and non-protein elements associated with the target 5M8Q structure, adding polar hydrogens to the protein structure, optimizing the protein structure via the CHARMm force field. Using Auto-DockTools-1.5.6, the ligands were prepared and saved in PDBQT format, maintaining the calculated flexible properties for each molecule after the DFT computations [38][39][40][41].

Result and Discussion
Zn-Q and Cu-Q complexes were prepared by solvent evaporation method and their confirmation was done by TLC and FTIR. Zn-Q and Cu-Q complex was confirmed by the color changes from light yellow to dark brownish yellow. Eighty percent and 85% yield of Zn-Q and Cu-Q complex respectively were confirmed by TLC. The physicochemical properties of quercetin, zinc sulfate, copper sulfate, and its Zn-Q and Cu-Q complex are shown in Fig. 3a and b. To check the compatibility of Zn-Q, and Cu-Q complex by using FTIR. Quercetin showed stretching and bending at different peaks by using different functional groups like -OH stretching at 3402 cm −1 peaks, C=O stretching at 2337 cm −1 , C=C stretching at 1510 cm −1 , and C-H bending at 1150 cm −1 . IR spectra of the quercetin-metal complexes indicate the formation of Cu-Q, and Zn-Q bonds at 3225 cm −1 , and 3390 cm −1 , respectively. By the interaction of Q with copper sulfate/zinc sulfate, it has been shifted to 603 cm −1 and 727 cm −1 , respectively, which can be explained by the coordination of carbonyl oxygen with Cu 2+ , Zn 2+ ions, respectively. Moreover, an increase in bond order from 1100 cm −1 in ligand to 1719 cm −1 , and 1845 cm −1 , respectively, after complexation with copper and zinc ions, respectively, indicates the involvement of O-H deformation vibration which coordinates in metal chelation. The results were attributed to the previously reported Rabaa et al. [42] who reported the metallic complex formation of polyphenols and found the same stretching peaks. Figure 3c shows the FTIR spectra of unloaded nanoparticles (without vitamin E). Figure 4e shows the zeta potential of the nanoparticles. Reduction of the DPPH was calculated for the inhibition of melanin synthesis by their antioxidant effect which was confirmed at 93.59±0.02%. Percentage vitamin E release in 100 mM of phosphate buffer pH 7.4 at 37°C was found to be 92 ± 2.021% after 8h. The permeation of vitamin E from nanoparticles on rat skin was observed within 3h was 88 ± 2.0124% and the maximum absorbable dose calculated at 17mg that was confirmed the SR behavior of (Zn-Q-PCL-NPs) and (Cu-Q-PCL-NPs). Interestingly, 80 ± 0.02% inhibition of melanocyte cells and 95 ± 0.03% increase of keratinocyte cells proved the tyrosinase enzyme inhibition of Cu-Q-PCL-NPs-E having 13 months shelf life. Conclusively, the use of pure quercetin and its copper complex in simple and its nanoparticles can provide better antioxidant properties with tyrosinase enzyme inhibition which can be used in the treatment of melanogenesis disease.

FTIR Analysis of Nanoparticles
FTIR spectrum of polycaprolactone showed stretching (C=O) mode at 1713 cm −1 , which can be shifted at 2933 cm −1 and 3728 cm −1 which were related to the C-H bond of saturated carbons in the case of nanoparticles. The FTIR spectrum of Cu-Q-PCL-NPs-E (Fig. 3d) showed additional peaks (compared to PCL-NPs) due to the presence of vitamin E in the blend matrix. The interaction between vitamin E and polycaprolactone nanoparticles occurred due to the characteristic peaks of vitamin E; 1092-1426cm −1 bands for C-C formation and 3720 cm −1 for C-O formation were detected in the spectrum of Cu-Q-PCL-NPs-E. Vitamin E was successfully incorporated into PCL nanoparticles due to the overlapping of the -C=O stretching of PCL at 1553 cm −1 for the Cu-Q-PCL-NPs-E. The vitamin E in the Cu-Q-PCL-NPs-E is stable, according to the spectrum analysis.

Scanning Electron Microscopy
SEM analysis was used to observe the morphological characteristics of the nanoparticles by using the alreadyreported method by Kumar et al. [43] including size, shape, and porosity. Figure 4a and b show the SEM image of blank NPs (Cu-Q-PCL-NPs, Zn-Q-PCL-NPs) and Fig. 4c and d show the drug-loaded NPs (Zn-Q-PCL-NPs-E, Cu-Q-PCL-NPs-E) that exposed the changes due to the lipophilic nature of vitamin E that was very stable during preparation of nanoparticles by using ultra-homogenization method. It was observed in SEM graphs of the nanoparticles that they have different sizes 324.5.02 ± 0. 88nm of Cu-Q-PCL-NPs-E and 994.9 ± 0.038 nm for Zn-Q-PCL-NPs-E and both contained pores, which facilitate the adherence of the vitamin E into the nanoparticles. The contrary nanoparticles formed have different shapes round, long, oblige, and spherical with a smooth surface. Zn-Q-PCL-NPs-E were smooth, spherical, and oval in shape, large at 994.9 ± 0.038 but in the case of Cu-Q-PCL-NPs-E the surface was very smooth and spherical and they have a small size of 324.5.02 ± 0. 88, so they can easily penetrate the skin [44]. f The 3D structure of nanoparticles with different parameter of surface roughness that were determined by atomic force microscopy (AFM)

Atomic Force Microscopy, Zetasizer, PDI and Zeta Potential
Zeta potential, AFM, zetasizer, and PDI are the main physicochemical parameters that can be used to check the ability of nanoparticles to penetrate the stratum corneum by crossing biological skin barriers and the size of nanoparticles that were observed by using the already-reported method by Schne ider et al. [45]. Zeta potential and charge through electrostatic repulsion affects physical stability of nanoparticles. Positive charge on Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E may be due to the presence of metal ions. As mucin is negatively charged, positive charge may increase adhesion property of nanoparticles that are showed in Fig. 4e [46].
In Fig. 4f, AFM result shows the 3D structure of nanoparticles with different parameters like height, width, and of surface roughness of nanoparticles. Length in X-axis and Y-axis was 5-10μm and the height of the nanoparticle was 75nm. Surface roughness of Q-PCL-NPs, Zn-Q-PCL-NPs-E, and Cu-Q-PCL-NPs-E gradually declined from 152.82 to 6.027nm that showed the increase in smoothness surface of Cu-Q-PCL-NPs-E due to the lipophilic nature of vitamin E and they can easily cross the skin barriers and reach to a targeted area (stratum corneum) for the inhibition of melanocyte cells. Blank nanoparticles have a small particle size (147.03 ± 0.02 to 796.07 ± 0.03) and PDI was 0.38 ± 0.03 to 0.74 ± 0.03 as compared to drug-loaded nanoparticles. Simple vitamin E has less molecular weight and a more lipophilic nature in Q-PCL-NPs-E that showed a decrease in particle size as compared to Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E. As the vitamin E was loaded in nanoparticles (Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E), the size and PDI gradually increased 324.50 ± 0.03 and 994.90 ± 0.06, and 0.36 ± 0.01to 0.88 ± 0.03, due to some swelling and entrapment of vitamin E within the nanoparticles as shown in Table I.

Entrapment Efficiency (%EE)
Greater EE of vitamin E up to 42.06-80.26% in all nanoparticles showed increased melanin inhibitory effect.
The percentage EE of all nanoparticles was observed to be directly proportional to the conc. of PCL and inversely proportional in the case of gelatin and PVA. The effect of PCL conc. on entrapment efficiency of vitamin E-loaded nanoparticles was studied by using 5% and 10% of Zn-Q-PCL-NPs and Cu-Q-PCL-NPs. More than 80% vitamin E loading was determined, which is due to the increasing polymer ratio. The increase in PCL conc. was due to its more stability, and increased EE of the nanoparticles (Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E). Nanoparticles used as a carrier of vitamin E are an attractive alternative for the treatment of a variety of disorders in which free radical is a factor that was involved in skin permeation and melanin inhibition. Figure 5a shows that the maximum cumulative drug release 85% of Zn-Q-PCL-NPs-E and 90.00% of Cu-Q-PCL-NPs-E was observed. To evaluate the drug release pattern, different kinetics models were applied, i.e., zeroorder, first-order, Higuchi, and Weibull models as shown in Table II. In the case of Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E, the values of the regression coefficient (r 2 ) values were 0.75 and 0.88, and Akaike information criteria (AIC) was 2.96 and 3.71 for zero-order release rate constants that were greater than the values obtained for firstorder release rate constants that are shown in Table II. It is thus linked because of vitamin E as a drug release from the nanoparticles (Cu-Q-PCL-NPs and Zn-Q-PCL-NPs) with various conc. of PCL following a zero-order release pattern. In the case of Cu-Q-PCL-NPs-E, the regression coefficient (r 2 ) 0.86 and AIC value 1.81 in the Higuchi model revealed that the drug release was diffusion-controlled, whereas no significant alteration was identified across all values [1] in the Weibull model. The Weibull model provided the best adjustment curve for Cu-Q-PCL-NPs-E since it had a higher α value 4.26 and a lower AIC value 0.48 as compared to Zn-Q-PCL-NPs-E [47].

Maximum Absorbable Dose in the Skin Membrane
MAD of Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E was calculated by using the already-reported method of Giovino et al. [48] as shown in Fig. 5B. Briefly, the drug permeability constant (k a ) value of 1.86 and drug concentration (20mg) presented on the exposed surface area within limited residence times (3h). The results obtained demonstrated that 84 ± 0.03% vitamin E permeate through the lipid layer membranes modeling and showed the influence of peptides on the dynamics of vitamin E diffusion. In addition, the estimated MAD for all selected compounds is less than 17 mg. This reflects the limited amounts of vitamin E that can be delivered across the skin membrane due to a limited surface area of 1.78cm 2 and residence time of 3h. All the above findings suggest that MAD may be used to estimate clinical transdermal absorption and the potential for transdermal delivery.

Stability Study
Many factors affect the stability of nanoparticles (Zn-PCL-NPs and Cu-Q-PCL-NPs) including the stability of the active ingredient(s), the potential interaction between active and inactive ingredients, the manufacturing process, the dosage form, the container-liner-closure system, handling, length of time between manufacture and usage, and sustained release of the drug. Shelf life for the optimized formulation was 11 months for Zn-Q-PCL-NPs-E and 13 months for Cu-Q-PCL-NPs-E which were calculated by using RGui software (R 4.1. 3 versions) in SR form for a longer time (∼2 years) as shown in Fig. 5c and d.

Non-cellular Antioxidant Activity
The studies of nanoparticles (PCL-NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, and Cu-Q-PCL-NPs) with vitamin E loading seem to be suitable transporters for these antioxidant compounds in the assessed conditions and these compounds play an important role against the epidermal layer of skin. The antioxidant activity of nanoparticles (PCL-NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, and Cu-Q-PCL-NPs) with vitamin E by using a UV-vis spectrophotometer was set to 370nm, and their hydrogen donating/ radical scavenging activity was determined. The absorbance of the stable DPPH decreases rapidly with increasing time (60-120s). The rapid step is defined as the removal of H-atoms from the antioxidant that is the most labile (3-OH, 4-OH in the case of quercetin). Furthermore, the delayed step indicates the oxidation-degradation products and residual activity, as well as the ability of nanoparticles to operate as antioxidants, which is dependent on their molecular structure. Cu-Q-PCL-NPs-E have higher antioxidant activity due to the considerable involvement of the 3 and 4 hydroxyl groups on the B-ring of quercetin. Furthermore, H-atom transfer reactions to DPPH are thought to be largely mediated by 3-OH and 4-OH. The DPPH scavenging activity of varying quercetin conc. of PCL NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, and Cu-Q-PCL-NPs is shown in Fig. 6a. The antioxidant activity of the Cu-Q-PCL-NPs-E was 93.30% that is greater than the free quercetin, their PCL-NPs, Q-PCL-NPs, and Zn-Q-PCL-NPs-E and their p value was 0.03 that is less than the significant value (p< 0.05) [49].

Cytotoxicity and Cellular Antioxidant Activity
In Fig. 6b, the cytotoxic activity of biological materials of different formulations like PCL-NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, and Cu-Q-PCL-NPs with vitamin E was evaluated by using a Resazurin assay [34]. The absorbance was taken at 6-h and 24-h time intervals and calculated percentage cell viability of PCL-NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, and Cu-Q-PCL-NPs using Eq. (10). We determined the percentage cell viability of eight different formulations of nanoparticles with or without vitamin E loaded for the cytotoxicity study at two different time intervals (6h and 24h) which is clear in Fig. 6b. The results showed that Cu-Q-PCL-NPs-E gave better results and provided an essential medium for the growth of MCF-7 cells, while Zn-Q-PCL-NPs-E also gave acceptable results and their p value was 0.04 that is less than significant value (p< 0.05).

Synergistic Effect Between Non-cellular and Cellular Antioxidants
Improved cell viability in the case of MCF-7 assay was observed after the use of the nanoparticle formulations with or without vitamin E loaded may be due to the presence of PCL [50]. Effective regulation and prevention of melanin formation by delivering vitamin E was maybe due to its nutritional effect which was already reported by Zhiping et al., by using the vitamin E and doxorubicinloaded nanoparticles for increased cell viability up to 50% [51]. In another study, Diao et al. reported that use of vitamin E-loaded nanoparticles promotes breast proliferation agent greater than 90% by reducing ROS production and p53 expression. In the reported study, improved cell regeneration by using vitamin E-loaded nanoparticles was observed which proved its biocompatibility and biodegradability [52,53].
In the case of non-cellular antioxidants, PCL-NPs-E, Q-PCL-NPs-E, and Zn-Q-PCL-NPs-E, and Cu-Q-PCL-NPs-E resulted in 52.33%, 72.65%, and 85.01%, and 93.59% inhibition due to the oxidation of nanoparticles induced by DPPH which was observed respectively in Fig. 6a showed a synergistic effect and these nanoparticles serve as a model to examine the hydrophobic-hydrophilic interactions in biological skin membranes. However, when vitamin E was added together with all types of nanoparticles (PCL-NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, and Cu-Q-PCL-NPs), the inhibition of vitamin E oxidation exceeded the additive contribution of the individual antioxidants by an additional 20% (p < 0.01) for Zn-Q-PCL-NPs-E, and 30% (p < 0.05) for Cu-Q-PCL-NPs-E. These results suggest a more synergistic effect was observed in the case of Cu-Q-PCL-NPs-E in comparison to these four antioxidants and vitamin E that are shown in Fig. 6(a.1).
In the cellular antioxidant study, the calculated degree of synergism between vitamin E and the nanoparticles (PCL-NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, and Cu-Q-PCL-NPs) was determined by the IF/IE values in Fig. 6

Superficial Rat Skin Model
In Fig. 7, melanin inhibition was observed by ex vivo analysis of Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E that showed keratinocyte and melanocyte responses. For the scoring of the melanogenesis response, 0 showed no melanocyte cell present, 1 represented little melanocyte cell, while score 2 was used for more melanocyte cell present but score 3 was considered moderate melanocyte cell and score 4 represented the presence of severe melanocyte cell. Similar trends of scoring were applied for keratinocyte cells. In the superficial rat skin model, most of the epidermis was removed, although a single layer of epidermal cells (keratinocytes and melanocytes) remained in its original form. When suspension of Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E was applied on the mice skin e Fig. 7 Melanin inhibition in rat skin was observed after 5 days treatment; indicating the tyrosinase enzyme inhibition in keratinocytes and melanocyte cells from the indicated experimental group; a untreated control group, b control group treated with Hyderquin plus (c) , c treated with Zn-Q-PCL-NPs-E and compared with d treated with Cu-Q-PCL-NPs-E (μg/mL) suspension and e was the graphical representation of keratinocytes and melanocyte cell response was observed treated with nanoparticles after 8h daily for 5 days, results revealed that increased occurrence of keratinocytes was greater than that of melanocytes which was confirmed by reduction of melanogenesis. Untreated control group 1 was washed with water and no any improvement of melanogenesis was observed while in group 2, treated with hyderquin plus pigmentation was observed which may be due to exogenous ochronosis process. Group 3 was treated with prepared Zn-Q-PCL-NPs-E nanoparticles which promoted keratinocyte proliferation that inhibit the tyrosinase enzyme which ultimately causes melanogenesis. Similar behavior with increased response was observed in group 4 treated with Cu-Q-PCL-NPs-E which may be due to the presence of Cu-Q complex.
In the case of Cu-Q-PCL-NPs-E, the histopathological results showed that more keratinocyte cells and less melanocytes were present as compared to the Zn-Q-PCL-NPs-E with their p value was 0.02 that is less than the significant value (p< 0.05). Results were attributed to the previously reported work of KC Park et al. [55] after discussing the effect of keratinocytes on melanogenesis [56].

Computational Study
During this simulation's procedure, we approximate the possible interaction pattern between the studied drug ligands (Zn-Q and Cu-Q complex as shown in Fig. 8 Table III shows optimization energies, dipole moments, polarizability, and (higher occupied molecular orbital and lower unoccupied molecular orbital) HOMO-LUMO gap values. The HOMO represents the highest energy level in the molecule that contains electrons, while the LUMO represents the lowest energy level that does not have any electrons. These orbitals are important in understanding chemical reactions and the properties of molecules. Using the HOMO-LUMO energy gap of Zn-Q and Cu-Q, optical, electrical, stability, and reactivity parameters are determined. Figure 8 displays optimized structures while Fig. 9 depicts contour plots of HOMOs and LUMOs for copper and zinc quercetin complexes. Chemical reactivity is vital in managing novel inhibitors and structures; the HOMO-LUMO energy gap, optimization, and descriptors give insight. The larger the energy gap, the more kinetic stability, less polarizability, and lower reactivity. The lower the energy gap, stability decreased, polarizability increased, and reactivity increased. Both Zn-Q and Cu-Q complexes have Eg of 3.07 eV and 3.06 eV. Hardness (η) and softness (S) assist explain chemical system behavior. Hard molecules have broad energy gaps, while soft molecules have tiny ones. Table III shows that these complexes are hard and soft in favor of the reactivity index. Table IV indicates the interaction of the synthesized complexes with 5M8Q protein structures.
After validating the kojic acid protein model, it will be utilized to investigate Zn-Q and Cu-Q binding ability. We test how efficiently ligand structures bind to active reference amino acid residues in the 5M8Q protein's  active binding region (kojic acid pocket). We choose the scoring algorithm's conformations with the lowest binding energies (highest possible stability). The obtained results, shown in Table III, showed that the binding energies of the docked ligands are all negative (−7.9 and −8.1). The Zn-Q-5M8Q and Cu-Q-5M8Q complexes were created using molecular docking, which is ideal for finding drug-molecule interactions with antioxidant capabilities and melanin suppression that may be utilized to treat melanogenesis disorder. 3D and 2D binding poses are also shown in Fig. 10.

Conclusion
Copper and zinc-quercetin complexes were prepared by a solvent evaporation method, confirmed by Fourier transform infrared spectroscopy (FTIR) and color changes of products from light yellow to brownish yellow. The results justified that vitamin E was incorporated into PCL nanoparticles (PCL-NPs, Q-PCL-NPs, Zn-Q-PCL-NPs, Cu-Q-PCL-NPs Zn-Q-PCL-NPs-E, and Cu-Q-PCL-NPs-E) that was prepared by ultra-homogenization method successfully. These nanoparticles showed a synergistic effect by using the  ----cellular-and non-cellular-based antioxidant effect that was evaluated by using the MCF-7 cell line and DPPH reduction method. According to the in vitro release studies, Zn-Q-PCL-NPs-E and Cu-Q-PCL-NPs-E showed burst release at the 5th minute after application; however, release lasted for 8h, and maximum absorbable dose (MAD) was calculated by using the permeation study of rat skin. In superficial rat skin model, ex vivo studies showed that the nanoparticles (Zn-Q-PCL-NPs and Cu-Q-PCL-NPs) containing vitamin E was a promising formulation for melanin inhibition due to the decreased melanocyte cells and increasing the no. of keratinocyte cells in the epidermal layer of the skin.