Enhanced Anti-Angiogenic and Anticancer Ecacy of Nanoformulated Catechin in the Chemoprevention of Skin Cancer

Aim: Nontoxic epigallocatechin gallate (EGCG) from green tea signicantly inhibits tumors in many cancers, including melanoma. Despite the benets, EGCG has poor stability therefore, to enhance bioavailability and anticancer eciency, we synthesized 3 nanoformulations. Methods: In Nano 1, ZnO nanoparticles were used for encapsulating EGCG. For Nano 2, EGCG nanoformulation was prepared with co-polymeric nanoparticle-encapsulated formulations of lycopene. Olive oil nano emulsion was used in EGCG Nano 3. Evaluated physical characteristics and anticancer ecacy in chick chorioallantoic membrane tumor model. Results: Nanoformulations were stable with high EGCG encapsulation eciency and loading. They inhibited melanoma growth effectively than free EGCG. Conclusion: Nanoformulations preparation methods eciently prevented the loss of EGCG activity and is a favorable approach for the treatment of melanoma. cells in the ex vivo chick chorioallantoic membrane (CAM) tumor model. Our data indicates that the EGCG nanoformulations enhanced cell death and reduced blood vessel formation in the melanoma tumors compared to free EGCG. Further studies of these nano EGCG formulations are required in the preclinical animal models to evaluate their ecacy. their anti-angiogenic effect against human skin cancer (A375) cells. We used the chick chorioallantoic (CAM) tumor model to assess the effect of the nanoformulations and free EGCG on tumor growth. The nanoformulations signicantly reduced the tumor weight and hemoglobin content in the CAM models of skin cancer. Our ndings suggest the combinational effect of EGCG with other dietary components like lycopene and olive oil enhanced cancer prevention


Summary Points
In melanoma the common skin cancer, the conventional therapies may lead to resistance to treatments. The limitations have supported the use of natural antitumor agents including the polyphenol epigallocatechin gallate (EGCG), which comprises most of the antioxidants in green tea.
Nontoxic EGCG showed skin protective effect and anticancer properties, however main shortcomings are low bioavailability due to malabsorption, high rate of metabolism, inactivity of metabolic products and rapid clearance from the body.
In this study, we prepared 3 nanoformulations of EGCG to overcome rapid metabolism, to increase antitumor e cacy.
To protect nano EGCG from oxidations, we applied co-polymeric nanoparticles utilizing ZnO, lycopene or olive oil nanoparticles in the preparation of our nanoformulations.

Introduction
Melanoma is the common skin cancer in humans, and the frequent therapy modalities include chemo or radiotherapy [1][2][3], surgery or more than one type of treatments [4][5][6]. These remedies may lead to radio and drug resistance, and these limitations have encouraged oncologists to develop different strategies for cancer prevention and treatment [7,8]. Novel approaches are currently being explored using natural antitumor agents including the polyphenol epigallocatechin gallate (EGCG), which comprises the majority of the antioxidants in green tea [9,10].
EGCG showed skin protective effect and antiproliferative, anti-angiogenic, and apoptotic properties in cancer cell cultures and preclinical studies [11][12][13][14][15]. However, the main shortcoming is its low bioavailability due to malabsorption, high rate of metabolism, inactivity of metabolic products and rapid clearance from the body [16]. Anticancer e cacy of EGCG was reported in a variety of cancer cell types including skin, colon, kidney, breast, brain, lung, and leukemia however, the concentrations of EGCG required are much higher than the peak plasma concentration [17,18].
As malignant melanoma is a heterogeneous disease, being the consequence of several molecular pathways and considering that the use of sunscreen does not entirely prevent skin cancer, additional natural chemo preventive approaches are advantageous. Melanoma and other skin cancer types are also caused by natural and arti cial ultraviolet (UV) radiation exposure [19,20]. Lycopene is a lipophilic C-40 carotenoid antioxidant present at high concentrations in tomatoes and has been reported to provide protection against acute and potentially longer-term aspects of photodamage and for chemotherapeutic e ciency in skin cancer [21][22][23]. Malignant melanomas and other skin cancer types are caused by natural and arti cial ultraviolet (UV) radiation exposure [19,20].
It has been shown that phenolic compound, when incorporated into polymeric nanoparticles, enhanced anticancer effects as observed in vitro, and in animal models via oral and intravenous administration [24]. Nanoparticles are useful strategies to overcome the poor absorption, rapid metabolism, and elimination inherent in most natural products; helping to increase their bioavailability [15,25,26]. Alari et al. [27] reported that zinc oxide (ZnO) nanoparticles kill melanoma cells directly through oxidative stress, DNA damage, and induction of apoptosis. Inorganic agents like zinc oxide (ZnO) particles exhibit a combination of absorptive, re ective, and light-scattering properties so that UV radiation does not reach and/or penetrate skin, and ZnO has been approved by the FDA [28,29].
Here, to enhance antitumor effect of EGCG in a nanoformulation, we used FDA-approved ZnO nanoparticles in the preparation of our nanoformulations. Here we synthesized 3 EGCG nanoformulations for the purpose of increasing its stability, anticancer and antioxidant e cacy in skin cancer by using copolymeric nanoparticle-encapsulated formulations of EGCG. We utilized the bene ts of ZnO nanoparticles as a UV blocker and used poly (lactic glycolic acid) (PLGA) as a capping agent for EGCG in the rst nanoformulation. In the second formulation, lycopene was used as an antioxidant, protecting EGCG from the oxidizing conditions caused by UV-exposure and olive oil was used to reduce oxidative damage. Also, we prepared a third nanoformulation using nano emulsion of EGCG-chitosan (core) encapsulated by an olive oil shell. We characterized these nanoformulations and assessed the loading capacity of EGCG in the nanoparticles. In cancer, the angiogenesis process accelerates solid cancer growth and thus we evaluated the 3 nanoformulations' inhibitory effect on skin tumorigenesis and their anti-angiogenic effect against human skin cancer (A375) cells. We used the chick chorioallantoic membrane (CAM) tumor model to assess the effect of the nanoformulations and free EGCG on tumor growth. The nanoformulations signi cantly reduced the tumor weight and hemoglobin content in the CAM models of skin cancer. Our ndings suggest the combinational effect of EGCG with other dietary components like lycopene and olive oil enhanced cancer prevention e cacy.

Preparation of different nano formulations encapsulating EGCG
Preparation of EGCG-chitosan EGCG-chitosan solution was prepared by a nanoemulsi cation method [30,31]. Brie y, 8 mg of ascorbic acid was dissolved in 8 ml of deionized water and 100 mg low molecular weight chitosan was added with continuously stirring for 15 min to get a clear, transparent solution. To this solution, 20 mg EGCG was added under stirring for 30 min to form EGCG-chitosan solution. Nano 1 Polymeric Nanoparticles: PLGA-EGCG/ZnO polymeric nanoformulation ZnO-PLGA particles were synthesized using a co-precipitation [32][33][34] method with slight modi cations as detailed in Fig. 1A. Zinc nitrate hexahydrate (2.97gm) was dissolved in 100 ml of deionized water, and 0.48 gm sodium hydroxide (NaOH) was dissolved in 60 ml deionized water. Then the zinc nitrate solution was added drop-wise to the NaOH solution under vigorous stirring for 12 h. White precipitate of zinc hydroxide, [Zn (OH) 2 ] nanoparticles formed and was centrifuged at 10,000 rpm for 20 minute and washed with deionized water three times to remove unreacted reactants. Zn (OH) 2 was annealed for 2 h at 500°C to form white powder of ZnO nanoparticles. The ZnO nanoparticles (10 mg) were dissolved in 1 ml of 10% PLGA solution (100 mg PLGA in 1 ml water) to form ZnO-PLGA suspension. This suspension was then added to the EGCG-chitosan solution under stirring for 1 h. Next, chitosan nanoparticles were prepared with ionic gelation using sodium tripolyphosphate (TPP) as crosslinking agent and one ml of. TPP (10 mg/ml in deionized water) was added drop-wise with constant stirring. The entire solution was then sonicated for ~ 30 sec using a probe sonicator and allowed to stir for ~ 4 h. This solution containing EGCG nanoparticles was dialyzed using a 100 kDa cutoff dialysis membrane to remove the impurities and the free EGCG. A milky nanoformulation of EGCG/ZnO formed and was used for further studies and characterization.

Nano 2 Solid Lipid Nanoparticles (SLNPs) EGCG/lycopene nanoformulation
SLNPs were prepared using a nano emulsi cation method [26,30,31] with modi cation (Fig. 1B). In brief, lipid phase lycopene solution (1%) was prepared by dissolving 10 mg lycopene in 1 ml olive oil under ultra-sonication (80% amplitude) with a Ultrasound (US) probe for 10 min at 60 mv, to form a clear dark red oil phase. To 1 ml of EGCG-chitosan solution, 500 µl of dissolved oil phase lycopene was added dropwise under ultra-sonication (80% amplitude) for 5 min and the then solution was stirred (600 rpm) overnight at room temperature. An orange colored EGCG/lycopene nanoformulation formed and was used for characterization and further studies.

Nano 3 SLNPs: EGCG lipid nanoemulsion
EGCG-chitosan solution was prepared using a nano emulsi cation method [30,31] by dissolving 8 mg ascorbic acid in 9.5 ml deionized water, then 100 mg low molecular weight chitosan was added and the solution was stirred for 15 min to get a clear, transparent solution. To 1 ml EGCG-chitosan solution, 500 µl of pure olive oil (lipid phase) was added drop-wise under ultra-sonication (80% amplitude) for 5 min. The solution was stirred (600 rpm) overnight at room temperature to form a milky nanoformulation (Fig. 1C).

Characterization of nanoformulations
Size measurement of nanoformulations Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS) techniques were used for measurement of the size distribution and zeta potential, respectively. Ten µl of each nanoformulation was resuspended in 1 ml of water and analyzed with a Malvern zeta sizer (Malvern Instrumentation Co, Westborough, MA).

Structure of the nanoformulations
The morphology and size of the nanoformulations were assessed using transmission electron microscopy (TEM). The dispersion of a nanoformulation was diluted with pure water and 20 ul was adsorbed onto carbon-coated grid lm and stained with 20% w/v uranyl acetate. Then the grid was attached to a platinum sample holder and the topographic images were obtained using high resolution TEM imaging under 200 KV.

Determination of encapsulation and loading e ciencies
The amount of EGCG encapsulated in the nanoformulations was determined by disintegrating the nanoparticles and measuring EGCG with ultraviolet-visible (UV-vis) spectroscopy. To prepare a standard curve, free EGCG (not encapsulated) solution was separated by ltering through a Millipore (Burlington, MA, USA) centrifugal device with a 3 kDa cutoff, assisted by centrifugation, at 28000 x g for 30 min. The concentration of EGCG in the nanoformulation was obtained by disintegrating the nanoformulation in acetic acid solution. The entire solution was passed through the Millipore ltration centrifugal device and centrifuged to separate the free EGCG. The EGCG concentration of the centrifugate/ ltrate was then determined using a Nanodrop 2000C UV-vis spectrophotometer (Thermo Fisher Scienti c, Waltham, MA). The EGCG concentration was calculated in comparison with the standard curve and expressed as mg/ml.
After lyophilization, a weighed nano encapsulated powder was analyzed for EGCG quanti cation using HPLC with an established standard curve [26]. The encapsulation e ciency was measured using the following equation: The loading ratio was determined by measuring the amount of EGCG in the nano capsules and the weight of whole nano encapsuled according to the following equation [26].
Evaluation of storage stability and in vitro release kinetics of the nanoformulations To con rm stability of the EGCG-nanoformulations, they were kept in sealed glass vials and stored at 37°C ± 2°C with a relative humidity of 65% ± 5% for 4 weeks. Samples were collected once a week to assess the concentration of EGCG as described above.
In vitro release kinetics The in vitro release kinetics of EGCG from the nanoformulations were investigated with a dialysis method with fresh release in PBS or fetal bovine serum (FBS). In brief, 1 ml of each nanoformulation or free EGCG was dissolved and added to a dialysis bag (molecular weight cutoff = 6-8 kDa) and dialyzed against release solutions PBS or FBS under gentle stirring at 100 rpm at 37°C. The entrapment e ciency of these formulations was measured at predetermined time intervals and compared with values obtained with the fresh formulations. At designated time intervals (0.5, 1, 3, 6, 12, and 24 h), 500 µL aliquots of external release solution was withdrawn. It was ltered through 30 kD Millipore separation membrane tubes and centrifuged at 28000 × g for 30 min at 4°C to separate the released EGCG from the nanoparticles. The amount of released EGCG in the supernatant was determined using a Nanodrop UV-VIS spectrophotometer against an EGCG standard calibration curve as described above. The proportion as a percentage of cumulative EGCG release was plotted against dialysis time, and each experiment was executed in triplicate.

Cell Culture
Human melanoma cancer cells (A375) were cultured in DMEM supplemented with 10% FBS, 100 U ml − 1 penicillin, and 100 mg ml − 1 streptomycin and incubated at 37°C in a humidi ed atmosphere of 95% and 5% CO 2 . Cells were passaged under sterile condition to maintain sub con uent level. To collect, cells were treated with 0.25% (w/v) trypsin/ EDTA to effect cell release from the culture ask. After washing cells with culture medium, they were suspended in medium and cells were counted th for further studies.

Ex vivo tumor growth in Chick Chorioallantoic Membrane (CAM) model
For tumor growth analysis, one-day-old chick embryos were purchased from Charles River (Norwich, CT) and maintained at 37°C and 55% relative humidity. A375 cells were used for ex vivo studies as described previously [35][36][37]. After 7 days of incubation, a hypodermic needle was used to make two holes on the eggshell and mild suction was applied at one hole so that the CAM dropped away from the shell. Using a Dremel drill (Racine, WI), a small window was cut in the shell over the false air sac. A375 cells (1×10 6 ) were placed on the CAM in 50% Matrigel with free EGCG 20 or 100 µg/CAM and its nano formulations containing 20 or 100 (EGCG equivalent) µg/CAM (n = 7-8 per group). The groups were: Control (PBS), EGCG, Nano 1, Nano 2, and Nano 3. After 8 days, the eggshell was removed carefully using a scalpel and scissors, and the tumors were excised from the CAM membrane and weighed.
Tumor hemoglobin (Hb): Hb content was indexed as a measure of tumor vascularity [38]. The antiangiogenesis activity of EGCG and the nano formulations were investigated in the CAM model of tumor angiogenesis by determining the relative Hb level in the tumor. Harvested tumors were collected in 2mlcentrifuge tubes and homogenized in 200 µl of distilled water, centrifuged and collected the supernatant. In a 96-well plate, 50 µl of the supernatant combined with 50 µl of Drabkin's reagent, and Hb absorbance was measured at 540 nm. Hb concentration was calculated in comparison with a standard curve and expressed as mg/dL.

Statistical Analysis
All analytical values shown represent the means of three replicates. Data were analyzed using one-way Anova. Mean separation test between treatments was performed using Tukey's test and P < 0.05 was regarded as signi cant.

Results
Chitosan nanoparticles encapsulating EGCG were synthesized by different methods. For Nano 1, nanosized inorganic compound ZnO was used along with polymeric nanoparticles of EGCG for preparations, and the physicochemical characterization showed an average particle size distribution of 170.5 nm (Fig. 2a) with very low poly dispersive index (PdI = 0.002) and zeta potential + 8.94 mV (Fig. 2b). Particle topography was examined with TEM and showed that clusters of ZnO PLGA coated nanoparticles were embedded in EGCG polymeric nanocomposite (Fig. 2c) For Nano 2, the SLNPs EGCG/lycopene nanoformulation used is the most frequently applied approach for enhancement of carotenoid stability, and the microencapsulation of hydrophilic SLNP, to creates a physical barrier and protects the pigment. The optimized EGCG loaded Nano 2 showed a particle size of around 234 nm and the zeta potential was + 18 mV (Fig. 3).
For Nano 3, the SLNPs EGCG/olive oil nanoformulation, olive oil was used to improve stability of the nanoemulsions of SLNPs EGCG. The zeta potential of nano 3 was found to be positive and the size was 236 nm in diameter with Zeta potential + 20 mV (Fig. 1C). TEM images showed spherical morphology (Fig. 4).

Encapsulation e ciency and loading ratio
The encapsulation e ciency of the EGCG in the nanoformulations was 22 mg/ml, and with the aid of UVvis spectroscopy, the amount of EGCG encapsulated in the chitosan nanoparticles was determined. EE was 99%, 99.6%, and 100% and loading ratios were 8.4%, 13.3%, and 16.6% for Nano 1, 2, and 3, respectively.
Effect of storage on the stability of nanoformulations As shown in Table 1, all 3 nanoformulations were stable after 4 weeks of storage. The released content of EGCG was in the range of 20 mg/ml and at the beginning and continued to be stable after 4 consecutive weeks with no change in the concentration.

Release kinetics
As depicted in Fig. 5, we observed similar in vitro release kinetics of all nanoformulations of EGCG. A very rapid and complete dissolution of EGCG (< 70%) was observed within 1 h, in Nano 1 and 2 in FBS and became stable at 40% over 24 h. Nano 3 showed gradual release and reached a peak of < 65% after 6 h and stayed in that range for 24 h in FBS. There was no or slow release of EGCG from the nanoformulations in PBS (Fig. 5).
Anticancer and anti-angiogenetic activity of EGCG nanoformulations in the CAM model Tumor weight Anticancer activity of the three nanoformulations was validated from tumor weight as a measure of tumor growth of A375 skin cancer, after exposure to these formulations at 20 µg/CAM or 100 µg/CAM. Untreated tumor proliferation and Hb values were compared to the treatment groups. There was a signi cant reduction in the tumor weight and hemoglobin levels of A375 tumors in the CAM model after treating with the EGCG nanoformlations at 20 µg/CAM (P < 0.01) and 100 µg/CAM (P < 0.001) compared to untreated group (Fig. 6A). Similar decrease was also observed for all three nanoformulations when compared with the free EGCG group (Figs. 4-6). Lower reduction was seen with free EGCG at 20 µg/CAM (P < 0.05) and 100 µg /CAM (P < 0.01) compared to the untreated group (Fig. 6B).

Tumor angiogenesis
Blood vessels were clearly observed in the tumors of the untreated group and in some of the free EGCG groups, but vessels were absent in the Nano 1, 2, and 3 treated tumors (Figs. 6C and 6D). Tumor hemoglobin level is a measure of tumor angiogenesis and treatments with nanoformulations exhibited an anti-angiogenesis effect against A375 as indicated in the reduction of Hb level of harvested tumors ( Fig. 6C and 6D) compared to untreated and free EGCG groups.

Discussion
EGCG is the main polyphenol in green tea and has been shown to have numerous health-promoting effects such as antioxidant and anti-in ammatory, and it is a suitable target for the development of novel cancer therapeutics [39,40]. Studies of skin cancer cells as well as human vascular epithelial cells demonstrated that EGCG promotes immunoregulatory/anti-neoplastic functions and anti-angiogenic effects [41,42]. A major challenge with EGCG is its low bioavailability, which is being pursued for improvement by encapsulating EGCG in nano-sized vehicles for delivery, and nanoformulations of EGCG have been reported in studies of different cancers including human and mouse melanoma [14,[43][44][45][46].
Most studies, however, have not revealed whether the nanoparticles could protect EGCG from degradation and oxidation. This would be particularly relevant because EGCG is very susceptible to oxidation, which starts during direct contact with air [47,48]. In addition, EGCG's strong antioxidative activity, it undergoes auto-oxidation to form reactive oxygen species, resulting in polymerization and decomposition that leads to ineffective bioavailability [39,46,[49][50][51]]. To answer the oxidation effect issue and enhance bioavailability, we tested several options and prepared nanoformulations of EGCG under three conditions to achieve a stable and bioavailable suspension and to prevent oxidization of EGCG as anticancer agents or when applied topically on the skin. In the present study, basic EGCG-chitosan nanoparticles were combined with ZnO PLGA, lycopene olive lipid, or olive oil lipid nanoparticles. One of the main aspects taken into consideration is UV light, the most preventable risk factor for many skin cancers [19,20], and scienti c evidence supports the bene ts of using ZnO, lycopene, or olive oil with high sunlight protection factor [28,52,53]. Additionally, EGCG is unstable under visible and UV light illumination by losing hydrogen atoms, leading to auto-oxidation [54]. Nanoparticles made up of the biodegradable and proven biocompatible polymer PLGA, albumin, or chitosan have been extensively utilized due their ability to control the time and rate of release of the incorporated compound [55,56].
Here, to prepare EGCG-chitosan solution, the compound was dissolved in ascorbic acid before adding to the biopolymer chitosan to improve EGCG bioavailability. It has been reported that ascorbic acid modulates catechin bioavailability from green tea and prevents oxidation [39,57]. Previously we reported that oral delivery of EGCG-chitosan nanoparticles increased the retention time and release of EGCG for a longer time in melanoma tumor xenografts [58]. EGCG loaded in nanoparticles can protect it from adverse environmental conditions, delay its degradation, and improve its bioavailability. Also, EGCG encapsulated in chitosan exhibited higher stability than free EGCG [26]. However, it is considered that the mechanism by which absorption of catechins is enhanced is likely due to the improved stabilization of catechins after encapsulation [59].
In the rst nanoformulation, EGCG-chitosan/ZnO PLGA, we combined the bene ts of ZnO nanoparticles as a UV blocker [60] and PLGA as a capping agent for EGCG [24,61]. In this nanoformulation, ZnO-PLGA suspension was added to the EGCG-chitosan solution with TPP solution because chitosan-TPP improves the EGCG stability in alkaline solution [59]. However, one disadvantage of using PLA-PEG nanoparticles is its unstable nature in acidic environment, and therefore it is not recommended for oral consumption [46]. It has been hypothesized that the charges on the chitosan and EGCG form matrices to overcome poor stability in the intestine [59,62].
In the second nanoformulation, chitosan EGCG was combined with lycopene lipid nano formulation to enhance the antioxidant activity of EGCG and protect the autooxidation. Lycopene, the bioactive red pigment that has the most potent antioxidant activity among carotenoids and plays a role in the protection against photooxidative processes, is an e cient singlet-oxygen quencher and can interact synergistically with other antioxidants [63,64]. Virgin olive oil improves the antioxidant activity and contains trace elements of a variety of phytochemicals, exerts bene cial effects on oxidative stress, and dissolves the insoluble crystalline form of lycopene to protect it from thermal oxidation [63][64][65]. Lycopene lipid nanoparticles were used as an antioxidant protecting EGCG from the oxidizing conditions caused by UV-exposure, and olive oil was used as a source of natural polyunsaturated fatty acids that attenuate oxidative damage [66].
In the third formulation, the EGCG-chitosan was encapsulated by an olive oil shell. It has been reported as it contains phytochemicals, which have proven to exert bene cial effects on oxidative stress and SLNPs with olive oil have been developed as a novel carrier [65,67]. Lipid carriers improves the encapsulation of EGCG and the stability in alkaline solution, resulting in better sustained release property for antioxidant activities [59].
We found that all three EGCG nanoformulations were in the range of 154 -233 nm and we previously reported similarly sized chitosan-EGCG nanoparticles [26]. We expected zeta potential of the nanoformulations of EGCG to be positive due to the abundant free amino acids of chitosan, and the formulations were spherical in shape as observed by TEM as reported earlier [26].
To establish proof of the concept, we tested the nanoformulations of EGCG of on the anticancer potential of melanoma in the CAM tumor model. Previously we reported the successful CAM engraftment from different cancer cell lines and that the xenografts maintained the same tumor growth pattern as observed in mice; the CAM model greatly shortens the time of tumor growth [35,37]. All three nanoformulations of EGCG showed signi cant anti-angiogenic effect and inhibition of tumor growth against A375 tumors in the CAM tumor model compared to free EGCG. The inhibition was observed even at the lowest concentration (20 µg/CAM) compared to free EGCG as reported by Siddiqui et al. [46] also reported that the concentration required to achieve inhibition of growth factor-induced angiogenesis was signi cantly reduced by EGCG nanoformulation compared to EGCG alone in the CAM angiogenesis model [24] and in mouse melanoma tumor angiogenesis [68, 69].
Here we observed inhibition of A375 cell tumor growth by the EGCG nanoformulations and we previously recorded reduction of another melanoma cell line (Mel 928) tumor xenografts by oral administration of chitosan-EGCG due to apoptosis and cell cycle inhibition [58]. Earlier, we reported remarkable inhibition of melanoma cell viability, with about 8-fold better e cacy of PLGA encapsulated EGCG, compared to free EGCG [58] and the growth inhibition was 5%-7% in the A375 cells treated with EGCG [70]. The mechanisms by which EGCG protects against skin cancers are due to the promotion of cell cycle arrest, apoptosis, or inhibition of angiogenesis as well as anti-in ammatory, immunomodulatory, and antioxidant effects [28]. Studies in mice have indicated the anticancer e cacy of EGCG against melanoma by oral or topical delivery [23].

Conclusion
Here we successfully prepared, characterized, and investigated the effects of different nanoformulations of polyphenolic compound EGCG as an antioxidant in melanoma. These nanoformulations were very stable and release kinetics revealed a constant release of EGCG, indicating the e ciency of the preparation method to prevent the loss of its activity. All these nanoformulations showed inhibitory effect on skin cancer growth and tumor angiogenesis. Our data suggest the need for further studies investigating the role that EGCG and other natural phytochemicals play in skin cancer interventions.
However, sustainable release of low dosage EGCG over a longer period is needed for treatment of chronic diseases like atherosclerosis or neurodegeneration.   Figure 1 Schematic diagrams of the preparation methods of the nanoformulations of EGCG. A) Nano 1 process wherein i) represents the preparation of ZnO PLGA coated nanoparticles, ii) preparation of EGCG chitosan coated conjugated structure iii) preparation of EGCG/ZnO polymeric nanoformulation. B) Nano 2 process where lipid phase (lycopene in olive oil) was formed by dissolving lycopene in olive oil. Water phase (EGCG in chitosan) was formed by dissolving EGCG in 1% chitosan solution. Emulsi cation of both phases by ultra-sonication formed EGCG/lycopene nanoemulsion. C) Nano 3 process where lipid phase (olive oil) and water phase (EGCG in chitosan) were formed by dissolving EGCG in 1% chitosan solution.
Emulsi cation of both phases by ultra-sonication formed EGCG-olive oil nanoemulsion.       In vitro release kinetics study of EGCG Nano 1, 2, and 3. a) EGCG standards calibration curve prepared by dissolving accurate concentrations of pure EGCG in water and measured with UV-Vis spectrophotometer. b) EGCG Release kinetics behavior from the three nanoformulations in FBS and PBS. Figure 6