Melatonin/Sericin Wound Healing Patches: Implications for Melanoma Therapy

Melatonin and sericin exhibit antioxidant properties and may be useful in topical wound healing patches by maintaining redox balance, cell integrity, and regulating the inflammatory response. In human skin, melatonin suppresses damage caused by ultraviolet radiation (UVR) which involves numerous mechanisms associated with reactive oxygen species/reactive nitrogen species (ROS/RNS) generation and enhancing apoptosis. Sericin is a protein mainly composed of glycine, serine, aspartic acid, and threonine amino acids removed from the silkworm cocoon (particularly Bombyx mori and other species). It is of interest because of its biodegradability, anti-oxidative, and anti-bacterial properties. Sericin inhibits tyrosinase activity and promotes cell proliferation that can be supportive and useful in melanoma treatment. In recent years, wound healing patches containing sericin and melatonin individually have attracted significant attention by the scientific community. In this review, we summarize the state of innovation of such patches during 2021–2023. To date, melatonin/sericin-polymer patches for application in post-operational wound healing treatment has been only sparingly investigated and it is an imperative to consider these materials as a promising approach targeting for skin tissue engineering or regenerative dermatology.


Introduction
The skin with subcutis constitutes the largest organ in the human body, exposed to external and internal aging factors.The stochastic process of skin aging implies functional and phenotypic variability in cutaneous and immune cells, which occurs along with functional and structural changes in extracellular matrix constituents, including collagen and elastin.Wrinkling, roughness, skin laxity, and decrease in elasticity are the main clinical features of the skin aging process [1][2][3].
The crucial external factor of skin aging is the excessive exposure to ultraviolet radiation (UVR) which is connected with hyperpigmentary changes [4] and wrinkling [5], and also induces common types of skin cancer such as basal cell carcinoma [6,7], squamous cell carcinoma [8][9][10], and malignant melanoma [11,12].The UVR spectra are: UVC (200-280 nm), UVB (280-315 nm) and UVA (315-400 nm), and radiation [13][14][15].The UVC and 280-290 wavelengths of UVB are absorbed by the ozone layer of the atmosphere; however, the stratum corneum is able to absorb the UVC radiation after the exposition to non-natural light sources [14].The UVA can penetrate the reticular dermis and induce biological effects, yet not as efficiently as the UVB radiation [16], which is absorbed by the upper layers of the epidermis and can penetrate the papillary dermis [13,15].The UVR has an influence on many complex processes in the human body and can also trigger systemic reactions [17,18].It can upregulate local and systemic neuroendocrine systems [18].The locally induced cytokines, urocortins, enkephalins, and melanocortins can impose systemic effects while released into the circulation, like the agitation of the central hypothalamicpituitary-adrenal axis, opioidogenic effects, and immunosuppression, as well as vitamin D synthesis and activation [17][18][19][20][21][22].
The UV lights have a tremendous impact on biological organisms and the origin of life on Earth [23][24][25][26].The energy of UVR is similar to the energy of covalent bonds, which means that any molecule electron excitation by UV light can disrupt or create a covalent bond.Organic molecules exposed to the energy of UV light commute to chemical bonds with high-energy levels, achieving molecular complexity [18,26].The cells can use their energy increasing the enthalpy of the system [26].UVB is crucial in photosynthesis, providing various forms of vitamin D which can be enzymatically activated at the local and systemic levels in different organisms [21,22,27].
In human skin, reactive oxygen species/reactive nitrogen species (ROS/RNS) production can be induced by UVR, thereby elevating the secretion of pro-inflammatory cytokines.Increased cell proliferation and oxidation processes can disrupt cell membrane integrity and induce DNA fragmentation [7].The topical delivery of antioxidants helps to maintain redox balance in epidermal cells, regulates pro-inflammatory cytokines release, and prevents oxidative damage and DNA fragmentation in these cells [28][29][30][31][32][33][34][35].
Melatonin (N-acetyl-5-methoxyindolamine) and sericin exert antioxidant properties and could be useful in topical wound healing patches by maintaining redox balance, cell integrity, and regulating the inflammatory response [36][37][38][39].In human skin, melatonin suppresses the damage caused by UVR through numerous mechanisms associated with ROS/RNS production and programmed cell death (apoptosis) [40][41][42][43].Melatonin derived from tryptophan constitutes an active pleiotropic molecule in the human organism, synthetized by cells of the pineal gland and also peripheral organs such as the skin, gastrointestinal tract, and lymphocytes [44][45][46].Melatonin influences biorhythms, scavenges free radicals, enhances DNA repair, influences the gene expression of anti-oxidative enzymes, and stimulates wound healing properties [47][48][49][50][51]. Sericin as an effective antioxidant exhibits skin protective activity against UVB and UVA radiation-induced damage [52].Chromophores' UVB absorption is predominant, whereas UVA is weakly absorbed by DNA and cellular chromophores with greater ROS/RNS generation, which leads to oxidative changes in the cells [13,53].Sericin is produced by silkworm's glands (e.g., Bombyx mori, Bombyx mandarins, and other species) and has been explored in biomaterial applications because of its biodegradability, anti-oxidative, and antibacterial properties [54][55][56][57][58][59].Furthermore, sericin inhibits tyrosinase activity and promotes cell proliferation, which can be supportive and useful in melanoma treatment [60][61][62][63][64].After surgery, the potential bacterial infections could exacerbate epidermal/dermal damage and delay wound healing.Thus, developing biomaterials for melanoma therapy that reduces the risk of infection, maintaining redox balance in skin cells and preventing cancer recurrence is essential for patient recovery [65][66][67][68][69][70].Sericin as a biomaterial has been used in the preparation of a variety of tissue engineering materials such as composites, hydrogels, membranes, nanofibers, and nanoparticles [71][72][73][74][75].However, poor mechanical strength and high production costs of sericin-based scaffolds make it difficult to introduce this molecule for medical use.Using a biopolymer with improved mechanical properties and with the incorporation sericin and melatonin as active compounds may be highly useful for wound healing in melanoma-affected patients [76][77][78][79][80].

Melatonin and Sericin against the Skin Aging Process
The process of aging has a prominent impact on the skin's healing function, mainly by prolonging the inflammatory phase and increasing ROS/RNS production [81,82].The skin constitutes a protective barrier between external and internal environments and has the sensory capacity to maintain body homeostasis in response to deleterious factors.Skin aging is a natural process with progressive functional and morphological changes, determined by the overall exposure to both intrinsic and extrinsic factors, which may vary depending on skin regions within diverse ethnicities.The clinical signs of skin aging are visible as wrinkles, a rough-textured appearance, a loss of elasticity, and laxity [83,84].
The physiological process of skin aging is characterized primarily by fluctuation changes in endocrine circadian rhythmicity, gene expression, and hormonal descent, which are the reasons for appearance of morphological and functional alterations [85][86][87][88].With increasing age, the occurrence of changes in the endocrine glands, the steroidogenic system, skin cholesterol synthesis, proopiomelanocortin (POMC) expression, and POMC-derived peptides production with the focus on the melanocortin receptor 1 (MC1R) and MC2R agonists, are more frequent and may lead to skin alterations and lesions [89][90][91][92].Vitamin D production, essential to maintain proper skin functions and immunity, also decreases with age [92][93][94][95].
Studies using dermal fibroblasts have shown that silk sericin stimulates collagen synthesis, which also indicates its anti-aging properties [144,145].Results have revealed that silk sericin activates collagen type I synthesis and suppresses oxidative stress, maintaining unaltered fibroblast growth kinetics and cellular structure [146].Next to ROS/RNSscavenging activity, sericin also exhibits anti-tyrosinase and anti-elastase properties.Recent studies have shown that particular sericin strains have an anti-proliferative activity on peripheral blood mononuclear cells; in vitro IFN-γ secretion was decreased, without affecting TNF and IL-10 release.Thus, sericin may be useful for dermatological use [62,147].
The recent inventions using sericin in anti-aging treatments include extract loadedsericin hydrogel as a topical agent [148], naringenin microemulsion-loaded sericin gel [149], and gold silk sericin/niacinamide/signaline complex [150].Extract-loaded sericin hydrogels in six formulations were examined for anti-melanogenesis on the B16F10 melanoma cell line, UVR-preventive properties of human keratinocytes (HaCaT), and anti-aging ef-fectiveness on normal human dermal fibroblasts.The study showed that the hydrogel increased the anthocyanin penetration through the skin.The most promising formulation using the purple waxy corn cob (Zea mays L.) extract demonstrated the highest tyrosinase activity inhibition, melanin pigment reduction, collagenase/elastase inhibition, collagen type I production, and elevation of cell viability.
fectiveness on normal human dermal fibroblasts.The study showed that the hydrogel increased the anthocyanin penetration through the skin.The most promising formulation using the purple waxy corn cob (Zea mays L.) extract demonstrated the highest tyrosinase activity inhibition, melanin pigment reduction, collagenase/elastase inhibition, collagen type I production, and elevation of cell viability.Thus, the purple waxy corn cob extract-loaded sericin hydrogel as a topical agent indicates a great potential in anti-aging products [148].Namely, naringenin microemulsion-loaded sericin gel showed an inhibition of UVR-induced photoaging and increased free radical scavenging.The in vitro cytotoxicity study on skin cancer cells enhanced antiproliferative activity by increasing ROS/RNS in cancer cells with caspase-3 (Casp-3) activation [149].The randomized study with the gold silk sericin/niacinamide/signaline complex have shown the efficacy of daily application in improving the condition of the skin with an antiaging effect [150].
UV light constitutes an important factor in skin aging and disturbances in skin proliferation.UV light up-regulates the nuclear factor kappa B (NF-κB) and releases pro-inflammatory cytokines, with a simultaneous increased generation of ROS/RNS.Next, free Thus, the purple waxy corn cob extract-loaded sericin hydrogel as a topical agent indicates a great potential in anti-aging products [148].Namely, naringenin microemulsionloaded sericin gel showed an inhibition of UVR-induced photoaging and increased free radical scavenging.The in vitro cytotoxicity study on skin cancer cells enhanced antiproliferative activity by increasing ROS/RNS in cancer cells with caspase-3 (Casp-3) activation [149].The randomized study with the gold silk sericin/niacinamide/signaline complex have shown the efficacy of daily application in improving the condition of the skin with an antiaging effect [150].
UV light constitutes an important factor in skin aging and disturbances in skin proliferation.UV light up-regulates the nuclear factor kappa B (NF-κB) and releases pro-inflammatory cytokines, with a simultaneous increased generation of ROS/RNS.Next, free radicals affecting DNA decrease protein tyrosine phosphates and up-regulate matrix metalloproteinase generation, which leads to collagen decomposition [151,152].Although, in healthy conditions urocanic acid, produced in the upper layers in the human skin, constitutes a natural skin UV absorber, excessive exposure to UV light should be avoided due to the harmful effects induced by UV light [153].

Melanoma: A Tumor of Melanocyte Origin
Melanoma, a tumor of melanocyte origin, constitutes one of the most formidable types of skin malignancy [154][155][156] described by local invasiveness, recurrence, early metastasis, and high mortality risk [157][158][159][160][161]. The standard treatment is surgical resection [162][163][164], while alternative therapies like chemotherapy, radiotherapy, or photodynamic therapy are focused on the elimination of the melanoma cells [165,166].Nevertheless, the limitations of the treatment can cause prolonged stress for patients.Insufficient light penetration depth using photodynamic therapy may be a barrier for reaching pigmented lesions [167].Furthermore, the complete tumor resection with residual tumor tissues may lead to major cutaneous defects [168,169].The risk of the wound infection in post-surgical treatment is an emerging issue in the wound healing process.Therefore, it is essential to promote skin regeneration during melanoma treatment [170].Throughout the years, various methods like autologous/allogenic skin grafts or tissue-engineered scaffolds were developed [171][172][173].However, wound healing patches have drawn attention; understanding the requirements of melanoma treatment are indispensable to develop a strategy with integrated wound healing and therapeutic effects.
To better understand the process of melanoma wound healing, it is important to focus on the molecular bases of tumor development and possible ways to impede them [174][175][176].Namely, the risk factors of melanoma induction are: UVR, burns, melanocytic nevi, fair skin or gene mutations (BRAF, NRAS, KIT) [177][178][179][180]. Also in the focus of attention is a tumor suppressor gene cyclin-dependent kinase inhibitor 2A (CDKN2A), and the encoding of p16INK4A and p14ARF proteins as a major genetic risk factor [181,182].In the cell cycle of p16INK4α, the transition from G1 to S phase is regulated and the cycle also acts as a CDK inhibitor, blocking the phosphorylation and inactivation of the Rb protein.However, p14ARF shows anti-proliferation activity, inhibiting the disintegration of the p53 tumor suppressor.During the G2/M phase in normal conditions, the expression of p53 stops the cell cycle or induces apoptosis, which can be a response, for example, to UVR-induced DNA damage [183].
This process can activate the heat shock proteins (HSPs) which protect the cells from physical or environmental stressors [184].In vitro studies have shown that after the exposure to UV light, melanoma cells release the HSP70 which activates anti-melanoma T cells [185][186][187][188]. Thus, HSPs play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and by preventing their aggregation [189].Additionally, HSPs have a protective function; they allow the cells to survive in otherwise lethal conditions.Various mechanisms have been proposed to account for the cytoprotective functions of HSPs.Namely, several of these proteins have been demonstrated to directly interact with components of the cell signaling pathways, for instance those of the tightly regulated caspase-dependent programmed cell death machinery, upstream, downstream and at the mitochondrial level.HSPs can also affect caspase-independent apoptosis-like processes by interacting with apoptogenic factors, such as the apoptosis-inducing factor (AIF) or by acting at the lysosome level [189].Melatonin is known to downregulate the expression of HSP40, HSP70, and HSP90 [32,190,191] to reduce oxidative stress.Thus, melatonin may constitute the additional defense in melanoma [32,[192][193][194][195].The anti-inflammatory properties of melatonin could also be an answer to the NF-κB activation, which is known to be the regulator in oncogenesis.NF-κB activation promotes cell proliferation and inhibits apoptosis and p53 in cancer development.
Recent studies have confirmed that melatonin and its indolic and kynurenic derivatives downstream the pathway of melanogenesis, causing a drop in the cyclic adenosine monophosphate (cAMP) level and the microphthalmia-associated transcription factor (MITF) and causing the resultant collapse in tyrosinase (TYR) activity and melanin content.These findings can be a breakthrough for the future studies on pigmentation in melanoma therapies [122,142,[196][197][198][199].Since the inhibition of melanogenesis in advanced melanomas can serve as an adjuvant strategy in the systemic therapy of melanoma [200], melatonin as a mitochondrial protector with anti-inflammatory properties and direct antioxidants should be explored in topical and transepidermal delivery, especially in skin damage after UVR exposition.It is also known to be very effective in alopecia and atopic dermatitis treatment.It may be also considered as a skin barrier protectant in chronopharmacology [42,45,130].In topical delivery, melanoma treatment using a combination of melatonin/sericin also indicates antioxidant and mitochondrial protection activity which may constitute a potential therapeutic strategy, including in the antibacterial properties of sericin that promote wound healing processes [201,202].We also acknowledge the potential limitation of the accumulation of melatonin in tumor cells, which may increase their resistance to pharmacoor radiotherapy, as discussed recently [203].

State of Innovation in Melatonin-Polymer Wound Healing Patches
Melatonin, a derivative of tryptophan, was initially characterized and isolated by Lerner et al. [204].Melatonin is an indoleamine, which is defined as a indole heterocycle that contains two chains, i.e., 5-methoxy and 3-amide group.The indole structure is rich in electrons and has high resonance stability and reactivity which is the reason for melatonin's free radical scavenging capacity [204].To date, it has been reported that melatonin neutralizes a high number of ROS including hydrogenperoxide (H 2 O 2 ), peroxyl radicals (ROO•), hydroxyl radical (•OH), singlet oxygen ( 1 O 2 ), and RNS, such as peroxynitrite (ONOO − ) or nitric oxide radicals (NO•) [205][206][207][208][209][210][211].The high antioxidant potential of melatonin enables the protection of the cells against oxidative stress more efficiently than other antioxidants.For instance, Figure 2 summarizes the cascade interaction between ROS/RNS and melatonin as well as its metabolites.Thus, melatonin has a capacity to detoxify numerous toxic ROS or RNS, whereas the scavenging ability of other antioxidants is significantly lower [212].

State of Innovation in Sericin-Polymer Wound Healing Patches
Sericin is a "glue-like" protein coiled around the protein core which keeps the fibroin filaments together.This macromolecule constitutes a globular protein built from coil and β-sheets [228].The coil structure for β-sheet can change in response to temperature, mechanical stretching properties, and moisture absorption.In 50-60 • C water solution, the protein acquires its soluble form.At lower temperatures, the coil structure converts into β-sheets resulting in the gel formation [229].Sericin exhibits a hydrophilic character and is composed of 18 amino acids with polar groups such as hydroxyl, carboxyl, and amino groups.The group formation is capable of forming crosslinks, co-polymerizations, and compositions with other polymers [230].The significant structure of sericin defines its biological properties that includes anti-bacterial activity, antioxidant, and biocompatibility [231].
The antioxidant and photoprotective potential of sericin against UVB in human epidermal keratinocytes was confirmed using the flow cytometry assessment [232].It was revealed that treatment with sericin significantly attenuated apoptosis by inhibiting the expression of pro-apoptotic proteins and upregulation of the anti-apoptotic Bcl-2 proteins family, as well as preventing Casp-3 activation [232].The role of sericin in preventing mitochondrial damage was also confirmed by the inhibition of hydrogen peroxide formation.As a consequence, the intracellular ROS/RNS and activation of poly-ADP-ribose polymerase enzyme (PARP) were distinctly reduced.Studies have shown that sericin is a potent antioxidant and anti-apoptotic agent [232,233].Additionally, the antioxidant properties of sericin are related to high serine and threonine content, whereas hydroxyl groups act as a chelator of trace elements such as copper and iron [234,235].
The studies have shown that nanofiber (NF) wound dressing loaded with 20% melatonin (NF + 20% MEL) indicated the highest mRNA expression of collagen type 1 (COL1A1) on the 14th day of treatment compared to Comfeel Plus, NF + 10% MEL, nanofiber dressing, and the non-treated group.Thus, NF + 20% MEL electrospun wound dressing could be used as an effective matrix for accelerating the wound healing process reducing the costs of wound repair [225].
Another study has shown that the melatonin-loaded hydrogel significantly increased the percent of effectiveness of wound closure, promoted tissue granulation and re-epithelialization, leading to accelerated collagen deposition, when compared to the control group and the hydrogel with no melatonin groups.Therefore, it is essential to conduct further research using melatonin in wound healing patches [223].

Conclusions, Challenges and Future Perspectives
Sericin-polymer patches have a variety of applications such as wound treatment [237,238], including acute [237] and infected large burn wounds [236], artificial skin [242], wound dressings [240,241], tissue engineering [247], regenerative scaffolds [246], and periodontal tissue engineering [251], whereas the main applications of melatonin-polymer patches in years 2021-2023 were wound healing and wound dressings [222][223][224][225]; they were also used in diabetic wound repair [215], skin delivery [218], and skin tissue engineering [221].During this period, melatonin-polymer patches were also used in cartilage repair, vital pump regeneration, tendon regeneration, membranes, nerve tissue engineer- The elevating secretion of pro-inflammatory cytokines, increased cell proliferation and oxidation processes can damage cell membrane integrity and lead to DNA fragmentation [7,27].Although UVR is one of the major melanoma risk factors it is also essential for initiating vitamin D synthesis, while active forms of vitamin D also have photoprotective properties [266][267][268].The topical treatment of large or non-resectable melanoma lesions including lentigo maligna using a combination of melatonin and sericin also shows that mitochondrial and antioxidant protection activity may lead to the development of a valuable additional strategy targeting melanoma, regarding the antibacterial properties of sericin that accelerate wound healing [269,270].

Conclusions, Challenges and Future Perspectives
Sericin-polymer patches have a variety of applications such as wound treatment [237,238], including acute [237] and infected large burn wounds [236], artificial skin [242], wound dressings [240,241], tissue engineering [247], regenerative scaffolds [246], and periodontal tissue engineering [251], whereas the main applications of melatonin-polymer patches in years 2021-2023 were wound healing and wound dressings [222][223][224][225]; they were also used in diabetic wound repair [215], skin delivery [218], and skin tissue engineering [221].During this period, melatonin-polymer patches were also used in cartilage repair, vital pump regeneration, tendon regeneration, membranes, nerve tissue engineering, skin tissue regeneration, and osteosarcoma treatment [213,216,217,220,226,227]. Research has shown that both melatonin and sericin exhibit high antioxidant potential that is beneficial in melanoma treatment [271].Melatonin/sericin-polymer patches have not been exploited for their application in post-melanoma wound healing treatment; this is the field that would likely benefit from further investigation.

Figure 2 .
Figure 2. The cascade reaction with ROS/RNS products (or metabolites) of melatonin which are involved in terms of attenuation of UVR-induced changes as described above and presented in Figure 1.

Figure 2 .
Figure 2. The cascade reaction with ROS/RNS products (or metabolites) of melatonin which are involved in terms of attenuation of UVR-induced changes as described above and presented in Figure 1.

Figure 3 .
Figure 3. Melatonin/sericin-loaded matrix systems and their potential impact on skin-related applications.

Figure 3 .
Figure 3. Melatonin/sericin-loaded matrix systems and their potential impact on skin-related applications.

Table 1 .
State of innovation in melatonin-polymer patches.

Table 1 .
State of innovation in melatonin-polymer patches.

Table 2 .
State of innovation in sericin-polymer patches.