Pathogenesis of Keratinocyte Carcinomas and the Therapeutic Potential of Medicinal Plants and Phytochemicals

Keratinocyte carcinoma (KC) is a form of skin cancer that develops in keratinocytes, which are the predominant cells present in the epidermis layer of the skin. Keratinocyte carcinoma comprises two sub-types, namely basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). This review provides a holistic literature assessment of the origin, diagnosis methods, contributing factors, and current topical treatments of KC. Additionally, it explores the increase in KC cases that occurred globally over the past ten years. One of the principal concepts highlighted in this article is the adverse effects linked to conventional treatment methods of KC and how novel treatment strategies that combine phytochemistry and transdermal drug delivery systems offer an alternative approach for treatment. However, more in vitro and in vivo studies are required to fully assess the efficacy, mechanism of action, and safety profile of these phytochemical based transdermal chemotherapeutics.


Introduction
Cutaneous carcinoma, or skin cancer, remains one of the highest occurring cancer types, with the number of incidences increasing globally. Although the number of cases differs significantly depending on the geographical region, it remains a major health care problem across the world, as it affects both men and women of every ethnicity/race [1].
There are two criteria that classify skin cancer; the cell from which they originate and their clinical behavior. Skin cancer is categorized into two major groups: non-melanoma skin cancer (NMSC) and malignant melanoma. The cases of non-melanoma skin cancer are generally substantially higher than melanoma cases and are often much less complex to treat compared to melanoma, as it has a lower metastatic potential [2]. Non-melanoma skin cancer, consisting of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), is often referred to as keratinocyte carcinoma, as these two subtypes are derived from the epidermal keratinocytes [3].

Methodology
A scientific literature search was performed during 2018-2020, using several databases (Science Direct, Google Scholar, Scopus, and Pubmed). The literature search focused on the following topics; KC, BCC, and SCC. Review articles linked to these topics contributed extensively to the structure of the current review. To understand the severity of KC and the increase in cases and mortality, various research papers, and cancer registries were analyzed. Previous research papers that reported on in vitro and in vivo studies regarding Figure 1. Estimated age-standardized world-wide incidence rates of non-melanoma skin cancer in 2020. Reproduced from [22]. Copyright 2021, International Agency for Research on Cancer 2021, World Health Organization.
Basal cell carcinoma is the world's most prevalent human cancer and is accountable for approximately 70-80% of all skin cancers [23,24]. A report by Dessinioti et al. revealed that Australia had the highest incidence of BCC (~1-2%) annually, with approximately 2448 of a 100,000 population diagnosed in 2011, followed by the US (450 per 100,000 in 2010) and Europe (220.1 per 100,000 yearly average) [25,26]. The South African National Cancer Registry reported approximately 14,414 BCC and 6950 SCC cases in 2016 [27]. Another study found that the lowest incidence rates of BCC were in Finland, in comparison to other European countries [28]. This trend is followed closely by Italy, with 88 out of 100,000 people diagnosed with BCC [29]. Squamous cell carcinoma has been reported to contribute approximately 20% of skin cancer cases [30]. Numerous reports indicate a significant incline in SCC incidence rates over the past three decades, with an approximately yearly increase of 3-10% [26]. Currently, SCC incidence rate is estimated to be between 15-35/100,000 people per year and is set to increase between 2-4% annually, due to chronic ultraviolet B (UVB) exposure and an aging population [31].

Diagnosis of Keratinocyte Carcinoma
The early diagnosis of KC, such as SCC, is crucial in controlling the risk of cancer becoming invasive and assists in minimizing the complexity of treatment plans. Approximately 70% of all BCCs are found on the head and neck, which can have detrimental effects such as facial deformities and the collapse of certain vital structures [32].
Dermatoscopy, an in vivo imaging diagnostic technique, is used to examine pigmented skin lesions under high magnification in order to distinguish between pigmented BCC and melanoma. Computer tomography or magnetic resonance imaging are used to determine the extent of abnormal cell tissue growth in cartilage, bone, large nerve, eyeball, or parotid gland, whereas skin biopsy aids in identifying KC subtypes and subsequent treatment plans [33]. Lesions that appear clinically abnormal can be tested via biopsy or excision. Abnormal lesions can be examined using incisional biopsy (incision, punch, or shave biopsy), where a section of tissue is removed, or excisional biopsy, where the entire lesion or tissue is removed. Appropriate treatment and prognosis is therefore based on tumor differentiation grade, histologic subtype, degree of dermal invasion, tumor depth, presence or absence of perineural, lymphatic, or vascular invasion, and extension of tumor cells to margins [34,35]. A tumor staging system, such as the Cancer Staging Manual, published by the American Joint Committee on Cancer (AJCC), is used to determine the prognosis and treatment plant based on the location and size of the tumor, whether it has spread to the lymph nodes, and the extent to which it has spread to other parts of the body [34]. There are two types of staging systems, the number staging system (Table 1) or the TNM (tumor, node, and metastasis) staging system. A full description of the TNM staging system has been described by Fahradyan et al. [35]. Table 1. The number staging system of keratinocyte carcinoma [34].

0
In situ carcinoma, cancer has developed but has not spread or grown into surrounding tissue 1 Tumor ≤2 cm in size, with less than two high-risk features 2 Tumor >2 cm in size, with two or more high-risk features 3 Tumor with invasion of the maxilla, mandible, orbit, or temporal bone or the tumor has spread to nearby lymph nodes (<3 cm in size) 4 Tumor with invasion of skeleton or perineural invasion of skull base or the tumor has spread to lymph nodes (>3 cm in size) or an internal organ High-risk features include >2 mm thick, cells are poorly differentiated or undifferentiated, has grown into the dermis, perineural invasion (space around a nerve), primary site non-hair-bearing lip, started to develop on the ear or lip.

Clinical Variants of BCC
Basal cell carcinoma accounts for approximately 70% of KC cases and most commonly develops on sun-exposed skin, predominantly on the head, but can also develop on the neck, trunk, and lower extremities. It is defined as a slow growing tumor, which rarely metastasizes but can cause facial deformities if left untreated. A characteristic feature of BCC is the formation of island or nests of basaloid cells found in the epidermis, which can invade the dermis depending on the variant of BCC [36,37]. There are numerous subtypes of BCC; however, several main variants are summarized in Table 2.

Actinic Keratosis as Precursor Lesions of SCC
Actinic keratosis (AK) is a principal precursor lesion for the formation of SCC. It is often found on parts of the body that are exposed to solar UV radiation such as the forearms, back, scalp, upper chest, face, neck, and back of the hands. Due to the correlation between the development of AK and exposure to UV radiation, it is often found in middleaged people and the elderly. It can be clinically identified by poorly formed borders, flaky erythroderma, and uneven papules or patches. It is also found on surface areas of the body that exhibit various pre-existing impairments such as uneven pigmentation, telangiectasias, and atrophy. The formation of AK can lead to the development of invasive SCC; however, it can also spontaneously regress or remain a benign AK lesion. Although most cases of SCC have been linked to AK, it has been reported that only 5-10% of AK lesions develop into invasive SCC. Histologically, actinic keratosis is the exponential growth of abnormal keratinocyte cells, which predominately occurs in the lower layers of the epidermis [38]. Additionally, these atypical keratinocytes display an assembly of distinct characteristics such as pleomorphic and hyperchromatic nuclei, polarity deficiency, cell size enlargement, and mitosis enhancement. Several subtypes of AK have been identified that exhibit a broad range of histologic patterns (Table 3) [38,39].
Reports indicate that the rate of progression from AK to SCC ranges from approximately 12% to 20% [40]. A study by Sahin et al. assessed the degree of AK advancement and its link to SCC formation. In this study, evaluations were performed on 115 lesions from 82 patients diagnosed with AK, over a period of eight years, in which the percentage of male patients was 51% and female patients was 48%. This study revealed that the highest percentage of AKs were located on the nose (30.4%), followed by the face (23.5%), lips (8.7%), and ears (7.8%) [41]. Furthermore, when comparing AK localization in males Molecules 2021, 26,1979 6 of 29 and females, it was observed that AKs frequently occur on the nose of males, whereas in females, they occur on the facial skin. When comparing the subtypes of AK, research indicates that proliferative AK has the highest rate of occurrence at 29.6%. Additionally, acantholytic AKs display a 4% progression rate to SCC [41].  Squamous cell carcinoma in situ (SCCIS) can be viewed as a fundamental transitional phase from AK to invasive SCC. Currently, the predominant trend in oncology research is the synonymity that exists between Bowen's disease and SCCIS, exclusively for lesions occurring on non-genital areas [38,39].
Squamous cell carcinoma in situ, which is also known as Bowen's disease, can be histologically identified by the following characteristics: atypia, which extends through the complete thickness of the epidermis, excluding the adnexal structures, and hyperparakeratosis, which can either be nominal or extremely abundant, which gives rise to a cutaneous horn. Atypical keratinocytes exhibit apoptosis, hyperchromasia, nuclear pleomorphism, as well as a "windblown" appearance when polarity is lost. It is generally defined as a skin disease that does not possess the ability to invade the dermis layer of the skin. Squamous cell carcinoma in situ can develop on any epidermal body site; however, studies indicate that approximately 72% of SCCIS cases occur on sun-exposed skin surfaces, namely the hands, neck, and head. It is therefore often diagnosed in elderly people aged over 60 years old and is rarely diagnosed in individuals under the age of 30. Other areas in which SCCIS can be found include the nail bed, soles of the feet, and palms of the hand. Squamous cell carcinoma in situ can be clinically distinguished by the following features; presence of a distinct plaque or scaly patch, which is benign and displays erythema. Lesions may develop unfavorable properties which include crustation, fissures, hyperkeratosis, and ulcerations. Reports indicate that the risk of SCCIS progressing to invasive SCC is approxi-Molecules 2021, 26,1979 8 of 29 mately 3-5%. Statistics further revealed that 20% of tumors that evolve to invasive SCC will ultimately metastasize [38,39].

Invasive Squamous Cell Carcinoma (SCCI)
Invasive squamous cell carcinoma is often referred to as standard SCC. Histologically, it is defined as the vertical invasion of abnormal cells, which originates at the basement membrane and advances into the dermis. It has been reported that approximately 97% of SCCI cases are linked to the malignant progression of AK [50]. There are several variants of SCC (Table 4).  Well-differentiated simplex SCC, in close proximity to an ulcer or scar • Develops on areas of the skin exposed to long-term disease or injury, commonly in lower extremities [58,59]

Recurrent and Metastatic KC
Tumor recurrence and incomplete excision of the primary tumor are major risk factors that can increase the possibility of developing metastatic SCC. Studies have revealed that aggressive tumors are susceptible to recurrence and are accountable for approximately 25-30% of SCC metastasis [60]. In a study by Clayman et al., 130 patients, which exhibited advanced or aggressive SCC tumors, displayed a recurrence of 27.5% [61]. Leading characteristics of recurrent tumors include large tumor size, invasion of lympho-vascular or perineural system, and tumor infiltration into subcutaneous tissue [62]. A study that comprised of 603 patients with cutaneous SCC revealed that 89% of patients died from distant metastasis, which is consistent with current SCC metastatic reports [63]. Key features that are linked to metastasis include cancerous cells that disseminate via the lymphatic, which are responsible for 80% of metastases [64], and the fact that the vast majority of metastatic SCC cases occurred on the head and neck of patients [60]. A report by Lazarus et al. presented a study in which 6900 patients had SCC; however, only 142 patients exhibited metastatic lesions. An evaluation of these lesions revealed that lymph nodes are the primary site for metastases occurrence (Table 5) [65]. On the contrary, metastatic BCC cases are rare, with a percentage rate of approximately 0.0028-0.55% [66]. Metastatic BCC follows the same trend as SCC, where 85% of cases originate from tumors that are located in the head and neck region of the patient. Moreover, statistics reveal that a minimum of two-thirds of metastasis cases arise from tumors that are exclusively situated on the face. Tumors that exhibit a specific set of characteristics can be categorized as having a high metastatic potential, such as tumors located in the mid-face or ear, a tumor that has been present for a long period of time, tumor diameter size (>2 cm), and previous radiation treatment [67]. A study by Freitas et al. evaluated 25 BCC cases between the time period of January 2012 and March 2017 and concluded that metastases occur most frequently in lymph nodes (Table 5).

Risk Factors Associated with the Development of KC
There are various environmental and biological factors that contribute to the development of KC, which are divided into external and internal factors. Identification of high-risk patients allows for early diagnosis and an efficient treatment regime to be established.

Solar UV Radiation
The main external risk factor for developing KC is exposure to solar UV radiation [68]. However, specific patterns of UV radiation exposure result in development of various types of KC. The development of SCC can be linked to long-term sun exposure, whereas BCC formation is associated with excessive sun exposure that transpired in early life as well as intermittent exposure [69]. Furthermore, Kim et al. highlighted that 90% of KC cases are linked to high levels of UV radiation exposure [70]. Solar UV radiation can be divided into three categories, according to a difference in wavelength, namely UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm) [71]. A study conducted by Grossman et al. showed that solar UV radiation induces KC development through DNA damage and immunosuppression [72]. Previously, UVA was reported to primarily be responsible for skin aging; however, recently, UVA has been coupled with UVB, where both are implicated in the development of cutaneous skin cancer. There are different mechanisms by which UVA and UVB cause DNA damage. UVB is known to play a greater role in KC development, due to wavelength penetration depth. UVB radiation is absorbed by cellular components that are present in the epidermis, whereas UVA radiation infiltrates into the basal layer of the epidermis and dermal fibroblasts [73,74]. A study conducted by Boukamp [75], confirmed the findings by Grossman et al. [72] with regards to UVB being a major contributing factor for KC development [75]. This study revealed that the formation of photoproducts, which damage the DNA present in keratinocytes, occurs due to long-term UVB exposure [75]. One of the major photoproducts that form is cyclobutene pyrimidine dimer (CPD). CPDs form thymine dimers (T/T) and pyrimidine-pyrimidone lesions (6-4PP), which, if unrepaired, are mutagenic. This specific type of DNA damage can be repaired by the nucleotide excision repair mechanism (NER); however, malfunction of this mechanism can result in multifocal skin cancer [76]. Additionally, UV radiation has been reported to cause mutations in the suppressor gene existing in the p53 protein.
The central function of the p53 tumor suppressor gene is to encode for a protein, which regulates the cell cycle and induces apoptosis [76]. Previous studies have indicated that mutations present in the p53 gene are associated with numerous human cancers and are most prevalent in KCs [77]. According to Kooy et al., UV radiation is able to induce immunosuppression by depleting epidermal dendritic Langerhans cells (CD1a+). As a result, T helper type-1 converts to T helper type-2 response, which inhibits the ability of cell-containing-antigens to induce antitumor immunity [78].

Indoor Tanning
Artificial sources of UV radiation have been categorized as cancer causing agents by the International Agency for Research on Cancer (IARC) in the year 2012 [79]. Indoor tanning generates similar adverse effects in human skin as exposure to solar UVB radiation; however, reports indicate that indoor tanning is 10-15 times stronger than exposure to solar UVA radiation [80]. A review conducted by Wehner et al. investigated approximately 9300 cases of keratinocyte carcinoma and was able to correlate the development of KC to indoor tanning. This report revealed a 67% higher risk of developing SCC and a 29% higher risk for BCC development when exposed to indoor tanning. Studies indicate that exposure to indoor tanning between 16-25 years leads to a greater risk of developing BCC [81].

Ionizing Radiation
The association between exposure to ionizing radiation and skin cancer formation was discovered by radiologists that were working with X-ray analysis. Furthermore, SCC development was observed in skin locations that exhibited dermatitis and ulceration, which was caused due to high-level ionizing radiation exposure, whereas formation of BCC was seen in low to moderate exposure to ionizing radiation [82]. Ionizing radiation is used as a form of treatment for various types of cancers; however, this is often associated with the development of radiation dermatitis, which occurs in approximately 95% of patients receiving radiation therapy. This is due to damage caused to the basal keratinocytes and hair follicle stem cells, which is followed by double-stranded DNA breaks and inflammation caused by the production of reactive oxygen species [83].

Arsenic Exposure
Studies indicate that the two leading characteristics that arsenic exhibits are its ability to act as a toxin and as a carcinogen. The mechanism of action is to target and negatively alter the cellular processes within various organ systems based on a dose and time-dependent manner. The toxic effects of arsenic are first displayed in the skin, which can include the development of BCC and SCC. Development of SCC from arsenic exposure is said to be more aggressive in nature when compared to chronic UV-induced SCC. Statistics reveal that 33% of untreated arsenic-induced SCC demonstrates metastatic behavior [84].

Age
Studies have shown that the development of KC is more prevalent in elderly patients with a median age diagnosis of 70 years and older [85]. There are several factors that potentially contribute to the frequent occurrence of KC in the elderly, namely prolonged exposure to solar UV radiation and repair mechanisms of the cell that are not functioning at optimum levels [86]. Transformations that occur in the immune system of elderly patients result in immune suppression, which can lead to opportunistic disease development, of which malignancies are frequently observed [87].

Skin Type
Skin type and pigmentation is a crucial factor regarding the development of KC. Skin type 1 is represented by people who have fair skin, light blue, and grey eyes, with light red and blonde hair, and are more susceptible to KC formation. The number of KC cases occur more frequently in fair-skinned individuals as compared to dark-skinned individuals. This is due to low levels of melanin present in the skin. Melanin, which is responsible for skin pigmentation, has photoprotective properties. It protects the skin by acting as a physical barrier to UV radiation and is furthermore able to absorb UV radiation, thereby limiting the amount of UV that penetrates the skin. Melanin is produced in the melanosomes, which is translocated to adjacent keratinocytes via dendrites [88,89].

Immunosuppression
Immunosuppression is a significant contributing factor for the development of KC, more specifically SCC. The development of KC occurs more frequently in patients that exhibit immunodeficiency, which includes patients that use immunosuppressive drug therapy in cases such as solid-organ transplantation, auto-immune inflammatory diseases, and human immunodeficiency virus (HIV) infection. Patients who have received organ transplants have an increased risk (20-200-fold) of developing SCC. A study revealed the incidence ratio of SCC caused by solid-organ transplantation to be 1355/100,000, whereas the incidence ratio of SCC in the general population was 38/100,000 [90].

Topical Pharmacotherapies Currently Used for the Treatment of KC
Topical chemotherapy is a form of non-surgical therapy used to remove or eliminate localized skin cancer cells. There are several topical treatments available (Table 6), which can induce cell death through the direct damage to DNA/RNA, such as 5-fluorouracil, or act as immune-modulators, such as imiquimod, which stimulates the production of various cytokines, thereby inducing antitumor activity. Topical treatments are often a consideration when patients are elderly or unhealthy; therefore, surgery may not be an option, or for individuals who have tumors/lesions on areas that cosmetically sensitive, and therefore surgery may result in a disfiguring scar [91].

Antiproliferative activity and induction of apoptosis in basaliomatous cells
Effective against undifferentiated BCC tumors, however not effective against keratotic BCCs (overexpression of p53 and cellular retinol binding protein-1) Mild erythema, edema, and local skin irritation [114][115][116]

Transdermal Delivery of Drugs
A fundamental approach used to enhance bioavailability of pharmaceutical drugs is developing novel drug delivery systems. Although oral delivery systems are preferred for pharmaceutical drug administration, these systems are linked to several disadvantages such as insufficient drug stability within the gastrointestinal tract, reduced drug concentration upon reaching its site of action due to metabolism, and decreased drug solubility in intestinal fluid resulting in poor permeability through the intestinal membrane [117]. Due to the large surface area and accessibility of the skin, extensive investigations of drug delivery via the skin have been conducted. Administration of drugs through the skin can result in the drug being confined within the skin (topical application) or the drug can penetrate through the skin, allowing it to reach the blood circulatory system (transdermal delivery). There are numerous advantages associated with transdermal drug delivery when compared to conventional drug administration routes, these include; non-invasiveness, increased patient compliance, enhanced drug bioavailability, more cost effective and decreased drug plasma fluctuations [118]. However, it is imperative to consider physiological and physiochemical factors, as these factors influence a drug's movement through skin. A drug's absorption rate is primarily controlled by skin age, moisture contents, and anatomical location. Aged skin has a decreased moisture contents, resulting in reduced and slower absorption when compared to younger skin. An increase in temperature leads to an enhanced absorption rate, whereas a reduction in blood flow results in a drug's influx being negatively impacted [119]. An ideal transdermal drug should possess characteristics such as a short half-life, low molecular weight (less than 1000 Da), bio-compatible, low melting point, and an affinity for both hydrophilic and lipophilic phases [120].

Targeted Delivery Through the Skin
The main objective of drugs that exert their pharmaceutical effect topically is to target a multitude of sites that exist in different skin layers, skin appendages, and underlying tissue. Studies indicate that the systemic circulatory system is predominately targeted by transdermal drug compounds; moreover, the anatomical structures of interest are hair follicles, nerves, Langerhans cells, keratinocytes, and melanocytes within the epidermis. It is imperative that drug candidates for transdermal delivery include the following characteristics: limited sites for hydrogen bonding, have a low molecular weight, average lipophilicity, and a low melting point [121]. The permeation of a drug occurs through the stratum corneum and can be calculated by Fick's second law: where J represents the transport flux, Dm is the diffusion coefficient of the drug present in the membrane, Cv represents the drug concentration that exists in the vehicle, P is the drug partition coefficient, and L represents the thickness of the stratum corneum [122].

Transdermal Drug Permeation Routes
Administration of drugs through the skin can follow two potential routes, transepidermal and trans-appendegeal ( Figure 2) [117]. The trans-epidermal pathway allows for molecules to pass through the stratum corneum and can be further sub-divided into intracellular and intercellular pathways [123]. The intracellular route depicts a pathway that permits the transport of hydrophilic or polar solutes through differentiated keratinocytes known as corneocytes, whereas the intercellular route allows the transport of lipophilic or non-polar solutes via intercellular spaces in the lipid matrix. The second transdermal drug route (trans-appendageal route) represents a pathway that allows the permeation of molecules via hair follicles that are associated with sebaceous glands as well as sweat glands [123]. This route is suitable for ions and large polar molecules, which experience difficulty in stratum corneum permeation [124].

Transdermal Delivery of Skin Cancer Drugs
The majority of chemotherapeutic drugs are distributed using the systemic circulatory system and are capable of generating cytotoxic effects on healthy cells. Therefore, transdermal drug delivery of anticancer compounds can be viewed as an attractive alternative, due to the two essential advantages of transdermal drug delivery systems, namely increased therapeutic benefit and enhanced drug targeting. However, there are several challenges that have been linked to this treatment method, including increased concentration, lack of bioavailability, and penetration of drug compounds possessing antineoplastic properties [125,126]. Due to the advancements in technology, anticancer macromolecules, inclusive of proteins and nucleic acids, that experience difficulty penetrating the stratum corneum can be delivered with the assistance of penetration enhancers, physical enhancement devices, and micro-carriers [127].
Studies have reported on two types of penetration enhancers, namely chemical and biological. Chemical penetration enhancers can be described as compounds that facilitate increased drug penetration through the skin, such as alcohol, terpenes, esters, fatty acids, polyols, and surfactants. Reports on various mechanisms of action for this type of enhancer include facilitating disturbances within the stratum corneum, intercellular protein interaction, and enhanced drug segmentation within the stratum corneum [128]. Biological enhancers are peptide-based and are capable of transporting an array of compounds, such as nucleic acids, proteins, polymers, and nanoparticles, throughout the skin with minimal toxicity. Physical enhancement devices can be characterized by the implementation of electric fields such as electroporation, iontophoresis, and sonophoresis [127]. Further literature explained that the fundamental purpose of these techniques is based on the ability to exert a momentary and reversible disintegration of the stratum corneum, which results in enhanced permeation of an antineoplastic drug at a tumor site [129].
In recent times, micro-nanocarrier systems that include inorganic nanoparticles, nano-emulsions, dendritic nanocarriers, and liposomes have attracted increased attention in the field of transdermal drug delivery, as they offer various advantages, such as increased skin penetration, enhanced solubility, and implementation of controlled release [127]. Within the micro-nanocarrier system, there exist several elements that greatly contribute to skin permeability, such as charge, shape, and size of nanomaterials [130].
This review explored several transdermal drug delivery and micro-nanocarrier systems for their application in skin disorders and skin cancer (Table 7).

Transdermal Delivery of Skin Cancer Drugs
The majority of chemotherapeutic drugs are distributed using the systemic circulatory system and are capable of generating cytotoxic effects on healthy cells. Therefore, transdermal drug delivery of anticancer compounds can be viewed as an attractive alternative, due to the two essential advantages of transdermal drug delivery systems, namely increased therapeutic benefit and enhanced drug targeting. However, there are several challenges that have been linked to this treatment method, including increased concentration, lack of bioavailability, and penetration of drug compounds possessing antineoplastic properties [125,126]. Due to the advancements in technology, anticancer macromolecules, inclusive of proteins and nucleic acids, that experience difficulty penetrating the stratum corneum can be delivered with the assistance of penetration enhancers, physical enhancement devices, and micro-carriers [127].
Studies have reported on two types of penetration enhancers, namely chemical and biological. Chemical penetration enhancers can be described as compounds that facilitate increased drug penetration through the skin, such as alcohol, terpenes, esters, fatty acids, polyols, and surfactants. Reports on various mechanisms of action for this type of enhancer include facilitating disturbances within the stratum corneum, intercellular protein interaction, and enhanced drug segmentation within the stratum corneum [128]. Biological enhancers are peptide-based and are capable of transporting an array of compounds, such as nucleic acids, proteins, polymers, and nanoparticles, throughout the skin with minimal toxicity. Physical enhancement devices can be characterized by the implementation of electric fields such as electroporation, iontophoresis, and sonophoresis [127]. Further literature explained that the fundamental purpose of these techniques is based on the ability to exert a momentary and reversible disintegration of the stratum corneum, which results in enhanced permeation of an antineoplastic drug at a tumor site [129].
In recent times, micro-nanocarrier systems that include inorganic nanoparticles, nanoemulsions, dendritic nanocarriers, and liposomes have attracted increased attention in the field of transdermal drug delivery, as they offer various advantages, such as increased skin penetration, enhanced solubility, and implementation of controlled release [127]. Within the micro-nanocarrier system, there exist several elements that greatly contribute to skin permeability, such as charge, shape, and size of nanomaterials [130].
This review explored several transdermal drug delivery and micro-nanocarrier systems for their application in skin disorders and skin cancer (Table 7). Table 7. Transdermal drug delivery and micro-nanocarrier systems in skin disorders and skin cancer.

Nano-Carrier Study Outcome Reference
Liposomes Synthesized elastic liposomes loaded with 5-fluorouracil (5-FU) investigated (in vitro and in vivo) for drug permeation enhancement across the stratum corneum of the skin.

Potential Therapeutic Effects of Phytochemical/Medicinal Plants against KC
Due to the considerable incline of KC cases worldwide, researchers have a vested interest in developing novel treatment options [139]. The fundamental purpose of an anticancer therapeutic strategy is the ability to selectivity target malignant tumor cells, that either inhibits their growth or induces cell death [140]. However, the vast majority of KC treatments that are currently available, also have a detrimental effect on non-tumors cells. In an effort to create cutting-edge, more effective and non-toxic anticancer treatments, researchers have explored the possibility of isolating compounds from natural sources, more specifically phytochemicals [141].
Studies have reported that phytochemicals and medicinal plants exhibit potential anticancer properties. From the year 1940 to 2014, approximately 50% of approved anticancer therapeutic agents were either obtained or derived from natural sources [142]. Several medicinal plants, and their bioactive compounds, have been investigated for potential anticancer activity. These have been tested against skin tumors, in both in vitro and in vivo studies, and have exhibited noteworthy activity by arresting the development and proliferation of skin tumor cells [143]. Phytochemicals have the potential to alter various molecular processes associated with the development of skin cancer and subsequently inhibit tumor proliferation [144]. Some of the reported studies on medicinal plants and phytochemicals have been summarized in Table 8. Additionally, emerging KC treatments that are currently in the clinical trial phase have been summarized in Table 9. Hypericin directly injected into affected tissue, 3-5 times weekly (SCC (40-100 µg) and BCC (40-200 µg)), showed no necrosis of surrounding tissue and was successful as a targeted delivery system Combination of hypericin and PDT resulted in pain and burning [145,146] Effect of 0.07% hypericin on BCC, AK, and Bowen's disease, followed by irradiation (weekly, for 6 weeks) All patients experiences pain and burning after irradiation, 50% complete clinical remission of AKs, 11% histological clearance of sBCC, and 80% histological clearance of Bowen's disease [147] Mice injected with SCC cells to develop tumors (3-15 mm diameter) were injected with 10 µL of DMSO containing 10 µg hypericin per gram of tumor and irradiated after 24 h Hypericin retained in tumors for a prolonged period of time was observed to be more effective in small sized tumors (<400 mm 3 ), whereas larger tumor displayed partial ablation followed by recurrence [148] Black salve (escharotic agent) Sanguinaria canadensis L. (bloodroot) Ointment containing 300 mg bloodroot, galangal, sheep sorrel, and red clover resolved suspected melanoma neoplasm of the left naris (63 year old male) Complete loss of the left naris and severe tissue damage [149] Application on BCC located on the nasal cavity (83 year old male) Complete loss of nasal ala [150] Application of black and "yellow" salve on micronodular BCC located on right nasal sidewall (65 year old female) Patient discontinued use due to pain and tenderness, formation of 12 mm ulceration with eschar formation. Secondary intention healing treated the wound [151] Application on 5 mm BCC lesion (51 year old male) Agonizing pain and formation of large eschar and formation of scar. Biopsy after 12-months showed no presence of BCC [152] Application on BCC located in the right-hand side of the neck (49 year old male) Development of triangular keloidal scar that had to be surgically removed and repaired. After reconstruction, no tumor was identified [153] Application to SCC on the right lower leg (55 year old woman) Formation of a thick escharotic plaque, which dislodged revealing normal granulation tissue. Histological examination revealed no residual SCC  Effect on B16 murine melanoma cells Induced apoptosis in melanoma cells through ERK1/2 signaling attenuation, upregulation of Bax, and down-regulation of Bcl-2 [158,159] Effect in normal human keratinocytes (NHK) after exposure to UVB radiation Enhanced survival rate of NHK through inhibition of the mitochondrial intrinsic apoptotic pathway and inhibition of inflammatory mediators IL-1α and prostaglandin-E2. However, did not inhibit malignant keratinocytes [160] Resveratrol-RV (polyphenol) Commonly found in Vitis vinifera L.   Betulin-based Oleogel-S10 Pentacyclic triterpenes isolated from Betula pubescens Ehrh.
Application to patients with actinic keratosis (157 patients) • Once and twice a day application resulted in complete tumor clearance in 3.9% and 6.8% of patients, respectively • Not significant clearance when compared to placebo [175] Molecules 2021, 26, 1979 22 of 29 Often, public perception is that herbal or medicinal plant treatments are deemed safe and effective, as is the case with the use of black salve. Black salve has not undergone any clinical trials in order to evaluate its safety or efficacy against skin cancer and is available to purchase over-the-counter or online. A review by Lim summarizes numerous case reports on the use of black salve for skin neoplasms; however, almost each of the cases reported side effects that included severe tissue necrosis and damage as well as scarring ulceration [176]. A major alkaloid compound present within bloodroot is sanguinarine, which has been linked to the potential carcinogenic effect of bloodroot; however, due to contradictory in vitro and in vivo results, the carcinogenic classification of sanguinarine has not yet been established [177].
Furthermore, there is a lack of quality control and quantification or standardization of the active ingredients within these "home" remedies. Black salve has been largely linked to the occurrence of facial deformities, which needs to be corrected through cosmetic reconstruction, which emphasizes the need for the correct safety and efficacy trials to be conducted before these types of remedies are made available on the market for use.
Similarly, in a pilot trial, betulin oleogel-S10 was initially shown to be effective in treating AK, with a clearance rate of 64%; however, this was a small trial and did not include the use of a placebo control [178]. Consequently, a larger study on the use of betulin oleogel-S10 was conducted by Pflugfelder et al. [175], including a placebo control group, which concluded that the topical use of betulin-oleaogel-S10 did not significantly clear AK when compared to the placebo group.
Camptothecin, an alkaloid isolated from Camptotheca acuminata Decne. is a cytotoxic compound that inhibits DNA topoisomerase I [179]; however, due to its insolubility in biocompatible solvents, it remains a challenge to effectively administer it intravenously. However, in a study by Lin et al. [132], α-MSH liposomes that encapsulate camptothecin showed an enhanced antiproliferative activity against melanoma cells, when compared to camptothecin alone, due to the targeted delivery and controlled release of camptothecin, which emphasizes the importance of using appropriate drug delivery systems. Similarly, a study by Kessels et al. [171] showed that a 10% sinecatechin ointment, which contained EGCG, was not effective in treating superficial BCC; however, it was concluded that this may be due to a lack of EGCG uptake by the cancerous cells and that using liposomes as a drug delivery mechanism may enhance the uptake and effectiveness of EGCG.

Conclusions
The number of KC cases is increasing at an alarming rate globally. Moreover, statistics revealed that South Africa is one of the leading countries in the world, with the highest rates of skin cancer cases. Although the current topical treatments available for KC have shown promising results, they have also been associated with numerous adverse effects. Therefore, the discovery, development, and treatment of KC using plants and their natural products are becoming increasingly sort after. Natural plant compounds, such as polyphenolics and alkaloids, have in numerous studies demonstrated increased antiproliferative and anticancer activity. Studies have shown that these natural compounds are capable of functioning both independently and interdependently. Various reports further indicated the emergence of nanocarrier and transdermal delivery systems and the numerous advantages they offer, specifically to overcome current challenges faced in cancer treatment, such as bioavailability, targeted delivery, and systemic toxicity. However, it is important to note that case studies alone are not considered conclusive to determine or evaluate the safety and efficacy of a test substance such as a plant extract or natural product. Large clinical trials are required that include a placebo control group and different skin types in order to determine the effectiveness of a test substance.
Although there have been numerous reports on the antiproliferative activity of plant extracts and their natural products on non-melanoma skin cancer cell lines, there have been relatively few studies that have evaluated the potential of these plants/natural products in clinical trials. Additionally, although some plant extracts or natural products may not have shown significant in vitro activity or anticancer activity in animal models when used alone, a suitable drug delivery system may provide for enhanced delivery and efficacy and therefore should further be explored.
The topical application of EGCG and caffeine, using different transdermal delivery systems, should be considered for further evaluation, as this may result in increased uptake of phytochemicals by the cancerous cells. In addition, the use of capsaicin against skin cancer should be further assessed, as it showed promising results in two patients but has not been assessed in larger clinical trials. The potential of synthesizing capsaicin derivatives may provide a source of novel compounds with decreased irritant effects and increased efficacy. Ursolic acid, which has extensively been studied for its activity against melanoma, has not been well documented for its activity against keratinocyte carcinomas and therefore should be considered for further assessment.
It is, however, important to note that upon identification of a plant extract or compound showing significant activity, the mechanism of action, as well as the efficacy and toxicity potential of the identified extract or compounds requires further evaluation using clinical trials, which includes the pharmacodynamics and pharmacokinetic properties of the plant extract/compound.

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