Recent Advances in Gene Delivery for Melanocyte-associated Disorders

Abstract

Melanocytes are cells present at the epidermal-dermal junction of the skin that produce pigment melanin, which provides color to the skin, eyes, and hair. Dysregulation in melanocyte function, viability, or differentiation can result in melanocyte-associated disorders that can be broadly classified based on etiology as melanocyte hyperproliferation and hyperactivation, defects in melanin synthesis, inflammatory alterations in melanin production/trafficking, melanocyte destruction, and defects in melanocyte migration. While most of these disorders are of benign origin, the cosmetic implications of these conditions are associated with significant psychosocial burden and cultural stigma, having a significant impact on affected individuals. These conditions are primarily driven by changes in underlying gene expression (both at the genetic and epigenetic levels). Targeting the underlying genetic and transcriptomic changes in melanocyte-associated disorders using gene replacement (plasmid DNA, mRNA), gene knockdown (siRNA), or miRNA replacement (miRNA) presents a promising strategy for developing treatments for these conditions. The delivery of naked nucleic acid molecules is challenging, and lipid- and polymer-based particles have been widely evaluated for the successful delivery of biologically active nucleic acids to the melanocytes. This review provides an overview of melanocyte-associated pigmentary disorders and their underlying genetic factors and examines current preclinical and clinical efforts using non-viral polymeric and lipid-based delivery systems for plasmid DNA and RNA-based therapeutics.

Graphical Abstract

1. Introduction

Acting as both armor and thermostat, the skin plays a crucial role in defending the body and preserving physiological balance [1]. Structurally, the skin is composed of three layers - the epidermis, dermis, and subcutaneous tissue [2]. Epidermal melanocytes are pigment-producing cells positioned at the epidermal-dermal junction (Figure 1). They are of neural crest origin, distinct from the majority of epidermal cells, such as keratinocytes, which arise from the surface ectoderm [3]. Melanocytes produce a pigment, melanin, which is transferred to the neighboring keratinocytes via dendritic extensions and protects against ultraviolet radiation (UVR)-induced damage. Moreover, through direct cell–cell contacts with dozens of other cells, melanocytes form a homeostatic regulatory structure of interacting cells.

Figure 1.

A schematic of skin showing presence of melanocytes at the epidermal-dermal junction and in the hair follicle. MSC- melanocytic stem cells.

The skin is vulnerable to a wide range of conditions, from common ailments like acne and eczema to more morbid disorders such as skin cancer [2]. Many of these conditions have a pigmentary component that either results from or contributes to dysregulation of melanocyte function, viability, or differentiation. These disorders may manifest as melanocytic malignancies, hyperpigmentation, or hypopigmentation and can be broadly categorized into five groups based on the underlying etiology: (i) melanocytic hyperproliferation and hyperactivation; (ii) inflammation-associated pigmentary alteration; (iii) defects in melanin synthesis; (iv) melanocyte destruction; and (v) aberrant melanocyte migration [3] (Figure 2 and Table 1). While most of these disorders are of benign origin, the cosmetic implications of these skin-related conditions can significantly impact the quality of life, primarily affecting the mental well-being of affected individuals due to social stigmas.

Figure 2.

Schematic representations (top) and clinical presentation (bottom) of different melanocyte-associated disorders and molecular mechanisms involved. Melanocyte hyperproliferation and hyperactivation (melanocytic nevi [31] melanoma [32], ephelides [33], solar lentigines [34]), defects in melanin synthesis (albinism [35]), inflammatory alterations in melanin production/trafficking (melasma [36], PIHP (post-inflammatory hyperpigmentation) [37], pityriasis alba [38], progressive macular hypomelanosis [39]), melanocyte destruction (vitiligo [40]), defects in melanocyte migration (nevus of Ota [41], nevus of Ito [41], piebaldism [42]).

Table 1.

Histological presentation, etiology, and genes involved with Melanocyte-associated disorders

Condition	Histological Presentation	Etiology	Genes Involved	Location for Gene Targeting	References
Melanocytic hyperproliferation & hyperactivation
Melanocytic nevi and melanoma	Melanocytic nevi- dermal-epidermal hyperplasia and rounded nests of melanocytes Melanoma-asymmetrical hyperproliferati on of melanocytes with cytologic atypia, invasion	Melanocytic nevi- growth-arrested melanocytes Melanoma-exposure to UV radiation and genetic alterations	BRAF, NF-1 and NRAS, CDKN2A, PTEN, TERT, MC1R R alleles, PRAME, AXL	Epidermal-dermal junction, epidermis, dermis	[32, 46-57]
Solar lentigines	Increase in the concentration of melanin Elongation of the epidermal rete	Chronic exposure to UV radiation leading to increased melanin production and epidermal thickening	TERT, FGFR3, PIK3CA, TYR	Epidermal-dermal junction	[43, 58-62]
Ephelides	Increased synthesis of eumelanin, larger and more dendritic melanocytes with increased melanosomes	MC1R gene triggered small hyperpigmented macular lesions following exposure to the sun	MC1R, ASIP, IRF4, EXOC2, TYR, BNC2	Epidermal-dermal junction	[43, 61, 63, 64]
Inflammatory alterations in melanin production and trafficking
Melasma	Increase in the concentration of melanin in the epidermis	Mutations in the tyrosinase gene, leading to increased melanin in the epidermis in sun-exposed areas	TYR, MITF, MC1R	Epidermis or dermal melanopha ges	[63, 65-71]
Post inflammatory hyperpigmentation	Increased melanin content in the hyperpigmented spots No loss of melanocytes	Overproduction of inflammatory mediators, causing increased melanin production and melanocyte hypertrophy	IL-37, MITF, DCT, TYR, and TYRP1	Epidermis or dermal melanopha ges	[43, 63, 72, 73]
Post inflammatory hypopigmentation	Reduced melanin content in the hyperpigmented spots No loss of melanocytes	Presence of inflammatory cytokines can impair melanocyte function, leading to hypopigmentation	TNF, IL1A/IL1B, or IL6	Epidermis	[43, 71, 73-75]
Pityriasis alba	Reduction in the number of melanocytes, melanin synthesis, melanosome size, and the number of melanosomes	Etiology unclear; Risk factors include UV exposure and poor cutaneous hydration	HMOX1, SOD1, IL6, and IFNG	Epidermal-dermal junction	[76]
Macular hypomelanosis	Reduced melanin content in the hypopigmented spots. No loss of melanocytes	Presence of bacteria Cutibacterium acnes or Propionibacterium species	Role of genetic predisposition or involvement is unknown	Epidermis	[73, 77]
Melanin synthesis
Albinism	Defects in melanocyte to synthesis or distribution of melanin, causing decreased or absent melanin pigment	Mutations in the tyrosinase gene, leading to inability in synthesizing eumelanin or melanosome biosynthesis	TYR (OCA1), OCA2, TYRP1 (OCA3), and MATP (SLC45A2 or OCA4), intronic mutation GPR143, T373K SNP mutation, MITF	Epidermal-dermal junction	[78-81]
Melanocyte destruction
Vitiligo	Absence of melanocytes	Targeted melanocyte destruction by CD8+ T cells promoting interferon-γ (IFN-γ) production	POMC, FGF2, EDN1, JAK1, TNF, IL6, IL1A/IL1B, IFNG	Epidermal-dermal junction and follicular melanocytes	[82-87]
Melanocyte migration
Piebaldism	Absence of melanocytes	Aberrant migration of melanoblasts in the embryo leading to lack of melanocytes	KIT gene, SNAI2	Dermis	[88-93]
Nevus of Ito/Ota	Dermal entrapment of melanocytes, leading to lesions having a gray-blue or brown hyperpigmentation	Dermal melanosis involving the distribution of trigeminal nerve branches (nevus of Ota) or lateral cutaneous brachial/the posterior supraclavicular nerves (nevus of Ito)	GNAQ, BAP1, TP53	Dermis	[94-97]

Underlying many pigmentary disorders are changes in gene expression, driven by both genetic and epigenetic causes as well as environmental stimuli. Gene therapy refers to the manipulation of gene expression for diagnostic, prophylactic, or therapeutic purposes [4]. Strategies for modulating gene function include traditional gene replacement approaches, gene silencing methods, and targeted gene editing tools [5]. Gene replacement approaches through plasmid DNA or viral vectors are employed to express a gene that is disrupted or silenced. Gene silencing methods often rely on RNA interference (RNAi), which involves the delivery of small RNA duplexes, such as antisense oligonucleotides (ASO), microRNA (miRNA) mimics, short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and dicer substrate RNAs (dsiRNAs) [5]. Most of these are used to silence or “switch off” specific individual genes, whereas miRNAs have the capacity to modulate entire transcriptional programs [6]. Targeted gene editing tools such as CRISPR/Cas systems and zinc-finger nucleases (ZFNs) offer precise engineering of the genome and can be employed to either disrupt or correct gene function.

To address skin disorders, gene therapy tools can be delivered either systemically, topically or via transdermal route by viral or non-viral vectors [5]. Adenovirus, adeno-associated virus, herpes simplex virus, retrovirus and lentivirus vectors, among others, have been explored as viral vehicles for gene delivery [7]. A wide range of non-viral delivery systems, including cationic liposomes, lipid nanoparticles, polymeric delivery systems, and niosomes have been evaluated for targeting melanocytes, offering promising platforms for the delivery of therapeutic nucleic acids in pigmentary disorders. In this review, we first highlight the biological role of melanocytes and melanin pigment and provide an overview of melanocyte-associated disorders. We then critically examine non-viral polymeric and lipid-based delivery systems for plasmid DNA and RNA-based therapeutics with a focus on the specific challenges associated with the delivery to melanocytes.

2. Melanocytes and the role of pigmentation

Melanocytes are cells that can produce the pigment melanin, which is synthesized through the process of melanogenesis and stored in lysosome-like vesicles called melanosomes [7]. Melanocytes are primarily located at the epidermal-dermal junction of skin (epidermal melanocytes) and within hair follicles (follicular melanocytes). Melanocytes are also present in the eyes, inner ears, leptomeninges, mucosa, and heart. In the skin, each epidermal melanocyte makes direct cell-cell connections with about 30-40 keratinocytes and other cells. This set of interacting cells is collectively referred to as the epidermal melanin unit [8, 9]. Mature melanosomes are taken up by the interacting keratinocytes, where they form a shield above the nucleus, protecting the keratinocytes from harmful UVR and other environmental stressors [9, 10]. Follicular melanocytes within hair follicles exist in three distinct lineages: melanocyte stem cells (MSCs), progenitor cells, and differentiated melanocytes [11-16]. MSCs reside in the bulge region of the hair follicle and serve as a long-term reservoir that replenishes the melanocyte population during successive hair cycles (Figure 1). These stem cells give rise to transient progenitor cells, which migrate to the hair bulb and further differentiate into mature melanocytes. The differentiated melanocytes localize to the hair matrix and are responsible for producing melanin, thereby imparting pigment to the growing hair shaft [9, 10, 17, 18].

Melanin is responsible for the strikingly polymorphic human traits of skin and hair color [9, 10, 19]. Melanin has two forms- pheomelanin (a yellow-red soluble form of melanin) and eumelanin (brown-black insoluble melanin) [20-22]. The overall pigmentation of the skin correlates with melanin density and is the collective outcome of: i) melanin synthesis in the melanocytes (including melanin amount and form); ii) melanosome maturation; iii) melanosome transfer to the neighboring keratinocytes; and iv) melanosome size, location and stability within the keratinocyte [9]. Similarly, hair pigmentation depends on the type and quantity of melanin produced by differentiated melanocytes in the hair bulb [8, 9, 20-25]. Many genes are involved in the process of melanogenesis. Tyrosinase (TYR) is a key enzyme in the melanin synthesis pathway and plays a central role in regulating skin pigmentation by catalyzing the rate-limiting steps of melanin production [10]. The melanocortin-1 receptor (MC1R) signaling axis, with antagonistic regulation by the agouti signaling protein (ASIP), plays a pivotal role in both determining skin and hair color and orchestrating the UVR-induced tanning response by regulating both the amount of melanin and the balance between eumelanin and pheomelanin production [26]. This regulation occurs in part through activation of the microphthalmia-associated transcription factor, MITF, which promotes the expression of melanogenic enzymes such as tyrosinase-related protein 1 (TYRP1) and dopachrome tautomerase (DCT), both essential for eumelanin synthesis [26-28]. In addition to melanin synthesis, pigmentation depends on genes that govern melanocyte development and survival or melanosome formation, trafficking, and transfer, including KITLG, GPR143, OCA2, SLC45A2, SLC24A5, AP3B1, MLPH, RAB27A, and MYO5A. Moreover, the BRAF gene is essential for maintaining MSC pools – the depletion of which causes hair graying – and somatic mutations in BRAF also drive the majority of melanocytic tumors [29, 30].

Dysregulation in melanin production or melanocyte function can lead to a variety of melanocyte-associated disorders. These conditions differ based on whether they primarily affect epidermal or follicular melanocytes, and effective gene therapy strategies must be tailored accordingly - both in terms of delivery route and target genes. The known genetic etiology of these disorders, along with potential therapeutic gene targets, are summarized in the following section.

3. Melanocyte-associated disorders and molecular mechanisms involved with these conditions

Melanocyte-associated disorders can be classified based on the melanocyte pathophysiology in these conditions including melanocytic hyperproliferation and hyperactivation (melanocytic nevi, melanoma, solar lentigines and ephelides); inflammatory alterations in melanin production and trafficking (post inflammation pigmentary alteration (PIPA), pityriasis alba, macular hypomelanosis, and melasma); melanin synthesis (albinism); melanocyte destruction (vitiligo); and melanocyte migration (Piebaldism and nevus of Ito/Ota) (Table 1 and Figure 2). With the exception of melanoma, most of these conditions are generally considered benign [43]. However, due to readily apparent alterations in skin pigmentation, many of these conditions have a significant psycho-social impact on affected patients [44, 45]. The genes involved in melanocyte-associated disorders can be categorized into six core molecular processes: MC1R/MITF signaling, melanosome biogenesis and trafficking, MAPK signaling, tumor suppression, EMT/dedifferentiation, and inflammation (Figure 3).

Figure 3. Summary of core molecular mechanisms involved in melanocyte-associated disorders.

Genes involved with melanocyte-associated disorders can be grouped into six categories: MCIR/MITF signaling; downstream melanosome biogenesis and trafficking; MAPK signaling; tumor suppression; inflammation; and EMT and melanocyte dedifferentiation. Lines indicate genes that are involved in each category of disorder.

3.1. Melanocyte hyperproliferation

Upon external or internal stimuli, melanocytes can proliferate to form a variety of pigmented neoplasms with distinct cellular, molecular, and clinical properties. These include melanocytic nevi, melanoma in situ, and invasive melanoma, among myriad others [52-54]. Solar lentigines are another condition that involves melanocyte hyperproliferation along the dermal-epidermal junction. It is a benign macular hyperpigmentation disorder triggered by chronic exposure to the sun [43, 61, 62] and involves the elongation of the epidermal rete [62, 64].

Melanocytic nevi comprise growth-arrested melanocytes and are caused primarily by the somatic BRAF^V600E mutation (present in approximately 80% of nevi) [55]. The majority of these lesions remain benign throughout the lifespan of an individual; however, upon further transformation, can give rise to melanomas [55]. In addition to arising from precursor lesions such as melanocytic nevi, invasive melanomas may arise from isolated melanocytes (de novo) [52, 98]. Melanoma is the malignancy of melanocytes, which is expected to be the fifth most commonly diagnosed cancer in both men and women in the United States (US) in 2025 [99]. Although melanoma accounts for only about 1–4% of all skin cancer cases, it is responsible for the majority of skin cancer–related deaths [48]. Cutaneous melanomas are commonly sub-classified into four major histopathological types: superficial spreading, lentigo maligna, nodular, and acral lentiginous melanomas [100]. Melanocytic neoplasms that are hyperproliferative yet restricted to the epidermal-dermis in their growth are referred to as melanoma in situ [56, 57]. Though non-invasive, the standard of care is surgical excision, given the potential for extensive spread, invasion and metastasis if left untreated. Treatment of melanoma in situ, particularly in sensitive areas such as the face and genitals, can result in significant morbidity [32]. Thus, strategies for the prevention or interception of melanocytic neoplasms, both benign and malignant, could substantially reduce morbidity and mortality as well as reduce overall healthcare expenditures.

Melanoma development is multifactorial and involves interaction between genetic alterations and environmental factors. The most common initiating genetic alterations of cutaneous melanoma occur in BRAF, NF-1 and NRAS [46], each of which are associated with activation of the RAS-RAF-MEK pathway [47]. Genetic alterations commonly selected for during melanoma transformation include loss-of-function mutations in the tumor suppressor genes CDKN2A (germline or somatic) and PTEN [48], as well as activating mutations in the TERT promoter that increase mRNA expression [49]. Another genetic risk factor for melanoma is certain germline variants in MC1R, a key driver for pigmentation programs as discussed above [50]. Referred to as “R variants”, a single copy is sufficient to increase risk of melanoma [51]. Melanocyte markers such as TYR and TYRP1 and genes associated with fatty acid metabolism or inflammation have been reported to be involved in solar lentigines [58]. The role of the TERT gene as well as FGFR3 and PIK3CA mutations in the development of solar lentigines has been reported [59, 60].

The primary environmental risk factor for melanocytic tumors is exposure to UVR. In addition to causing extensive DNA damage, UVR also induces substantial epigenetic, immunologic, and structural changes to the skin, each of which affects the frequency, presentation, growth, and progression of melanocytic nevi through modulation of gene expression [52, 101, 102]. The genes PRAME and AXL are commonly up-regulated in melanoma, whereas our group reported that miR-211-5p is most highly expressed in melanocytic nevi and is necessary and sufficient to permit growth arrest in the presence of the BRAF^V600E oncogene [6, 55, 103, 104].

Collectively, these are just a few of the prominent genetic and transcriptional changes associated with melanocytic neoplasms, demonstrating that modulating gene function through gene replacement (i.e., CDKN2A, PTEN), microRNA replacement (i.e., miR-211-5p), or gene silencing methods (i.e., MC1R R alleles, mutant BRAF, PRAME), could be powerful tools for prevention or interception. Current prevention strategies include avoidance of sun or use of sunscreens to prevent the development of benign lesions or their transformation to melanoma [105], but presently there are no effective chemoprevention strategies to prevent the transformation of melanocytes [106].

Ephelides, also referred to as freckles, are small hyperpigmented macules that appear in early childhood after exposure to the sun and are associated with common variants of the MC1R gene [43, 61]. While not hyperproliferative, melanocytes in ephelides are larger and more dendritic, with increased melanosomes and melanin production [43, 64, 71]. In the case of ephelides, apart from MC1R, other genes involved in their development include ASIP, IRF4, EXOC2, TYR, and BNC2 [64]. Solar lentigines, which appear similar to ephelides but are more persistent, result from both hyperproliferation and hyperactivation of melanocytes. Therapeutic options to reduce hyperpigmentation in solar lentigines – which include topical treatment with hydroquinone, retinol, kojic acid, or tretinoin [70, 107] as well as chemical peels, cryotherapy or laser therapy [43] - often yield incomplete or temporary results. Gene delivery platforms for the prevention and treatment of melanocyte hyperproliferation is therefore being widely explored preclinically and clinically [108, 109].

3.2. Inflammatory alterations in melanin production and trafficking

Skin inflammation can result from a defensive response to acute or chronic stimuli, whether endogenous or exogenous, including UVR, allergens, pathogens, hormonal changes, physical or chemical injuries, mechanical stress, or adverse drug reactions [71, 75, 110, 111]. Recruitment of inflammatory mediators can alter melanogenesis, leading to hyper- or hypo-pigmentary disorders such as melasma, ephelides, pityriasis alba, macular hypomelanosis, and post-inflammation pigmentary alteration (PIPA) [75, 110]. PIPA can be further classified into post-inflammatory hyper- or hypo-pigmentation conditions.

3.2.1. Inflammatory alterations causing hyperpigmentary disorders

Melasma is a hyperpigmentation skin disorder that affects sun-exposed areas, primarily the face, and can histologically be epidermal, dermal, or mixed [70]. It is triggered by a combination of genetic predisposition, hormonal influences, and UVR exposure [71]. Alterations in skin barrier properties, such as a reduction in total surface lipid content, can also initiate the development of melasma lesions [112]. Epidermal melasma results from increased epidermal melanin concentration and the number of melanosomes in the epidermis [113, 114]. Post-inflammatory hyperpigmentation disorder is a common outcome of skin inflammation or trauma [43, 71]. The hyperpigmentation condition is associated with the appearance of dark pigmented spots due to the aberrant distribution of melanin in neighboring keratinocytes [43, 73]. It is also a side effect of cryo-, light-, or laser- therapy [43]. Some hyperpigmentary disorders are associated with the presence of melanophages. These are melanin-containing inflammatory dermal macrophages, solitarily distributed in the superficial dermis around vessels or capillaries [115-117]. Melanophages have been observed in post-inflammatory hyperpigmentation disorder and dermal melasma, where they localize to the deep dermis [113, 118].

Each of these conditions are highly associated with alterations in inflammatory mediators. Overproduction of inflammatory mediators such as prostaglandins, leukotrienes, and thromboxanes can enhance tyrosinase activity, leading to melanocyte hypertrophy, causing hyperpigmentation [43, 71, 74]. MITF is involved in several processes that lead to hyperpigmentation, from stimulating tyrosinase for melanogenesis to regulating melanocyte proliferation and survival [63]. A bioinformatic analysis reported that increased melanin content and tyrosinase activity in post-inflammatory hyperpigmentation was associated with MITF, DCT, TYR, TYRP1, and inflammatory cytokines, including IL-37 [72]. Several miRNAs targeting MITF (miR-25, miR-429, and miR-137) or causing skin lightening or treating skin pigmentation (miR-141-3p, miR-200a-3p, miR-675, and miR-1299) have been identified in vitiligo and melasma [65-69]. Further, miR-145 and miR-125b have been shown to regulate melanogenesis [119, 120]. Kim and coworkers reported an inverse correlation between miR-1299 levels and Arginase-2 levels, which is expressed in skin fibroblasts and keratinocytes, leading to the development of melasma [65].

Hyperpigmentation disorders are often treated with pharmacologic agents that inhibit tyrosinase [107]. Current first-line therapies for pigmentary conditions such as melasma or ephelides typically involve topical application of hydroquinone, either alone or in combination with other agents like corticosteroids, retinol, kojic acid, tazarotene, or tretinoin [70, 107]. Despite the availability of these treatments, many cases are refractory and can relapse, making long-term management challenging [70, 121]. Ablative strategies such as chemical peels, cryotherapy or laser therapy can also be used to reduce hyperpigmentation [43]. However, dermal melasma often shows poor response to topical treatments, as most agents fail to adequately penetrate the deeper dermal layers [43, 122]. Effective treatment of dermal melasma also requires dermal remodeling, which is a delayed process [122].

The use of lipid-based nanocarriers to silence key genes in the pigmentation pathway (i.e., MC1R, MITF, TYR) holds potential for alleviating the symptoms of melasma. The use of siRNAs targeting MITF as depigmentation agents has been evaluated [71, 107]. Silencing the tyrosinase gene using RNAi compound, RXI-231 (a self-delivering siRNA targeting tyrosinase) could offer a potentially more effective alternative for treating various hyperpigmented conditions, including melasma, solar lentigines, or post-inflammatory hyperpigmentation disorder [123].

3.2.2. Inflammatory alterations causing hypopigmentary disorders

Similar to post-inflammatory hyperpigmentary disorders, post-inflammatory hypopigmentation disorders are also associated with trauma or inflammation [43, 71]. In the hypopigmented condition, the spots appear to have reduced pigmentation due to loss of melanin in the keratinocytes [43, 73]. Examples of hypopigmentation resulting from inflammatory alterations include pityriasis alba and progressive macular hypomelanosis. Pityriasis alba is a benign, self-limiting, non-contagious skin condition primarily affecting children [76, 124, 125]. The hypopigmentation in the lesions is associated with a reduction in the number of melanocytes, melanin synthesis, melanosome size, and the number of melanosomes transferred to the keratinocytes [126-128]. Elevated reactive oxygen species (ROS) levels also affect melanosome maturation, leading to abnormal transport of melanosomes to the keratinocytes [73]. While the condition's full etiology remains unclear, several risk factors have been identified, including UVR exposure and poor cutaneous hydration [125, 126]. Progressive macular hypomelanosis presents as ill-defined hypopigmented patches mainly appearing due to the presence of bacteria Cutibacterium acnes or Propionibacterium species [73, 77]. Histological evaluation suggests reduced melanin in the hypopigmented macules with no loss of melanocytes [129].

The presence of inflammatory cytokines such as TNF-α, IL-1, or IL-6 can impair melanocyte function, leading to hypopigmentation [74, 75]. The change in the expression levels of HMOX1, SOD1, IL6, and IFNγ in pityriasis alba tissues has been reported [76]. The role of genetic predisposition or involvement in the development of progressive macular hypomelanosis is currently unknown and warrants further investigation.

While these conditions can gradually resolve and restore normal skin pigmentation, the initial lesions often raise concerns due to the cosmetic impact on the patients. Skin-tone matching makeup [71], laser therapy, phototherapy, protection from sun exposure around the lesions, the use of sunscreens, topical corticosteroids or calcineurin inhibitors and topical vitamin D (calcitriol) are used to treat hypopigmented lesions [43, 124, 130]. The use of chemical peels or laser therapy has also had promising results in normalizing pigmentation in these conditions [43, 71]. Treatment options for progressive macular hypomelanosis mostly include effectively treating the bacterial infection using oral or topical antibiotics, benzoyl peroxide, or UVA or narrowband UVB phototherapy [73, 77]. The use of topical 1% clindamycin and 5% benzoyl peroxide has been reported as an effective treatment strategy [39].

Similar to disorders of hyperpigmentation, understanding the role of genetic alterations in hypopigmented disorders is also essential to developing gene delivery systems for targeting these disorders. Modulation of inflammatory cytokines involved during hypopigmentation could regulate mediators and receptors such as TYR, miR-211, MITF, HMOX1, SOD1, IL6, and IFNG to restore pigmentation in the depigmented spots.

3.3. Melanin synthesis (Oculocutaneous albinism)

Albinism is an autosomal recessive hereditary condition resulting from defects in melanocytes to synthesize or distribute melanin, causing decreased or absent melanin pigment [78, 79]. Oculocutaneous albinism (OCA) is the most common presentation of albinism involving the skin and eyes [78]. Individuals with OCA struggle with eye abnormalities, including nystagmus, reduced iris or retinal pigment, and photophobia [131].

There are seven different types of non-syndromic albinism identified (OCA1-OCA7), which are associated with genetically distinct mutations, including TYR (OCA1), OCA2, TYRP1 (OCA3), and MATP (SLC45A2 or OCA4) [80, 81]. These lead to an inability to synthesize eumelanin or disruption of genes involved in melanosome biosynthesis and integrity. Computational analysis identified 37 single-nucleotide polymorphisms (SNPs) in the 3’UTR of mRNAs from five OCA and OA genes, which are predicted to contribute to hypopigmentation [132]. Several miRNAs have been shown to target genes associated with OCA including tyrosinase (miR-330-5p, miR-326, miR-382-5p), TYRP1 (miR-145, miR-128, miR-155, miR-365), OCA2 (miR-101-3p), MITF (miR-25, miR-96, miR-204, miR-211, miR-182, miR-218, miR-137, miR-186-5p, miR-145) and cellular processes (miR-876-5p) [133-136].

Currently, albinism has no cure [137]. However, gene therapy is actively being explored as a strategy to treat albinism, both through gene replacement and by targeting downstream alterations associated with OCA. For example, OCA2-specific siRNA has been reported as a potential skin-brightening agent by reducing melanin content, thereby improving skin tone and diminishing dark spots [138]. Intraocular administration of the human tyrosinase (TYR) gene using adeno-associated virus (AAV)-based vectors in a rat model reversed the ocular anomalies associated with OCA1, along with inducing pigmentation and increased melanin production [139, 140]. CRISPR-Cas ribonucleoprotein technology has also been used to correct intronic mutations (i.e. GPR143) in ocular albinism type 1 (OA1) patient-derived induced pluripotent stem cells (iPSCs) [141]. GPR143 plays a role in melanosome maturation and maintaining melanosome size [27]. Rescue of melanin production in rabbit albinism has been demonstrated by editing the p.K373T SNP in TYR [142]. Future approaches could additionally include gene silencing and miRNA replacement to restore pigmentation.

3.4. Melanocyte destruction

Destruction of melanocytes caused by CD8+ T cells leads to a complete loss of pigmentation in the skin causing vitiligo, an acquired autoimmune disorder [45]. While the epidermis becomes devoid of melanocytes, the melanocyte reservoir in the hair follicle bulge is preserved. This niche harbors melanocyte stem cells and melanoblast-like populations, though mature melanocytes are typically absent, consistent with the bulge’s role as a regenerative source of repigmentation [82, 83].

In vitiligo, CD8+ T cells infiltrate the skin and secrete interferon-γ (IFN-γ), which induces chemokines that recruit additional T cells and sustain local inflammation, ultimately leading to the targeted destruction of melanocytes [84, 85]. Additionally, neighboring keratinocytes secrete IFN-γ-induced chemokines as well [85]. Oxidative stress in vitiligo skin also contributes to melanocyte destruction through the accumulation of ROS which may be exacerbated by inflammatory cytokines such as TNF-α, IL-6, and IL-1 [86, 87]. In vivo evaluation of JAK1 siRNA in a vitiligo mouse model showed reduced CD8+ T cells in the skin, preventing epidermal depigmentation and modulation of autoimmunity [85]. Different miRNAs are involved in vitiligo pathogenesis with some upregulated including miR-9, miR-25, miR-99b, miR-125b, miR135a, miR-145, miR-155, miR-199a-3p, miR-330-50, miR-493-3p, and miR-2909, and others downregulated such as miR-21-5p, miR-145, miR-200c and miR-211 [86, 143-145].

Clinical treatment of vitiligo includes the use of topical calcineurin inhibitors, corticosteroids, and JAK inhibitors. UVA or narrowband UVB therapy have also shown promising results, though long-term use of UVA has been associated with carcinogenesis and is no longer widely recommended. Topical therapy can also stabilize the lesions in the absence of exposure to sunlight. Cosmetic coverage of vitiligo-affected regions with skin-tone matched makeup can be used to make vitiligo appear less noticeable. Surgical grafting or depigmentation therapies can also be utilized. However, relapse upon discontinuing these therapies is common [45]. Topical application of immunomodulators (pimecrolimus 1%) has an improved synergistic effect on the repigmentation of lesions with narrowband UVB therapy [146].

Clinical evaluation of ruxolitinib cream was well-tolerated for a period of three years, and led to significant repigmentation of vitiligo lesions, which was maintained up to six months after the treatment was stopped [147, 148]. However, there is high inter-patient variability to these treatment options and repigmentation is lost in 40% of cases [87, 149]. There is a need to develop effective strategies for successfully reintroducing pigmentation in the skin without loss of pigmentation post-treatment. Therapeutic approaches to either relocate stem cells or reduce skin inflammation could be potential strategies for vitiligo treatment. Recruitment of precursor populations from the hair bulges with migratory and proliferative capabilities to differentiate into mature melanocytes and assist with repigmentation in the affected areas could be a potential strategy [82]. The regeneration of melanocytes might be achieved by introducing migratory factors like α-MSH, bFGF, or EDN-1 using gene delivery strategies [87]. Gene delivery strategies aimed at silencing IFN-γ signaling, such as targeting IFNGR1 with siRNAs, or reducing CD8⁺ T cell recruitment, could help restore pigmentation in the epidermis of individuals affected by vitiligo[145]. Vitiligo-associated melanogenesis targets, including long noncoding RNAs (lncRNAs) or miRNAs (miR-21, miR-211-5p) could also be a novel diagnostic or treatment strategy for vitiligo [150, 151]. To target gene delivery to the MSCs to promote skin repigmentation a transfollicular pathway for cargo delivery would need to be investigated. This differs from melanoma treatment strategies wherein the melanocytes reside around the epidermal-dermal junction or dermis, and the delivery systems need to penetrate through the stratum corneum and viable epidermis to elicit a response [150, 151].

3.5. Melanocyte migration

Aberrant migration of melanoblasts in the embryo can lead to piebaldism (a hypopigmented condition) or nevus of Ito/Ota (associated with localized hyperpigmentation) [152]. Piebaldism is a rare pigmentation disorder involving an autosomal dominant genetic anomaly leading to stable depigmentation or white patches on skin (leukoderma) and hair (poliosis) due to a lack of melanocytes [88-90]. Nevus of Ota is a benign, periocular, dermal melanosis primarily involving the distribution of trigeminal nerve branches on the face [94, 95]. Due to the deep entrapment of melanocytes, the lesions have a gray-blue or brown hyperpigmentation [94, 95]. Around half the cases also involve hyperpigmentation of the eyes [94]. Individuals with this condition can develop sensorineural deafness and are at a higher risk for glaucoma or uveal melanoma [94, 153]. Nevus of Ito is a benign condition pathologically similar to nevus of Ota; however, in this case, the lesions appear around the neck, shoulders, axilla, upper arms or deltoid, which are associated with the lateral cutaneous brachial or the posterior supraclavicular nerves [153, 154].

Piebaldism is most commonly associated with mutations on the KIT gene, and 99 KIT variants associated with the disease have been identified [90]. KIT is a proto-oncogene encoding the receptor tyrosine kinase and is required for the distribution and survival of melanoblasts during embryogenesis [90-92]. Additionally, some patients may have mutations in other genes such as SNAI2 [93]. The severity of the disease can also be influenced by additional factors such as the color of the skin and hair, and modifications in the MC1R gene [152]. The initial activation of GNAQ in the development of Nevus of Ota/Ito and subsequent activation of other mutations, primarily BAP1 and TP53, in primary or recurrent tumors, are associated with the transformation of these benign lesions into malignant melanoma [96, 97].

There is no cure for piebaldism, though cosmetic products such as makeup or hair coloring products can provide temporary solutions [92]. Procedures to repigment depigmented skin areas, including using epidermal autografts [155-158], punch grafting [159], and dermabrasion [160] as well as the use of dihydroxyacetone (1,3-dihydroxydimethyl ketone: DHA) [161] or Erbium:YAG laser [162] have been attempted. In the case of Nevus of Ota/Ito, the condition can also be treated with lasers that target pigmentation [70, 153]. However, recurrence of the lesions after laser treatment has been reported and can also result in complications like scarring or non-uniform appearance [153, 154].

To date, no gene therapy strategies have been evaluated to directly target KIT mutations in Piebaldism. However, nanoparticles complexed with anti–c-KIT siRNA have been shown to reduce inflammation in models of allergic asthma, suggesting potential for therapeutic modulation of KIT expression [163]. Studies for targeting GNAQ for uveal melanoma suggest that developing strategies for inhibiting protein kinase C (PKC) could potentially be an effective strategy [164]. Exploring gene therapies for potential targets for treating Nevus of Ota/Ito or the KIT mutation in Piebaldism could be an exciting avenue for preventing the transformation of benign lesions into malignant melanoma or for reintroducing pigmentation.

4. Challenges with nucleic acid delivery to melanocytes

Nucleic acid delivery to melanocytes can be done by delivering plasmid DNA, mRNA, siRNA, miRNA or antisense oligonucleotides. Plasmid DNA and mRNA both encode therapeutic proteins, leading to gene replacement, while exogenous miRNA delivery is helpful for miRNA replacement and siRNA delivery for gene knockdown (Figure 5). Plasmid DNA has higher stability than mRNA and demonstrates sustained gene expression. DNA vaccines targeting tumor antigens can also induce systemic immunological responses against cancers through antigen-specific responses, which have also been explored as a vaccine strategy for melanoma [165]. An in-depth review of various melanoma tumor-associated antigens explored for DNA vaccine strategies can be found elsewhere [165]. However, DNA plasmids have high molecular weights and need to penetrate the nucleus to elicit the desired response, which raises safety concerns due to potential host genome integration [166, 167]. Gene editing techniques like CRISPR-Cas9 offer the opportunity for inserting, deleting, or modifying DNA for treating genetic disorders [166]. For melanocyte-associated disorders, these nucleic acid molecules can be delivered topically or systemically.

Figure 5.

Cellular internalization and endosomal escape of carriers for gene delivery.

4.1. Topical delivery to melanocytes

Most melanocyte-associated disorders are associated with genetic or transcriptomic alterations and most of these conditions lack effective preventative or therapeutic treatment options. Even those that are not malignant are often associated with a significant psychosocial burden and cultural stigma and have a significant impact on the lives of affected individuals [45]. Targeting the underlying genetic and transcriptomic changes in melanocyte-associated disorders presents a rational strategy for developing treatments for these conditions using gene therapies.

The gene delivery strategies to melanocytes must be selected on a condition-specific basis, depending on the type of gene being delivered and the type of melanocyte targeted. As previously mentioned, epidermal melanocytes reside around the epidermal-dermal junction and are sparse in comparison to keratinocytes and fibroblasts in the skin. Since most of the melanocyte-associated conditions are benign and occur around the skin surface, localized topical treatment strategies are preferred for treatment around the localized lesions. Topical delivery of nucleic acids offers several advantages over systemic application, including targeted, localized delivery with limited systemic effects. Additionally, as melanocytes reside around the epidermal-dermal junction, treating benign lesions and conditions locally is a more effective strategy for improving efficacy and reducing systemic toxicities.

The primary challenge of topical delivery to the melanocytes is the permeability barrier function of skin due to the presence of stratum corneum and the epidermis[168, 169]. Additionally, the thickness of the stratum corneum across the body is heterogeneous, which affects absorption into the skin [170]. Transport across the skin can take place via two pathways- transepidermal (which is further divided into paracellular and transcellular) and transappendageal (further subdivided into delivery via hair follicles or sweat glands) (Figure 4) [170, 171]. Paracellular transport refers to transport through the gaps or interspaces between the cells. Transcellular pathway refers to transport across cell membranes via active or passive transport processes or by endocytosis [170]. Whereas melanocytic stem cells and follicular melanocytes in the bulge area of the hair follicles can be targeted vis transappendageal routes; melanocytes in the epidermis and dermis must by targeted transepidermally [8].

Figure 4.

Transport across the skin can take place via two pathways- transepidermal (which is further divided into paracellular and transcellular) and transappendageal (further subdivided into delivery via hair follicles or sweat glands). Follicular melanocytes can be targeted via the transappendageal pathway whereas epidermal or dermal melanocytes must be targeted via transcellular or paracellular routes.

Another challenge with targeted delivery to melanocytes is their scarcity in the skin: the number of melanocytes is much smaller compared to keratinocytes. Each melanocyte is associated with and transfers melanosomes to 30-40 keratinocytes in the skin [8, 172, 173]. Moreover, melanin abundance and type represent important biological variables. Beyond its well-known protective roles against UV radiation and oxidative stress, melanin is a chemically diverse and highly reactive compound that can influence cellular susceptibility to chemotherapy, radiotherapy, and photodynamic therapy [174-177]. Therefore, for any given therapeutic delivery system, it is essential to evaluate whether cargo uptake or efficacy is affected by melanin content or composition. This can be assessed by comparing uptake efficiency across lightly, moderately, and darkly pigmented normal human melanocytes, as demonstrated in a recent in vitro study from our group evaluating the delivery of miRNA-complexed ultradeformable cationic liposomes [178].

These biological and physical factors make it necessary to tailor delivery methods for each condition. In the case of vitiligo, for example, where follicular melanocyte stem cells can repopulate affected areas to overcome depigmentation challenges, genome editing techniques like CRISPR-Cas9 (for DNA-RNA interactions) or zinc-finger nucleases (ZNF) and transcription activator-like effector nucleases (TALENs- for protein-DNA interactions) might be used as an efficient repigmentation strategy [179, 180]. The delivery of CRISPR-Cas9 can be either carrier-independent using physical methods like electroporation or osmosis, mechanical methods like microfluidics, or using a viral or non-viral carrier system [179, 180]. Combination delivery strategies for the treatment of vitiligo may be of benefit to target CD8+ cells around the epidermal-dermal junction and preferentially targeting melanocytic stem cells and follicular melanocytes through the transappendageal route in the hair follicles to replenish melanocytes in the epidermis to induce pigmentation. However, in porcine and human skin, the stratum corneum and barrier-forming tight junctions pose a challenge for effective delivery [181-183], though delivery through the transfollicular route has been previously demonstrated using various strategies [184-189].

In contrast, in the case of disorders of epidermal melanocytes, such as melanoma in situ, viral and non-viral vectors must be delivered via trans- or paracellular routes, where they can be internalized by the cells for gene knockdown (siRNA) of oncogenes, or gene replacement (plasmid DNA or mRNA) or tumor suppressors, or miRNA replacement (miRNA) therapy (Figure 5).

The delivery of nucleic acids across the skin is particularly challenging. Naked nucleic acid molecules are negatively charged, have a high molecular weight, a stiffer structure, and are unstable due to degradation by nucleases. Finally, even when successful, once the nucleic acid reaches the target cell, it must then be internalized, typically via the endocytotic pathways, and transported, without degradation to the correct cellular compartment [50]. Thus, an efficient delivery strategy for nucleic acid-based therapeutics that will reach the target site and release the payload must overcome several barriers; protection from degradation, localization and penetration in the target tissue, cellular uptake, endosomal escape, and localization in the target subcellular compartment [50].

Strategies to augment nucleic acid delivery to the melanocytes through topical, systemic, intralesional or intramuscular delivery have been widely explored. These include overcoming physiological barriers, including improved circulation in the blood for reaching the target site (in case of systemic delivery), localized delivery (via topical application), and increased retention at the target site. Different techniques, including physical methods, chemical methods, or structural modification, have been explored for delivering nucleic acids to melanocytes [190-192]. The principle of physical penetration promotion is based on the initiation of diffusion through artificial microchannels, which are produced by physical intervention using microneedles [123, 193], laser or electroporation [123, 194-196], iontophoresis [197-199], or low frequency ultrasound [196, 200, 201] for a limited period of time. The use of chemical methods includes delivery by various nanoparticle systems systemically or topically [202]. Cationic lipids or polymeric carriers assist in complexation of the nucleic acids, cellular targeting and internalization, and delivery to the target subcellular compartment. Surface modification by attachment of poly(ethylene glycol) (PEG) can further improve circulation time by protecting from uptake by the reticuloendothelial system [203]. PEGylation may also affect the pathways for cellular internalization as reported for PEG-lipid nanoparticles (LNPs) compared to conventional LNPs [204]. Digiacomo and coworkers demonstrated preferential use of clathrin-mediated endocytosis and caveolae-mediated endocytosis by PEG-LNPs compared to conventional LNPs, which were internalized by micropinocytosis [204]. However, PEGylation can also reduce cellular uptake and induce anti-PEG antibody immune responses [204].

Chemical modifications of the oligonucleotide backbone have been shown to provide increased stability and enhanced delivery in vivo [205-207]. These strategies assist in nucleic acid delivery to the melanocytes, including site-specific bioavailability leading to accumulation at the disease site, improved circulation, reduced renal clearance, internalization by the cells, and gene vector release [208]. Additionally, the size, shape, and surface properties such as charge, surface modifications, or attachment of targeting moieties can influence the distribution of nanoparticles, and optimization of these parameters can improve localization at the target site [209].

Once the carrier is internalized by the cells, the nucleic acid molecules also need to undergo endosomal escape to elicit the desired response [210]. Endosomal escape can presumably be facilitated by the proton sponge effect [211-213] or by pore-forming sequences [214-216]. Ionizable lipids in the LNP lead to an influx of protons due to increased membrane potential and activation of proton pumps due to the acidic pH in the endosomes. This leads to increased osmotic pressure due to increased chloride ions in the endosomes to neutralize the membrane potential. This causes swelling and subsequent bursting of the endosomes, allowing the nucleic acid to escape [211-213]. These interact with the endosomal membrane, creating hexagonal structures that destabilize the membrane, releasing the nucleic acid payload in the cytoplasm [211, 213, 217]. In addition, peptide sequences have been shown to mediate membrane destabilization by either barrel-stave or toroidal pores formations [214-216].

Once in the cytoplasm, the nucleic acid molecules must reach the desired site, i.e., cytosolic or nuclear, to exert the expected biological response, namely miRNA replacement (miRNA), gene knockdown (siRNA), or gene replacement (plasmid DNA or mRNA) (Figure 5). The plasmid DNA, once inside the cell, needs to undergo nuclear internalization and cross the nuclear barrier to initiate the transcription process, which poses a secondary barrier. The mRNA undergoes translation to proteins in the cytoplasm. In the case of siRNA and miRNA, they also need to be incorporated into the RISC complex in the cytosol to either silence and knockdown genes or to regulate genes, respectively.

4.2. Systemic delivery and tumor microenvironment

In the case of advanced melanomas, generalized vitiligo, and systemic albinism, systemic delivery of therapeutic agents may be more effective. There are no approved treatments for albinism, though topical or systemic-delivered experimental therapies could be explored. Systemic delivery of naked nucleic acids is associated with the induction of inflammatory responses, enzymatic degradation due to endogenous nucleases, reduced cell-specific targeting, systemic toxicity, off-target effects, and rapid clearance [210, 218].

Additionally, in melanoma, the tumor microenvironment and drug resistance limit the effectiveness of therapeutic strategies [219, 220]. The melanoma tumor microenvironment is complex, including molecular factors, immune cell infiltration, and checkpoint expression. The tumor microenvironment mainly comprises extracellular matrix, regulatory T cells, tumor-associated macrophages, cancer-associated fibroblasts, dendritic cells, lymphocytes (CD4+/CD8+), and endothelial cells [221, 222]. However, based on the presence or absence of immune cell infiltration, the tumors can be classified as “hot” (with immune phenotype) or “cold” (exclusion of immune cells) [220]. As such, a case-by-case evaluation is important to determine whether immune checkpoint inhibitor-based therapies will be effective therapeutic strategies in such patients or not. Response to therapies is also dependent on the stromal confinement and spatial architecture within the tumors [220]. The tumor extracellular matrix presents as another biological barrier that can hinder therapeutic efficacy [50, 223]. For a delivery system to efficiently deliver genes to all the cells, it must also be able to penetrate the core of hyperproliferative tumors or lesions, whether in the case of melanoma or other melanocyte-associated hyperproliferation conditions. Once the delivery system reaches the target cell, it needs to follow the same mechanisms for cellular internalization and cargo release as described above to exert the expected biological response.

As melanomas are heterogeneous, precision medicine approaches by expressing specific biomarkers, developing stimuli-responsive particles to endogenous triggers like acidic or hypoxic tumor microenvironment, or exogenous triggers like light or radio frequencies, or the use of immunomodulators can assist in the elimination of cancerous cells [209, 221]. Using nucleic acids targeting the underlying mechanisms also assists in overcoming challenges associated with multidrug resistance [224, 225].

It is important to note that the majority of existing studies on targeted delivery to melanocytes focus on melanoma, particularly advanced metastatic melanoma. In many of these cases, even when tumors are injected subcutaneously in model systems, they do not recapitulate the unique anatomical and biological barriers involved in delivering cargo to epidermal melanocytes. Thus, while our focus in this review is on both topical and systemic delivery strategies targeting epidermal melanocytes, the majority of existing delivery research has been conducted in melanoma models. Consequently, much of the literature we reference below is skewed toward systemic delivery in the context of melanoma, and we note the limitations this poses for direct translation to melanocyte-targeted therapies.

5. Critical evaluation of current gene delivery strategies for melanocyte-associated disorders

A variety of systems have been explored for the delivery of nucleic acids to prevent or treat some of the melanocyte-associated disorders. In this review, we will focus on nonviral organic systems, i.e., liposomes, solid lipid nanoparticles, ionic liquids, polymeric carriers, and niosomes. For inorganic nanoparticles, readers are referred elsewhere (Figure 6) [226-228]. Table 2 encompasses the advantages, limitations, biocompatibility, and current clinical status of nonviral organic delivery systems for melanocyte-associated disorders.

Figure 6.

Schematic of representative organic nanoparticles evaluated for gene delivery to melanocyte-associated disorders, including carrier systems, the cargos that have been evaluated for delivery, and the delivery route for these carriers. ASO: Antisense oligonucleotide

Table 2.

Advantages, limitations, biocompatibility, and current clinical status of nonviral organic delivery systems for melanocyte-associated disorders

	Cationic liposomes	Lipid nanoparticles	Exosomes	Peptide- based nanoparticles	Polymeric nanoparticles	Niosomes	Ionic liquids
Advantages	Biodegradable, biocompatible, high nucleic acid encapsulation efficiency. Enhanced topical penetration (with deformable liposomes) [229]	Biocompatible, biodegradable, less toxic, improved stability, facilitates endosomal escape, can deliver genes to non-dividing cells, can be produced at large-scale, high transfection efficiency [230-233]	Cell derived, biocompatible, target specific cells, deliver nucleic acids, low immunogenicity [234-236]	Physicochemical properties of the nanoparticles govern bioavailability, cytotoxicity, systemic toxicity and half-life [237-239]	Biodegradable, reduced systemic toxicity and accumulation (in case of biodegradable polymers), scalable production, cellular targeting, uptake and rapid dissociation post internalization, physicochemical properties of the nanoparticles govern cytotoxicity and immunogenicity [240, 241]	Biodegradable, biocompatible, low immunogenicity, low toxicity, high stability, and easy production [242, 243]	Ionic liquids offer properties like high solubility, inherent charge, tunability, high polarity, thermal and chemical stability, and miscibility, making them useful for dissolving drugs with poor solubility or delivering macromolecules such as siRNA and miRNA [244-246]
Limitations	Positive charge of these vesicles can lead to cytotoxicity or inflammation and requires optimization. Cationic liposomes can have low blood stability due to opsonization, rapid clearance by the reticuloendothelial system and can have poor tumor penetration after systemic administration.[247, 248]	Reduced biodegradabil ity under some circumstances, rigid morphology, storage temperature and stability, immunogenic [231, 249, 250]	Lack of standardized isolation and purification protocols, scalability, stability, limited loading capacity, risk of contamination, potential for immune rejection and clearance, heterogeneity [234, 235, 251, 252]	Physical instability, low cell specificity, difficulty crossing cell membranes, immunogenicity, poor specificity, rapid systemic degradation, Various factors such as size, surface charge, and homogeneity govern the stability of these nanoparticles during circulation and require optimizations [237, 239, 253, 254]	Physical instability, systemic toxicity and accumulation (in case of non-biodegradable polymers), reduced cellular uptake, oxidative degradation, low loading and delivery efficiency, high production costs [240, 241]	Physical instability, scale-up issues (including high production time and costs), low drug loading [410, 411], storage temperature, stability, topical application of niosomes is limited [243, 255, 256]	High production cost, cytotoxicity, extent of ionic pairing, rapid enzymatic degradation [257, 258]
Biocompatibility	Yes, however, the positive charge of these vesicles can lead	Yes	Yes, however, factors like source and purification can affect biocompatibility [235]	Depending on the physicochemical properties of the peptides, they can be biocompatibile	Yes, but dependent on the physicochemical properties	Yes	Yes
Clinical translation	Yes	Yes	No	Yes	Yes	No	Yes

5.1. Lipid-based nanoparticles

Lipid-based nanoparticles primarily include cationic liposomes and solid lipid nanoparticles. While both the carriers comprise lipids, the orientation of lipids in these carriers differ. Some of the lipids commonly used in gene delivery for synthesizing cationic liposomes or lipid nanoparticles include DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), MVL5 (N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide), SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate), ALC-0315 (6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium), and Dlin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) (Figure 7) [259, 260]. Of these, SM-102, ALC-0315, and Dlin-MC3-DMA have been used in commercially available LNP-gene delivery systems, including the COVID-19 vaccines and patisiran (the first siRNA-based therapy to obtain FDA approval) [260]. Along with the lipids, these carriers also include neutral helper lipids such as cholesterol, DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) or DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) to improve the physicochemical properties of the vesicles and transfection efficiency [259, 260]. PEGylated lipids are also included to improve circulation and reduce immunogenicity for systemic delivery. These carriers can be tuned for targeted delivery and improved pharmacokinetic profiles.

Figure 7.

Structures of commonly used cationic lipids, ionizable lipids, helper lipids, cholesterol, and edge-activating agents.

5.1.1. Cationic liposomes

Cationic liposomes have been widely explored and offer a promising strategy for delivering genes to the target sites. They offer several advantages due to their biocompatibility and biodegradability [259]. Liposomes consist of a hydrophobic lipid bilayer and an aqueous core [260]. Various factors, including lipid composition, size, and surface properties of the cationic liposomes, affect the therapeutic efficacy of these vesicles, and these can be optimized for improving their efficacy and better cell selectivity for gene delivery [259, 261]. Endosomal escape of nucleic acids from the cationic liposomes is also variable based upon the physicochemical properties of the liposomes [262]. Increasing the concentration of pH-sensitive lipids like DOPE in cationic liposomes and the removal of cholesterol demonstrated higher knockdown due to increased release of siRNA [262]. However, the higher surface charge of the cationic liposomes is correlated with increased cytotoxicity [263]. Additionally, there are still some challenges associated with the clinical use of cationic liposome systems, including rapid clearance by the reticuloendothelial system (RES), opsonization, poor tumor penetration, endosomal escape, or lysosomal degradation (due to the positive charge of these vesicles) [259]. Various strategies are employed to overcome these challenges.

Cationic liposomes have been evaluated for targeting melanoma and other melanocyte-associated skin disorders. They have been shown to regulate MITF upon α-MSH stimulation by delivery of miR-141-3p and miR-200a-3p, leading to skin whitening due to suppression of melanogenesis and tyrosinase activity in mouse melanocytes [66]. This can be effective for targeting hyperpigmented disorders. Cationic liposomes complexed with DNAs or siRNAs have been explored in in vitro or in vivo melanoma models [264, 265]. These have also been evaluated as an imaging modality for understanding biodistribution, tumor growth, and tumor localization with bioluminescence imaging [265]. The efficacy of cationic liposome-based delivery systems in the treatment of melanomas has been investigated clinically. An early phase 1 study was initiated in Argentina in 2020 to evaluate the safety of combined immunotherapy and gene therapy in histologically confirmed melanoma in humans [266, 267]. The liposomes consisted of equimolar amounts of cationic lipid DMRIE and helper lipid DOPE, were complexed with plasmid DNA (HSVt k- HSV thymidine kinase) and co-administered with ganciclovir as a prodrug to patients intra/peritumorally [267, 268]. The objective for this combination therapy was to induce immune cell infiltration leading to tumor regression. The treatment did not show significant adverse effects; however, in two patients, the treatment was withdrawn due to disease progression [267]. The study did not meet the primary objective and was hence terminated [266].

Immunotherapy has demonstrated striking improvement in reducing the risk of recurrence and overall survival in patients with advanced melanoma [269, 270]. Various immune checkpoint blockade inhibitors have received clinical approval from the FDA as treatment options for advanced metastatic melanomas or as adjuvants. Some of these treatments include monoclonal antibodies targeting cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1 (PD-1) receptors. Adaptive immunity against cancer cells can additionally be regulated by targeting dendritic cells (DCs), which are a heterogeneous group of antigen-presenting innate immune cells [271, 272]. DCs promote activation of potent anti-tumor T cell response by redirecting cytotoxic CD8⁺ T cells against melanoma and influencing response of macrophages or natural killer cells [272, 273]. Cationic liposomes have been evaluated for transfecting dendritic cells using plasmid DNA and mRNA to suppress melanoma metastasis and activate dendritic cell-mediated anti-tumor response [274]. They showed a dendritic cell-mediated anti-tumor response, increased cytokine levels including IL-6, IL-1α, IFN-γ, and TNF-α, and reduced lung metastasis in vivo. They also demonstrated higher transfection efficiency with polycation lipids that were linked with spermine than with commercially available Lipofectamine 2000. Natural polyamines like spermine improve transfection efficiency by forming dense small particles for transporting nucleic acids and facilitating endosomal escape [275].

A clinical trial was conducted by BioNTech, targeting four tumor-associated antigens using a tetravalent mRNA-lipoplex vaccine system, for patients with advanced melanoma [276]. The liposomes comprised cationic lipid DOTMA or DOTAP, and helper lipids DOPE or cholesterol [277]. The vaccine targeted tumor-associated antigens, including NY-ESO-1, tyrosinase, MAGE-A3, and TPTE, through the activation of the antigen-presenting cells for subsequent induction of inflammatory cytokines and T-cell responses. The liposomes were designed to protect the naked RNAs from serum degradation and in vivo targeting [276]. The findings suggested the induction of strong CD4+ and CD8+ T cell immunity upon vaccine administration with synergistic effects with anti-PD1 therapy in checkpoint-inhibitor (CPI) experienced patients with melanoma [278]. The role of these tumor-associated antigens (NY-ESO-1, tyrosinase, MAGE-A3, and TPTE) was also evaluated by analyzing the protein and transcript expression of these in children for the feasibility of the tetravalent mRNA-lipoplex vaccine in pediatric melanomas. All four antigens were activated in pediatric melanoma, however, to a lesser extent compared to adult melanoma, except tyrosinase [279]. Further evaluation of tumor-associated antigens in pediatric melanomas is required for developing targeted vaccine systems.

While most in vitro studies involve the use of 2D cultures, 3D melanoma spheroid models developed using the hanging drop method offer a great opportunity to understand melanoma progression, invasion, and in vitro drug efficacy testing [280-282]. 3D cultures allow a better option for recapitulating and mimicking the tumor microenvironment compared to 2D cultures [281]. Triculture spheroids additionally offer a tool to understand crosstalk between melanocyte, keratinocytes, and fibroblasts. A 3D early melanoma spheroid model composed of SK-MEL 28 melanoma cells, CCD-1137Sk fibroblasts, and HaCaT keratinocytes was used to evaluate the transfection efficiency of an mRNA-cationic liposomal system consisting of DOTMA and DOPE (as helper lipid). The monoculture melanoma spheroids showed the highest eGFP expression compared to the other two cell lines, but a lower eGFP expression in triculture spheroids was observed with transfection limited to the outer layers of the cells [283].

For an effective therapeutic strategy, the combination delivery of nucleic acid molecules with small molecules has also been evaluated, which demonstrated a stronger anti-tumor synergistic effect. Co-delivery of siRNA targeting Bcl-xl and XY-4, Aurora Kinase-A inhibitor using cationic liposomes demonstrated enhanced efficacy in vitro and higher anticancer efficacy and apoptosis in vivo in a B16 melanoma mouse model [284]. XY-4 is an AURKA inhibitor that caused G2/M cell cycle arrest while silencing Bcl-xl induced apoptosis, offering a system with dual targeting effects.

Various stimuli have been explored for releasing molecules, including temperature, light, redox reagents, ultrasound, pH, and enzymes, etc. Since tumors tend to have an acidic microenvironment, carriers that are sensitive to pH have been explored for effectively delivering drugs or genes that are sensitive to acidic pH for releasing their payloads to tumor cells [285-290]. As previously described, the endosomal escape of nucleic acids is a critical step for the release of the molecules into the cytoplasm. The pH range of endosomes lies around 5.5-6.5, and various groups have demonstrated that cationic lipids with a pH-sensitive imidazole head group initiate osmotic swelling of the endosomes. This leads to the rupture of the endosomes and improved delivery efficacy of the cationic liposomes [291-294].

To evaluate the potential of pH-sensitive cationic liposomes for the delivery of paclitaxel and siRNA (Bcl-2) in the melanoma tumor model, a kojic acid-imidazole-based lipid was synthesized through a four-step process [295]. The kojic acid backbone was functionalized with an endosomal pH-sensitive imidazole ring. Kojic acid is a widely used and well-tolerated skin lightening agent that prevents melanin production by inhibiting tyrosine synthesis in melanocytes and hence was utilized for designing the lipid backbone. The inhibition of cell proliferation and reduction in tumor growth was demonstrated along with the potential of a synergistic effect of anticancer drug potency and multidrug resistance reversing ability of siRNA in the tumor [295]. Additionally, melanocytes have a neutral pH; however, the pH of melanosomes alters. The rate-limiting step for melanin synthesis- tyrosinase, is pH sensitive and requires acidic pH, while during the later steps, L-DOPA requires alkaline pH [296]. This causes active pH regulation within the melanosomes. The transfer of melanin from melanocytes to keratinocytes involves vesicle-mediated and phagocytic processes influenced by local pH. pH-sensitive drug carriers can potentially provide an effective therapeutic strategy, as there is a reversed pH gradient in melanoma with high internal pH.

A combination of nucleic acids complexed with cationic liposomes and metallic or peptide-based nanoparticles consisting of small molecules is another dual targeting strategy which has been explored. The co-delivery of MCM4 siRNA and cisplatin complexed in liposome and metal-organic frameworks (MOFs) based co-delivery system demonstrated pH-dependent release, strong anti-tumor activity, and induction of ferroptosis. MOFs have a porous structure for adsorbing drug and facilitating controlled release. The hybrid nanocarrier system, combining liposomes complexed with siRNA and metal-organic frameworks consisting of cisplatin, offer a dual-targeting system for treating malignant melanoma[297].

Combining cyclic RGD with cationic liposomes promoted cellular internalization of siRNA into melanoma cells through interaction with α_vβ₃integrin receptors [298]. Integrins are cell adhesion receptors consisting of α and β subunits. They play a vital role in normal physiology as well as tumor growth and metastasis. Studies have reported altered integrin expression in normal, benign nevi, and malignant melanocytes [299]. Thrombin receptor, PAR1, is also associated with the activation of angiogenic (VEGF, IL-8), invasive (MMP-2), or adhesion factors (integrins), and cationic liposome-based PAR-1 siRNA delivery demonstrated reduced metastasis, angiogenesis, and melanoma growth in vivo [300]. PAR1 also has a higher expression in metastatic melanoma compared to nevi or primary melanomas [301, 302]. It promotes migration, survival, and anti-apoptotic activity of melanoma cells [302].

Another active targeting strategy explored for targeting malignant melanoma is the use of folate-decorated cationic liposomes for delivering siRNA targeting hypoxia-inducible factor-1α (HIF-1α)- a regulator of oxygen homeostasis and associated hypoxic cellular responses and influences melanoma progression [303]. Folate receptors are upregulated in cutaneous malignant melanoma, and hence folate-decorative liposomes offer a promising approach to selectively target melanoma cells [304]. Delivery of HIF-1α siRNA showed significantly enhanced transfection efficiency, reduction in HIF-1α production, and anti-melanoma activity in vitro by inducing apoptosis in hypoxia-induced A375 melanoma cells [305]. Peritumoral delivery of folate-decorative liposomes for miR-21-targeted modified mesyl phosphoramidate (or μ-) oligonucleotide inhibited tumor growth in an in vivo xenograft tumor model with minimal toxicity [206]. This modification allowed improved and specific RNA binding, resistance to nucleases and reduced toxicity [206, 207].

Intact liposomes without any surface modifications or without the aid of any external stimuli cannot readily permeate the skin layers due to the barrier properties of stratum corneum and stratum granulosum. Melanocytes reside around the epidermal junction, and for effective delivery of nucleic acids, the active compounds must permeate through the stratum corneum and the viable epidermis. Combination of physical techniques such as non-invasive topical iontophoresis, low frequency ultrasound assisted or sponge spicules based-microneedle systems along with cationic liposomes or lipid nanoparticles have been explored for enhanced penetration in the skin for efficient delivery of nucleic acids to skin associated cancer cells [200, 201, 306]. The sponge Haliclona sp. Spicules (SHS) creates microchannels in the stratum corneum, permitting delivery of siRNA to deeper skin layers and further facilitating internalization [306, 307].

For minimal- or non-invasive delivery of cationic liposomes by topical application, researchers have evaluated ultradeformable cationic liposomal systems (also referred to as transferosomes, elastic liposomes) that penetrate the stratum corneum and retain nucleic acid molecules at the basal epidermis where melanocytes reside [308-310]. These differ from traditional liposomes as they include edge-activating agents, which improve membrane flexibility. Edge-activating agents are single-chain surfactant molecules that can be non-ionic, such as Tweens or Spans, cationic, including sodium cholate or sodium deoxycholate, or anionic, such as oleic acid [309, 311]. Such carriers can pass across the skin layers through the microlamellar spaces intact presumably due to the deformability characteristic that facilitates their permeation through the skin [309, 311]. Cevc et. al. proposed a hydration gradient-driven transport mechanism for the penetration of these vesicles in the skin layers [312]. Various groups have demonstrated the potential of ultradeformable liposomes to cross the stratum corneum, permeate the epidermis, and deposit in the upper dermis or effectively internalize into the basal epidermis and knock down the expression of target proteins [313, 314]. In a recent study, we demonstrated successful delivery of biologically active miR-211-5p complexed with ultradeformable cationic liposomes (UCL-211) in vitro and ex vivo [178]. We evaluated the role of different levels of pigmentation on cellular uptake of UCLs in melanocytes [178]. We confirmed delivery of biologically active miR-211-5p using fluorescent sponge reporter assay and phenotypic changes using quantitative phase imaging, as well as delivery of biologically active miR-211-5p in vitro. We also showed stabilization of BRAFV600E+ nevi melanocytes in the benign state in vivo using a transgenic nevus growth-arrested mouse model following transdermal delivery of UCL-211 [178].

While cationic liposomes offer several advantages for nucleic acid delivery, various studies highlight that these systems can be cytotoxic due to non-specific interactions with anionic host cell membranes, reduce immunomodulator secretion by depleting macrophages (and therefore can be immunogenic), and have lower in vivo transfection efficiency [229, 263, 315].

5.1.2. Lipid nanoparticles

Lipid nanoparticles differ from liposomes primarily based on their structural organization. LNPs consist of an outer lipid monolayer with multiple reverse micellar structures inside [327]. Lipid nanoparticles can protect the encapsulated molecules from degradation, providing increased circulation time, especially if they have surface modifications, and can also be tuned to control the release of drugs at tumor sites. Lipid nanoparticles can be synthesized using various techniques, including thin-film hydration, ethanol injection, T-junction mixing, and microfluidic mixing [328]. Multiple LNP-based therapeutics have been approved or are currently in clinical trials for drug and gene delivery [329]. BioNTech SE recently completed a Phase 1 study evaluating a liposome-based naked tetravalent RNA vaccine system targeting tumor-associated antigens in advanced melanoma patients [189]. Another study conducted by ModernaTX, Inc. is currently recruiting patients in Clinical Trial Phase 2 for individuals after complete resection of high-risk melanoma to assess recurrence-free survival upon treatment with individualized neoantigen therapy of mRNA-4157 formulated in LNP and intravenous infusion of pembrolizumab[316, 317].

Co-delivery of two oncosuppressor miRNAs, miR-199-5p and miR-204-5p, the main regulators of MAPKi therapy, by co-encapsulating them in lipid nanoparticles resulted in a reduction of melanoma cell proliferation and inhibition of tumors in BRAF-mutant melanoma cell lines A375 and M14 [318, 319].

High-throughput screening of lipids is another strategy being used to identify chemically diverse biodegradable ionizable lipids with high yield and improved transfection efficiency for nucleic acid delivery. This technique can be utilized to modify functional groups strategically to obtain diverse lipids. Using this strategy, Chen et al. presented a study wherein they identified an ionizable lipid iso-A11B5C1, which was comparable to SM-102, the lipid utilized in the COVID-19 vaccine formulation by Moderna, for its mRNA delivery potency. They utilized a Ugi-based three-component (amine, isocyanide, and aldehyde) reaction platform for the synthesis of the lipid. The lipid structure comprised aldehyde and isocyanide tails with an amine head group. These LNPs were synthesized by using helper lipids DOPE, C-14-PEG2000, and cholesterol to deliver mRNA to the muscle, minimizing off-target delivery. A potent anti-tumor effect in a B16-F10 melanoma vaccine mouse model was also demonstrated. LNPs synthesized using the chemically modified lipid had more localized effects at the target site, while SM-102 had a broader distribution, including high expression levels in the spleen and liver, comparatively [330].

Active targeting for melanocytes can be achieved through targeting specific receptors on the cell surface. One example is KIT, which is the most specific melanocyte cell surface receptor in the skin. Intradermal injection of KIT receptor-targeted LNP of NRAS targeting siRNA (siNRAS^Q61K) in transgenic Tyr::NRASQ61K mouse model to preferentially target nevus cells demonstrated selective knockdown of the mutant NRAS[331]. The LNPs consisted of cationic lipid DOTMA, neutral lipid DOPE, PEGylated lipid, and a targeting peptide with 27 amino acids combined with siRNA at a ratio of 1:4:1 (lipid:peptide:siRNA) for specifically targeting nevus melanocytes.

As previously mentioned, immunotherapy to activate T cells is an attractive strategy for the treatment of melanoma. CD3, a protein expressed on the T cell surface, leads to their activation and subsequent antitumor activity. Role of LNPs in delivering mRNAs for targeting macrophages expressing CD3, cytokines, or induction of T-cell-mediated immune responses to inhibit melanoma growth has been well demonstrated [332-335].

Dual targeting of mRNA encoding anti-TYRP1 CAR and anti-CD3 antibody for targeting T-cells using an LNP-based system demonstrated tumor infiltration, tumor volume and progression reduction, and enhanced immune response in an in vivo B16F10 melanoma model, along with high TRP1 expression levels [336]. Previous studies have highlighted that co-expression of CAR T cells with cytokines or immune checkpoint inhibitors can increase tumor infiltration and overall survival [337, 338]. Single peptide-modified anti-PD-L1 mRNA-loaded LNPs demonstrated reduced tumor burden and improved survival in a B16F10 melanoma model in mice. The single peptide construct was utilized as a virtual mRNA zip code, to control posttranslational protein localization by directing cells to secrete target proteins into blood circulation, improving the circulation time of the therapeutics[323].

Peptidomimetics are compounds that can mimic a natural peptide or protein. Studies have highlighted that the configuration of hydrophilic peptidomimetic headgroup of cationic lipids enhances the biocompatibility of the resulting lipid nanoparticles. Lipid functionalized peptidomimetics are another class of lipid nanoparticle systems explored for preferentially delivering siRNA to the tumor tissue in a B16F10 mouse melanoma model for inhibiting tumor growth [321]. These consist of amines conjugated to the peptidomimetic headgroup of the cationic lipids. The presence of an amine from ornithine in the peptidomimetic headgroup resulted in improved gene delivery with reduced cytotoxicity [339, 340]. The LNP system comprised lipids DoGo4, Chorn3, and mPEG DSP, where increased concentration of DoGo4 improved siRNA binding efficiency and reduced cytotoxicity [321]. The addition of amine groups lead to significantly reduced cytotoxicity of the LNP/nucleic acid carrier as well as enhanced target gene knockdown [339, 340]. Optimizations of the number of free amines conjugated played a role in nucleic acid delivery efficiency and biocompatibility. Short alpha amine peptidomimetics, DoGo2 and DoGo3 had enhanced siRNA delivery efficiency but moderate plasmid DNA delivery efficiency due to its larger size [339, 341]. A combination of amide bonds formed by either the alpha or delta amines on the head groups of ornithine provided higher plasmid DNA delivery efficiency with significantly reduced cytotoxicity [339]. Additionally, the ratio of free alpha or delta amines along with a linear delta amide linkage could be modulated for enhanced cellular penetration as well as intracellular siRNA delivery [339].

Combination therapy using lipid nanoparticles with small molecules or PROteolysis-Targeting Chimeras, which degrade target proteins, allows co-targeting with distinct molecules, enhancing therapeutic effectiveness [320]. Concomitant delivery of PTEN plasmid-loaded lipid nanoparticles and BRD4-targeted PROteolysis-TArgeting Chimera (ARV) loaded nanoliposomes targeting BRAF inhibitor-resistant melanomas showed substantial tumor growth inhibition and apoptosis in A375V BRAFi-resistant melanoma cells and 3D tumor spheroids [320]. BRD4 is overexpressed in melanoma cells and leads to resistance to BRAF inhibitors, therefore selective targeting can resensitize the melanoma cells to BRAF inhibitors, and it also targets c-Myc, an oncogenic protein upregulated in resistant melanomas [320, 335]. Upon co-delivery of PTEN plasmid and ARV, downregulation of c-Myc levels was also observed [320]. Co-delivery of a siRNA (siCD47) and etoposide (potent topoisomerase inhibitor) using lipid polymer hybrid nanoparticles in a B16F10 melanoma lung metastatic in vivo model showed higher reduction in tumor growth with the co-delivery in vivo following systemic administration. Activation of the immune response following siCD47 administration was confirmed by the increase in a number of macrophages and T-cells [322].

5.2. Exosomes

Extracellular vesicles (EVs) or exosomes are nano-sized biovesicles secreted by cells released into the extracellular environment. As they are biologically derived, they are less immunogenic [342-344]. EVs have been shown to carry proteins and genetic materials from the cell of origin, and can be taken up by neighboring cells. EVs have high potential as diagnostic or therapeutic biomarkers [344]. They can range widely in their size from 30 nm to 2 microns [342]. Reduction in hyperpigmentation in melasma patients can be achieved by downregulating melanogenesis by targeting MITF or tyrosinase. These are critical transcriptional factors for melanogenesis and can be potential targets for hyperpigmented melanocyte-associated conditions. The role of exosomes released by keratinocyte cell culture supernatant in vitro carrying miR-675, a H19 miRNA targeting MITF in patients with melasma demonstrated downregulation of H19-induced melanogenesis [324]. Crosstalk between keratinocytes and melanocytes through exosomal delivery of miR-330-5p, targeting tyrosine, led to the suppression in melanocyte pigmentation upon exposure to miR-330-5p [325]. These studies suggest that exosomes play a crucial role in cross-talk between different cells in the skin and understanding these mechanisms can offer effective strategies for targeting hyperpigmented disorders, which include melasma, ephelides, or solar lentigines.

The role of bioengineered exosomes in immunotherapy for targeting T cells has been evaluated [326, 342]. Targeting metastatic melanomas using BRAF siRNA encapsulated in mature dendritic cell-derived exosomes to silence BRAF mutation demonstrated serum stability to the siRNA with significantly smaller tumor volumes, inhibiting melanoma growth [326].

Gene editing techniques such as CRISPR-Cas9, Zinc Finger Nucleases (ZFNs), and Transcription Activator-Like Effector Nucleases (TALENs) allow precise modifications to the DNA [345, 346]. Delivery of BRAF-specific CRISPR/Cas9-ribonucleoproteins to melanoma cells for the purpose of gene editing using cationic LNP system has showed high transdermal delivery efficiency, anti-tumor efficacy and gene editing capability of the LNPs in a B16F10 melanoma in mice and reduced BRAF expression after treatment [347].

5.3. Peptide-based nanoparticles

Cell-penetrating peptides are positively charged, short peptide sequences that offer a promising strategy for nucleic acid delivery [348]. The hydrophobic and electrostatic interactions between the peptides and nucleic acids lead to the formation of self-assembled peptide-based nanoparticles [253, 349, 350]. To further improve the properties of these peptide-based nanoparticles, strategies have been employed to decorate the peptides with PEG, modify peptides or graft long-chain fatty acids or fusogenic moieties to improve their properties [253, 349]. Various factors such as size, surface charge, and homogeneity govern the stability of these nanoparticles during circulation and require optimizations [253].

Dual targeting strategies for the treatment of cancers have been widely explored. A peptide-oligonucleotide conjugate was developed using click chemistry by linking a PD-L1-binding peptide to anti-miR-21 for targeting tumor cells, and PD-L1 overexpressing macrophages were evaluated for dual targeting in melanoma. The PDL1-binding peptide had an azide-modification with a sequence of N₃-NYSKPTDRQYHF (Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe). There was a reduction in tumor growth, a reduction of endogenous miR-21 in the tumor, higher TUNEL-positive cells, confirming apoptosis, and a reduction in tumor-associated neovascularization with increased infiltration of T-cells in the tumor microenvironment [351]. Most clinical studies utilize only immunotherapies (checkpoint inhibitors); however, these show variable therapeutic responses. Dual targeting of tumor cells and tumor microenvironment in melanoma offers synergistic tumoricidal effect overcoming limitations of current strategies [351].

Peptide sequences with high specificity can be developed by generating libraries of peptides or proteins on bacteriophage surfaces [352-354]. Using the phage display technology, a short peptide, SPACE (skin penetrating and cell entering peptide- AC-TGSTQHQ-CG (Ala-Cys-Thr-Gly-Ser-Thr-Gln-His-Gln-Cys-Gly (Disulfide bridge: Cys2-Cys10))), was discovered for delivering nucleic acids across the skin barrier, which can facilitate penetration across the stratum corneum and epidermis.

Various studies have demonstrated the use of SPACE, a peptide penetration enhancer, for enhancing the permeability of siRNAs in the skin for the treatment of subcutaneous melanoma or other skin disorders [355, 356]. These peptides penetrate through the transcellular pathway and upon interaction with skin proteins, cause alterations in the protein secondary structures with negligible effect on skin integrity and cell toxicity [357]. Local high SPACE peptide concentration (over 4mg/mL) is an important factor for maximizing nucleic acid delivery in the cytoplasm through topical application [356]. Skin penetrating peptides non-covalently bind to keratin and transport across the skin through the transcellular pathway [357, 358].

Another short synthetic cyclic peptide identified using phage display was TD1 for effective transdermal delivery of biomolecules through intact skin [359]. Transdermal peptide-based MITF-siRNA cream with a TD1-R8 (ACSSSPSKHCG-oligoarginine) peptide was evaluated for efficient delivery through intact skin as an effective therapy for the treatment of facial melasma. Arginine-rich peptides enhance cellular penetration and the guanidine groups of arginine improve endocytosis, however, arginine residue length requires optimizations for improved therapeutic efficacy and reduced toxicity [360, 361]. Clinical evaluations in 31 patients with pigmented skin lesions showed significant lightening of the lesions upon application of the developed cream, which was confirmed by performing dermoscopy to demonstrate skin lightening [107]. However, this formulation only targeted epidermal melasma. Further optimizations or more efficient carrier systems are required for treating melanophages in dermal melasma. TD1 peptide has also shown follicular penetration and can be further evaluated for targeting the follicular melanocytes in vitiligo skin [359].

Most carriers provide siRNA transfection into cells via membrane fusion, including cationic liposomes and poly(ethylenimine) (PEI)-based nanoparticles. Another technology for delivering RNAi molecules is the combination of cell-penetrating peptides (CPP) or utilizing a permeation enhancer along with poly(amino acids), which provides improved efficacy for cellular internalization and low cytotoxicity. Various cell-penetrating peptides, including amphipathic peptide Mgpe9, cRGD or multiple arginine residue-rich peptides, have been evaluated for plasmid DNA or siRNA delivery in melanoma [193, 362-364]. All these peptides were arginine-rich due to their cell-penetrating properties, enhancing cellular internalization. However, due to toxicity, limited selectivity for target cells, and inherent instability during circulation and hence reduced efficacy have hampered clinical translation of these systems [365-367]. Mgpe9 comprises 9 arginine residues, 5 hydrophobic amino acid residues, 4 histidine residues, and 2 terminal cysteines, allowing stable nano complex formation between the peptide and the target plasmid DNA, which followed a non-follicular pathway for permeation through the skin [362].

5.4. Polymeric nanoparticles

Polymeric carriers have been widely explored for drug and gene delivery. The tunability of the composition, size, architecture, and surface chemistry of these carriers allows for the development of tailor-made systems. Biological barriers such as blood clearance, evading the immune system, tumor microenvironment, cellular internalization, and the release of nucleic acids during endocytosis are important parameters to be considered while designing these systems [368]. These nanoparticles rely on electrostatic interactions with the nucleic acids to entrap them in the core [369]. In-depth information showcasing different structures for polymeric nanoparticles can be found in previously published reviews [231, 368, 370, 371].

5.4.1. Polysaccharides

Different polysaccharides have been studied for nucleic acid delivery, of which chitosan-derived NPs and alginate have shown to be the most beneficial. These polysaccharides have demonstrated biodegradability, low toxicity, muco- and bio-adhesiveness, and biocompatibility. Polysaccharides explored include chitosan, alginate, dextran, and hyaluronic acid-based systems (Table 3) [372].

Table 3.

Lipid-based preclinical and clinical gene delivery strategies.

Carrier System	Disease	Nucleic Acid	Preclinical / Clinical	Strength	References
Cationic liposomes	Hyperpigmented disorders	miR-141-3p and miR-200a-3p	Preclinical	Regulated MITF upon α-MSH stimulation	[66]
Lipid Nanocapsules	Melanoma	pHSV-tk	Preclinical	Imaging modality for understanding biodistribution, tumor growth, and tumor colocalization with bioluminescenc e imaging	[265]
Cationic liposomes	Melanoma	Plasmid DNA	Clinical	Combination therapy with immunotherapy and genetics induced immune cell infiltration leading to tumor regression	[266, 267]
Cationic liposomes	Dendritic cells in melanoma	Plasmid DNA and mRNA	Preclinical	Dendritic cell-mediated antitumor response, increased cytokine levels including IL-6, IL-1α, IFN-γ, and TNF-α, and reduced lung metastasis	[274]
Cationic liposomes	Melanoma	Tetravalent mRNA	Clinical	Vaccine targeting tumor-associated antigens, through the activation of the antigen-presenting cells for subsequent induction of inflammatory cytokines and T-cell responses.	[276]
Ultradeformable Cationic liposomes	Melanoma	siGLO (RISC-independent control)	Preclinical	Passive delivery of siRNA-complexed ultradeformable cationic liposomes through the stratum corneum and delivery around the epidermal-dermal junction	[313, 314]
Ultradeformable Cationic liposomes	Melanocytic nevi	miR-211-5p	Preclinical	Demonstrated stabilization of BRAFV600E+ nevi melanocytes in the benign state in vivo	[178]
Lipid nanoparticle	High-risk melanoma	mRNA-4157	Clinical	Phase 1 studies showed improved neoantigen-specific T-cell response with mRNA monotherapy as well as combination therapy with pembrolizumab	[316, 317].
Lipid nanoparticle	Melanoma	miR-199-5p and miR-204-5p	Preclinical	Reduction of melanoma cell proliferation and inhibition of tumors	[318, 319]
Lipid nanoparticle	Melanoma	PTEN plasmid	Preclinical	Combination therapy with PROteolysis-TArgeting Chimeras, which degraded target proteins, allowed enhanced therapeutic effectiveness, and tumor growth inhibition	[320]
Lipid functionalized peptidomimetics	Melanoma	siRNA against STAT3	Preclinical	Inhibition of tumor growth, improved siRNA binding efficiency and reduced cytotoxicity	[321]
Lipid nanoparticle	Melanoma	siCD47	Preclinical	Co-delivery with potent topoisomerase inhibitor showed higher reduction in tumor growth	[322]
Lipid nanoparticle	Melanoma	Single peptide-modified anti-PD-L1 mRNA	Preclinical	Single peptide construct acted as a virtual mRNA zip code, controlling posttranslational protein localization, improving the circulation time of the LNPs	[323]
Exosomes	Melasma	miR-675	Preclinical	Downregulating melanogenesis by targeting MITF	[324]
Exosomes	Hyperpigmented disorder	miR-330-5p	Preclinical	Suppression in melanocyte pigmentation	[325]
Exosomes	Metastatic melanoma	BRAF siRNA		Mature dendritic cell-derived exosomes for silencing BRAF mutation leading to melanoma growth inhibition	[326]

5.4.1.1. Chitosan

Chitosan is a carbohydrate polymer that can complex and transfer oligonucleotides to cancer cells. Chitosan has been classified by the US Food and Drug Administration as Generally Recognized as Safe (GRAS). It is used as an excipient in several marketed wound dressings and devices [373-375]. Positive charge of chitosan at physiological pH facilitates the binding of negatively charged siRNA molecules through electrostatic interactions. Chitosan nanoparticles complexed with oncosuppressive miRNA, miR126, and functionalized with melanoma-specific marker, chondroitin sulfate proteoglycan 4 (CSPG4) antibody demonstrated synergistic inhibition in reducing the proliferation of BRAFV600E-resistant melanoma cells in combination with phosphatidylinositol 3-kinase (PI3K) inhibitor (PIK-75). The combination treatment with miR126 and PIK-75 remained effective after the development of resistance to MAPK/ERK inhibitors like vemurafenib or dabrafenib [376].

Chitosan-dependent photocytotoxicity in B16F10 melanoma cells has also been demonstrated using topical noninvasive photodynamic therapy strategy [377]. TAT peptides have been widely explored for their role as cell-penetrating peptides for delivering anti-cancer drugs to solid tumors [378, 379]. They can improve the solubility of compounds and their cellular uptake [379]. Trimethyl thiolated chitosan conjugated with HIV-1 derived TAT peptide and hyaluronic acid nanoparticles for downregulation of PD-L1 and STAT3 genes in melanoma have been evaluated. The dual PD-L1 and STAT3 siRNA delivery demonstrated enhanced effects compared to monotherapy of the siRNAs and led to an arrest in tumor progression with increased survival in vivo [380]. The membrane-permeable TAT peptide and the hyaluronic acid in the nanoparticles played a role in nanoparticle stabilization, provided protection during circulation, assisted in targeting the tumor cells and with siRNA release in the cells.

Ethosomes comprising mannosylated chitosan have been explored for the development of an mRNA vaccine system for transcutaneous immunization. Ethosomes are phospholipid, ethanol, and water-based soft vesicles with enhanced permeation properties across the skin, owing to the high ethanol concentration, which acts as a permeation enhancer [381]. Mannosylation can provide stealth properties due to the hydrophilic properties of the mannose sugars, improving permeability and bioavailability [382-384]. Additionally, macrophages and dendritic cells express high levels of surface mannose receptors, and using mannosylated chitosan for gene delivery induces mannose receptor–mediated endocytosis [385]. The mRNA used in this study encoded for three melanoma neoantigens, leading to enhanced maturation of dendritic cells. A transdermal patch in combination with the mRNA ethosomes and PD-L1 siRNA was further evaluated for the antitumor effects and immune response in a melanoma mouse model. A significantly higher antitumor immune response with the combination therapy was observed with higher TNF-α, IL-12, and IFN-γ levels and CD4+ and CD8+ T cell infiltration in the tumors [386]. This suggested vaccine system, comprising mRNAs and siRNAs, can be further explored for tumor treatment, specifically targeting the immune response in the tumor microenvironment.

Chitosan-shelled nanobubbles for nuclear factor E2-related factor 2 (Nrf2) siRNA delivery for overcoming cisplatin resistance in melanoma, along with external stimuli using ultrasound, showed improved transfection efficiency of the siRNA-loaded nanobubbles [387]. An oligomer of chitosan, chito-oligosaccharide (COS), has also been evaluated for the delivery of RNAi molecules. COS nanoparticles provide several advantages over chitosan-based nanoparticles as they are more flexible for controlling charge, size, and other physicochemical properties. COS has also exhibited several biological properties such as anti-tumor, anti-inflammatory, immunostimulant, and antioxidant effects. Liu et al. used COS to develop nanoparticles to improve the delivery of siRNA for the treatment of melanoma [388]. They synthesized nanoparticles comprising phenylboronic acid (PBA)-modified COS, and the siRNA was loaded into these nanoparticles by electrostatic complexation and chemical cross-linking. PBA targets sialic acid residues overexpressed on cancer cells improving selectivity and efficacy of the nanocarriers for cancer cells [389]. PBA is also stimuli-responsive, specifically to pH and ROS with low cytotoxicity and holds potential for further evaluation [389]. These nanoparticles significantly inhibited the proliferation of melanoma in cell studies and inhibited metastasis and tumor growth in mice models. COS-based nanoparticles loaded with siRNA showed improved physicochemical properties as promising nanocarriers for siRNA delivery in anti-tumor therapy [388]. Some of the challenges with chitosan-based nanoparticles include variable reproducibility due to variations in their batch to batch physicochemical properties, such as purity, molecular weight, and degree of deacetylation [390, 391]. There is additionally a lack of standardized classification for characterizing chitosan, leading to clinical translation challenges [390, 391]. Another limitation of chitosan-based nanoparticles is the issue with scalability and reproducibility for large-scale synthesis for commercialization. There is also a lack of long-term safety analysis in preclinical studies with chitosan-based particles [392].

5.4.1.2. Hyaluronic acid-based nanoparticles

Hyaluronic acid, a negatively charged anionic polysaccharide comprising non-sulfated glycosaminoglycans, is a biodegradable, non-immunogenic and biocompatible biomaterial, abundantly found in the skin, being the main component of the extracellular matrix [394, 395]. It comprises repeating units of N-acetyl-D-glucosamine and D-glucuronic acid, which are bound by beta-linkages [395]. The release of siRNA from HA-based nanoparticles intracellularly is triggered by the HA degradation due to intracellular hyaluronidase (Hyal) [395]. Hyaluronic acid nanoparticles coated with cationic liposomes (liposome-protamine-hyaluronic acid (LPH) nanoparticles) have been evaluated for delivering siRNAs in melanoma and metastatic lung models [393]. Protamine, an arginine-rich peptide, assisted in the condensation of HA and the siRNA, forming a stable core, while the cationic liposome coating protected the core from degradation and assisted in cellular delivery and internalization of the nanocomplexes [396, 397].

Redox-responsive, tumor-targeting smart anionic calcium phosphate (CaP) based nanoparticles stabilized with disulfide cross-linked HA (HA-ss-HA) loaded with siRNA have been explored in xenograft melanoma models in vivo. The thiolated hyaluronic acid (HA-SH) derivative provides an anionic outer "shell" creating a smart redox-responsive delivery system [398]. CaP nanoparticles have shown rapid and uncontrollable crystal growth, along with issues with tissue specificity, limiting the clinical applications of these systems.

5.4.2. Poly(ethylenimine) analogs

Cationic polymers such as poly(ethylenimine) (PEI) have been utilized for synthesizing polymeric nanoparticles for nucleic acid delivery with branched PEI having superior nucleic acid delivery properties than linear PEI [399-404]. The high cationic charge density of PEI increases its tendency to electrostatically interact with the negatively charged nucleic acid molecules with high transfection efficiency to form nano-sized complexes for gene delivery [400]. PEI delivers nucleic acid molecules into the cells following the proposed endosomal proton sponge effect [399].

Various studies have evaluated the feasibility of using siRNA-PEI complexes both in vitro and in vivo for delivery to a variety of cancers [403, 405, 406]. Branched PEI-siRNA complexes have been evaluated in melanoma in combination with stearic- or oleic acid derivatives, demonstrating tumor growth reduction with increased levels of Caspase 3, an apoptotic marker, and immune cell infiltration [407-410]. Stearic and oleic acid acted as condensation agents due to their structural characteristics, forming stable core nano-complexes and improving PEI-siRNA complexation and protecting from degradation [409]. However, the high cationic charge of PEI is associated with cytotoxicity, and hence, tolerability evaluations and tuning of PEI molecular weight to achieve efficient intracellular nucleic acid delivery with minimal cytotoxic effects are required [401, 411].

Cyclic RGD peptide-based cRGD–R9–cholesterol–PEI–PEG (RRCPP) nanoparticles using low-molecular-weight poly(ethyleneimine) (LMW PEI), poly (ethylene glycol) (PEG), cholesterol, and a cell-penetrating peptide conjugate cRGD (R8–cRGD) showed high specificity to the target cells and enhanced penetrating abilities [363]. Cyclic RGD with high-affinity ligands (arginine-glycine-aspartic) specifically bound to the integrin αvβ3 receptor on the tumor cells, allows targeted delivery to melanoma cells [205, 364]. Octa arginine (R8) peptide-based nano complexes with siBraf combined with coated microneedles demonstrated effective delivery at the tumor site and inhibition of tumor growth. Microneedle systems involved small micron-sized needles that can facilitate percutaneous delivery by creating microchannels in the skin [193].

5.4.3. Biodegradable copolymers

Other commonly utilized polymers for synthesizing nanoparticles for drug or gene delivery applications are poly(d, l-lactide-co-glycolic acid) (PLGA) or poly(ethylene glycol)-b-poly(d,l-lactide), which are biodegradable [402-404]. These nanoparticles protect the nucleic acid from degradation and allow controlled release [369]. PLGA-based polymeric scaffolds or nanoparticles have been evaluated preclinically and clinically in a phase 1 clinical trial for oligonucleotide or mRNA delivery in metastatic melanoma and have demonstrated dendritic and immune cell recruitment and activation [412, 413]. Clinical results demonstrated a favorable safety profile with immune activation and no autoimmune or inflammatory toxicity. However, the sample size was limited, and the authors suggested combining immune checkpoint blockade in future clinical trials as the T cell checkpoint markers were observed in treated patients [412].

Sirnaomics, Inc., a biopharmaceutical company, recently conducted a Phase 2b study for treating basal cell skin carcinoma (BCC) with their drug candidate STP705. They evaluated a histidine-lysine co-polymer-based polypeptide nanoparticle (PNP) for enhanced delivery of STP705, a siRNA therapeutic with dual-targeted inhibitory property to knock down both TGF-β1 (with pixofisiran) and COX-2 (lixadesiran) gene expression directly via localized injection [414-418]. The histidine-lysine copolymers (HKP) comprise H3K4b peptide, consisting of a lysine core with four branches that contain multiple repeats of histidines and lysines with a 4:1 w/w ratio for carrier: siRNA payload [417, 419]. These polymers had pH-buffering properties, leading to enhanced transfection and efficient nucleic acid delivery. Different histidine-lysine copolymers were evaluated for their effectiveness in carrying nucleic acids. The presence of histidine in this copolymer aided in the endosomal escape of nucleic acid molecules as it has a pKa of 6.0, and acted as a buffer, while lysine enhanced the transfection efficiency by complexing with the nucleic acids [420]. The linear HK polymers showed negligible transfection efficiency, while the degree of branching positively correlated with the transfection efficiency of DNA with the polymers, suggesting that the branched polymers condensed strongly with the DNA [421]. Higher histidine concentration had a higher effect on endosomal buffering, allowing higher transfection; however, higher lysine in the branched polymers was associated with increased toxicity, making optimization of the ratios and configuration an important factor to consider during optimizations [421]. Polymer H2K4b was effective for carrying plasmids, but not for siRNAs. H3K4b, a four-branched polymer, was more effective for intratumoral siRNA delivery compared to H3K8b, eight-branched polymer. Overall, the four-branched carriers were more effective and also easier to synthesize compared to the eight-branched. Increased polymer branching led to higher nucleic acid transfection, and polymers less branched than four were ineffective for siRNA or plasmid delivery [419, 422, 423]. However, eight-branched polymers were difficult to synthesize and expensive, and provided similar siRNA transfection efficiency to four-branched systems. Additionally, the introduction of glycine in H2K4b led to reduction in siRNA delivery due to reduced α-helical structures, affecting interaction and siRNA release into the cells [419]. There was a correlation between the endosomal pH and optimal polymer (linear or branched), with the branched polymers binding strongly to the DNA at acidic pH [423]. These studies demonstrated the importance of polymer structure and architecture for the successful delivery of nucleic acids.

A natural crosslinking polymer, genipin, has also been utilized for developing self-fluorescent cationic polymeric nanoparticles for safe and efficient RNA transfection for imaging purposes in B16F10 murine melanoma cells in vitro [424]. Genipin inherently fluoresces upon crosslinking and is less cytotoxic than glutaraldehyde which is the conventionally used crosslinking agent [425]. Cationic poly(amino acid) based gene carrier particles comprising poly(lysine) grafted with hyaluronic acid (HA) and tosyl-protected arginine residues (PLL-RT) for co-delivering two DNA plasmids, pshVEGF-A and pshPD-L1 have also been explored for tumor microenvironment reprogramming and for inhibiting tumor recurrence by provoking immune memory in B16F10 melanoma mouse model [426].

5.4.4. Dendrimers

Another class of polymers used for the delivery of either conventional molecules or oligonucleotides are dendrimer-based system. Encapsulation of small dendrimers in lipid vesicles, like liposomes, offers improved stability and cellular internalization properties to these systems. Dendrosomes complexed with nucleic acids have been explored for the melanoma and skin papillomas [427, 428]. DNA complexed with poly(amido amine) (PAMAM) dendrimers loaded into liposomes offered increased transfection efficiency with negligible toxic effects for melanoma treatment [427]. Physical strategies such as topical iontophoresis have also been used for the localized delivery of PAMAM dendrimers complexed with antisense oligonucleotide, bcl-2 in in vivo skin cancer models. Improved reduction in bcl-2 protein expression, tumor volume and apoptosis in the tumor were observed with iontophoretic delivery compared to intradermal administration of the Bcl-2-dendrimer complex [429]. Overall, these studies highlight that dendrimers could potentially be used in gene silencing approaches for melanocyte-associated disorders.

5.4.5. Micelles

Micelles are self-assembled amphiphilic polymers which have been extensively explored for the delivery of chemotherapeutic agents in cancer. Several hydrophobic-hydrophilic polymeric combinations have been studied for optimal loading, systemic circulation, stability, and delivery to the target cancer tissues. Some of the widely accepted hydrophobic polymers are poly(lactide) (PLA), poly(lactide-co-glycolide) (PLGA), polyesters, poly(caprolactone) (PCL), and poly(amino acids). The hydrophilic polymers used to wrap the hydrophobic core are poly(ethylene glycol), poly(oxazolines), chitosan, dextran, and hyaluronic acid. The micelles can be designed in a way that are stimuli-sensitive, which allows effective delivery of the therapeutic cargo. Some of these stimuli include pH, ultrasound, hypoxia, light, and temperature. pH-switchable cationic micelles as transdermal siRNA delivery nanoplatforms using cationic polymers (SCP-HA-PAE) by conjugating skin/cell-penetrating peptide (SCP) and hyaluronic acid (HA) to the amphipathic polymer (poly β-amino esters, PAE) were developed for treating melanoma. The micelles could penetrate through the stratum corneum and target melanoma cells. The pH sensitivity of these micelles stimulated endosomal escape in the cells, leading to enhanced siRNA delivery [430]. Targeting of immune responses by a significant increase in antigen-specific CD8+ T cells or NF-κB, leading to suppression of tumors using micelles has been evaluated [431, 432]. Micelles have also been utilized for developing skin-depigmentation conjugate system comprising antisense oligonucleotide (ASO) and polymerized prodrug, phenylethyl resorcinol. Phenylethyl resorcinol is a potent inhibitor of TYR and, as a result, has skin-depigmentation properties while ASO acted as a carrier, improving prodrug solubility and facilitating transdermal delivery. The micelles retained their bioactivity in vitro and reduced tyrosinase activity and amount of melanin. In an in vivo mouse model, the loaded micelles reduced melanin content leading to skin depigmentation upon topical application, confirming they were able to penetrate through the stratum corneum [433]. However, critical challenges with micelles still exist, making their clinical translation difficult. These include their instability during circulation, limited tumor cell uptake, sub-optimal loading capacity and release, and elicitation of immune responses [434, 435].

5.4.6. Nanoworms

Another class of polymeric nanoparticles is filomicelles and nanoworms. These particles have a nonspherical morphology that resemble fibers or worms. Worm-like biomimetic nanoerythrocytes for delivery of charge reversible siRNA showed effectiveness as a melanoma gene therapy strategy. These nanoworms were developed by membrane cloaking RBCs which protected siRNA from enzymatic degradation. The tumor targeting, anti-tumor efficacy, and tumor tissue apoptosis with these carriers was demonstrated in BALB/c mouse model of melanoma [208].

5.5. Niosomes

Niosomes, are nonionic surfactant molecules that form a similar structure to liposomes with a bilayer membrane enclosing an aqueous core. They offer some advantages over liposomes, including cost and stability. Several non-ionic surfactants such as Tweens, Spans, and Brij have been used for synthesizing niosomes which are stabilized by the addition of cholestrol [436]. The entrapment efficiency of niosomes is dependent on the various factors like hydrophilic-lipophilic balance, critical packing parameter, physicochemical properties of the payload, cholesterol content, phase transition temperature of the surfactants and hydration temperature [436, 437]. These vesicles can also squeeze through the small pores of stratum corneum and penetrate into the skin [438]. Synthesizing cationic niosomes using microfluidic mixing for transfection of melanoma cells with antiluciferase-siRNA consisting nonionic surfactant poly(oxyethylene) sorbitan trioleate (Tween 85)) and cationic lipid (didodecyl dimethylammonium bromide (DDAB)) have been explored in vitro in B16-F10 mouse melanoma cells and an in vivo mouse model with intratumoral administration [439, 440]. Studies have highlighted superior efficacy with improved biological activity and prolonged release with elastic cationic liposomes compared to conventional niosomes [438].

Elastic cationic niosome system loaded with tyrosinase plasmids (pMEL34 or pAH7/Tyr) have also been evaluated for vitiligo treatment [438, 441, 442]. The niosome system demonstrated higher expression of tyrosinase, storage stability, and transdermal delivery compared to conventional non-elastic niosomes [441, 442].

Some of the limitations with niosomes are their physical instability, and scale-up issues, including high production time and costs, as well as low drug loading [443, 444]. Additionally, storage temperature for niosomes also needs to be controlled to maintain stability. The gel-liquid transition temperature of the surfactant incorporated also plays a role in the drug retention properties of these vesicles [443]. Topical application of niosomes is limited, but can be overcome by incorporation in a gel-based system [256].

5.6. Ionic liquids

Ionic liquids also referred to as green liquids, have been evaluated for various applications specifically in the pharmaceutical and biomedical areas [244, 445]. They alter barrier function of the skin, significantly improving transdermal delivery [246]. These are mostly salt combinations consisting of cationic and anionic salts, which improve transdermal transport by acting both as enhancers or solvents [244, 246]. They can be used for antibody, small molecule drugs or gene delivery [245]. Ionic liquids offer properties like high solubility, inherent charge, tunability, high polarity, thermal and chemical stability, and miscibility, making them useful for dissolving drugs with poor solubility or delivering macromolecules such as siRNA and miRNA. [244-246]. Recent studies have demonstrated the use of ionic liquids, which alter the skin's barrier properties, enhancing the drug's permeation and improving overall efficacy. Ionic liquids as carrier system for delivering nucleic acid molecules have been explored for the treatment of melanoma [258, 446]. These systems have demonstrated improved cellular uptake and lysosomal colocalization in vitro with B16F10 cells and showed gene silencing and apoptotic activity in vivo[446].

Robed siRNA (siRNA-ionic liquid moieties) is another concept currently being explored for transdermal nucleic acid delivery. A biocompatible benzyl dimethyl alkyl ammonium ionic liquid moiety robed with GAPDH siRNA-IL demonstrated therapeutic GAPDH knockdown. Ionic liquid-siRNA complexes were synthesized following cation/anion exchange and then acid-base neutralization for treating skin diseases.[447]. These complexes can penetrate intact through the stratum corneum and be internalized by disease cells in the viable epidermis. The robed-siRNA complexes are independent of the nucleic acid sequence and hence can be explored for other skin conditions. However, the extent of ionic pairing and cytotoxic effects of ionic liquid-based systems needs to be tuned to improve the biocompatibility of these systems.

Liquid crystalline or lyotropic liquid crystal systems are also used for delivering small RNAs and can be synthesized by combining solvents with amphiphilic molecules, followed by ultrasonication or high-pressure homogenization to form nanodispersions. They have been evaluated for sustained release of low molecular weight drugs, nucleic acids, peptides and proteins [448]. Liquid crystalline nanodispersions have been explored in vitiligo for knockdown of TYRP-1 in melan-A cells. TYRP-1 is associated with melanin synthesis and leads to depigmentation due to autoimmune melanocyte destruction in vitiligo[449]. A combination system of ionic liquid and cationic liposomes has also been explored for treating melasma with MITF siRNA (IL-CL/p-siM), which showed downregulation of tyrosinase and MC1R levels, leading to inhibition of melanin synthesis in melanocytes in vitro. Ionic liquid was incorporated for its permeation-enhancing properties to improve transdermal delivery, while the cationic liposomes protected the siRNA from degradation and facilitated cellular internalization. The anti-pigmentation effects after treatment with IL-CL/p-siM were also evaluated in a clinical trial conducted on 34 volunteers with no epidermal pigmentation, and observed skin lightening and reduced hyperpigmentation of the test area [450]. Ionic liquid-based solid-in-oil (S/O) dispersion has also been explored for the delivery of nucleic acids in vitro and in vivo in B16F10 melanoma cell lines for improving skin permeation and cellular internalization [451].

6. Current limitations with gene delivery approaches for melanocyte-associated disorders

While most of the melanocyte-associated disorders are influenced by alterations in the sequence or expression of the genes discussed in Section 3, the vast majority of existing and current research in the field focuses on melanoma therapy, specifically on systemic delivery. Some exceptions include recent studies focusing on topical delivery strategies using lipid-based nanoparticles for melanocyte-associated disorders, primarily focused on melanoma and vitiligo. Exploration of the potential therapeutic value of gene therapy-based approaches for melanoma chemoprevention and non-melanoma melanocyte-associated disorders is limited and requires expansion, both through additional efforts towards target identification as well as gene delivery approaches to target those identified pathways or genes. Similarly, addressing therapeutic delivery to melanocytes in the skin remains a major challenge, largely due to the lack of tractable experimental systems as well as limited knowledge regarding the underlying disease mechanisms and targets.

Topical delivery could potentially be an attractive strategy for melanoma chemoprevention or for melanoma in situ, though it is still underexplored with no approved gene-therapy-based preventive options. Systemic neoadjuvant and adjuvant immune checkpoint therapy with pembrolizumab and nivolumab has achieved regulatory approval for stage IIB-IIC melanomas; however, no gene delivery therapy has received similar approval [452-455]. Once melanomas metastasize, systemic delivery strategies are required for treatment and to prevent further progression. Similarly, other melanocyte-associated disorders, although socially debilitating, are extremely under-evaluated, with limited focus on gene and drug delivery for treating these conditions, including vitiligo, albinism, and melasma.

7. Challenges, opportunities, future directions

With the notable exception of melanoma, melanocyte-associated disorders are mostly benign though the prominent clinical appearance can make them socially debilitating for patients. Various strategies have been explored for targeting melanocyte-associated disorders. In this review we focused on lipid and polymer-based delivery systems for targeted gene delivery to melanocytes. Apart from lipid-based and polymeric nanoparticles, viral vectors, physical techniques and metal or inorganic nanoparticles have also been evaluated for nucleic acid delivery in melanocyte-associated disorders [7, 191, 197-199, 456-459]. Microneedle devices, along with penetration enhancers, have been evaluated to reduce hyperpigmentation through the delivery of tyrosine-targeted self-delivering RNAi molecule, which can permeate through the stratum corneum and the dermis [123].

Additionally, the epidermis is primarily comprised of keratinocytes, while the number of melanocytes in healthy skin is approximately 5% of the overall cell population. Targeting melanocytes for melanocyte-associated disorders can be achieved by targeting melanocyte-enriched receptors like MC1R, c-Kit, Grm1, receptor protein kinases, N-cadherin, αvβ3 integrin, and endothelin receptor, among others [460-463]. Nanoparticles functionalized with a melanoma-specific marker, chondroitin sulfate proteoglycan 4 (CSPG4) antibody, have also been evaluated [343]. Photosensitive peptide derivatives of α-MSH have shown to bind to MC1R on melanoma cells, further leading to receptor-mediated internalization with minimal damage to the neighboring keratinocytes [462]. Additionally, in a study from our group, we demonstrated in vitro that normal human melanocytes had the highest cellular uptake of ultradeformable cationic liposomes (UCLs) compared to human epidermal keratinocytes and human dermal fibroblasts [178]. Although the exact reason is unknown, some studies suggest melanocytes are susceptible to higher lipid uptake due to the process of melanogenesis.

For many pigmentation-related conditions, fully accurate models are lacking, and most have limited in vitro or in vivo platforms for preclinical evaluation. Existing models also present additional challenges, including differences in skin structure, particularly relevant for studies targeting topical delivery. Most preclinical studies rely on mouse models; however, despite similarities in the basic architecture of human and mouse skin (epidermis, dermis, and hypodermis), there are significant differences in overall thickness, permeability, and complexity as well as the location and function of melanocytes. Additionally, the immune system in humans versus mice has several differences, which can lead to challenges in clinical translation [464-466]. As a result, the development of effective delivery strategies and reliable assessments of efficacy will first require the establishment of more accurate systems for evaluating both delivery efficiency and biological impact.

In vitro skin models offer a promising opportunity for preclinical evaluation of delivery systems. Various model systems have been explored for mimicking the skin architecture, including 2D monolayer cell culture systems with keratinocytes, fibroblasts, and melanocytes or complex in vitro 3D models, such as spheroids, microfluidics, organ-on-chip models, transwell-based assays, or bio-printed skin models, which can also include extracellular matrix to recapitulate the skin structure [467-470]. While 2D models offer information on cellular cytotoxicity, uptake, and pathophysiology, they lack the intricacies involved with cell-cell communications and altered states in disease conditions. 3D models can be generated with multiple cell types and are able to recapitulate some anatomical structures and can closely mimic some diseased cell behavior, especially in melanoma; however, they lack vasculature and immune cells, have a poorly developed stratum corneum, lack tight junctions, hair follicles, sweat glands, and are often complex to develop [469, 471]. Some human skin equivalent 3D models are also commercially available for drug testing; however, they primarily recapitulate healthy skin for drug screening or sensitivity testing, but lack intricacies associated with disease conditions [469, 472]. Further in-depth detailed information on in vitro skin models can be found in excellent recent reviews [469, 471-473].

3D models recapitulating melanocyte-associated disorders could offer potential insights into disease development, progression, as well as for in vitro gene and drug testing for developing targeted therapies [467]. Development of effective models needs to be further explored to understand these complexities and the dynamic interaction between the diseased cells and microenvironment, specifically in the case of melanoma [280-282, 474].

In the case of melanoma, human melanoma arises from the epidermis and then invades the dermis as the condition progresses; however, in mice, melanomas are of dermal origin. Most preclinical studies evaluating melanoma utilize the B16F10 metastatic melanoma cell line for in vitro and in vivo testing. This cell line is of murine origin, and although widely used, it does not recapitulate the hallmarks and complexities of human melanomas, including the tumor microenvironment, the genetic mutations associated with human melanomas, or the precise cell of origin [475, 476]. Additionally, these cells are implanted and not present within the standard architecture of the skin. This model rapidly develops lung metastasis and is advantageous for evaluating metastasis. However, for developing the metastasis model, the B16 cells are often injected intravenously, which does not recapitulate metastasis development from the primary tumor. This is a significant disadvantage of this model, as the initial processes involved in metastatic transformation from the primary tumor cannot be evaluated [475]. Findings from B16-based models have historically failed to consistently predict human outcomes. Given the biological differences between B16 cells and human melanoma, as well as structural disparities between mouse and human skin, the preclinical utility of this model, particularly for evaluating topically applied compounds, should be interpreted with caution.

To improve the translational capabilities of murine models, genetically modified models, including transgenic or knockout models, or xenograft-induced humanized models, could provide higher translatability [477-484]. Further in-depth information for various next-generation melanoma models has been discussed elsewhere [478]. A study also developed the use of xenograft models with full-thickness human skin grafted onto nude mice [485]. In a study from our group, we demonstrated the arrest of melanocytes in the nevus state upon transdermal delivery of miR-211-5p using ultradeformable liposomes by coupling both a transgenic nevus growth-arrested mouse model and adult human skin explants [178].

Although significant advances have been made in developing carrier systems for gene delivery to melanocytes, further progress is needed to overcome key challenges, including targeted delivery, physicochemical stability, safety upon administration, efficient cellular internalization, and successful transfer of nucleic acid cargo to melanocytes.

Lipid-based nanoparticles for gene delivery have received clinical approval. However, their cold storage temperature requirement as well as susceptibility to degradation during storage affect their shelf life. To overcome this issue, lyophilization of the nanoparticles is often performed and the type of cryoprotectant utilized plays a role in long-term stability [259, 486]. While cationic liposomes have been widely explored for gene delivery for melanocyte-associated disorders, their positive surface charge can cause immunogenicity and cellular toxicity and requires optimization [259, 486]. These lipids can additionally stimulate immune activation and can induce pro-inflammatory cytokine secretion [486, 487]. Surface modifications with peptide headgroups, hyaluronic acid derivatives, or PEGylation have shown reduced toxicity and enhanced transfection efficiency [488-490]. Various factors like lipid composition, and nitrogen to phosphate (N/P) ratio can influence the physicochemical properties, cellular internalization, and cytotoxicity [229, 262]. Incorporation of helper lipids like DOPE, and DPPC can also reduce toxicity associated with cationic liposomes [489]. Additionally, ionizable lipids offer a safe alternative to cationic lipids to overcome these limitations [491].

One of the primary challenges for polymeric delivery systems is their lack of tumor cell selectivity [492]. Additionally, natural polymers are associated with batch-to-batch variability and lack tunability [368]. PEI is associated with cytotoxicity, while PLGA can be recognized and rapidly cleared by the reticuloendothelial system. PEGylation of these systems has been shown to improve the safety profile [492, 493]. Incorporation of targeting moieties, modifications, or a combination of multiple carrier systems with different physicochemical properties also provides an opportunity to overcome challenges with transfection efficiency, systemic stability, targeted delivery, cellular uptake, controlled cargo release and overall improved therapeutic efficacy.

Overall, to develop safe and effective targeted nucleic acid delivery strategies, more effort is needed to develop better carrier systems and improved in vitro and in vivo models to fully explore these platforms' clinical potential. Further evaluation of underlying mechanisms, genes involved, and targeting strategies for melanocyte-associated disorders is required.

Figure 8.

Successful delivery of biologically active miR-211-5p complexed with ultradeformable cationic liposomes (UCL211) in vitro and in vivo in a transgenic nevus growth model. a. Experimental design for in vitro studies with fluorescently labeled UCL-211. FITC- F-DOTAP from the UCLs; LP- light pigmented melanocytes; MP- medium pigmented melanocytes; DP- dark pigmented melanocytes. b. Effect of pigmentation on UCL-211 uptake. Presence of melanin pigment in normal human melanocytes (NHM) did not affect the uptake of UCL-211. c. Comparing uptake of UCL211 in different skin cells. NHM had the highest UCL-211 uptake compared to human dermal fibroblast (HDF) and human epidermal keratinocytes (HEK). d. Delivery of biologically active miR211-5p by UCL-211 in vitro was confirmed by determining if miR replacement of miR211-5p caused differentiation in melanocytes using Phasefocus LivecyteTM System. Phenotypic switching was observed over 72h with changes in cell morphology in UCL211 and PMA (positive control) treated groups. e. NHM with BRAFV600E mutation can form melanoma (malignant) or nevi (benign). f. In vivo evaluation in a transgenic nevus growth-arrested model demonstrated miR replacement with UCL-211 drives BRAFV600E melanocytes to the nevus state. 4HT- 4-Hydroxytamoxifen. Figure adapted from [178].

Table 4.

Polysaccharide-based preclinical gene delivery strategies.

Carrier System	Disease	Nucleic Acid	Preclinical/ Clinical	Strength	References
Chitosan nanoparticles	BRAFV600E-resistant melanoma	miR126	Preclinical	Nanoparticles complexed with miRNA and functionalized with melanoma-specific marker for synergistic inhibition of melanoma cells	[376]
Trimethyl thiolated chitosan, HIV-1 derived TAT peptide and hyaluronic acid nanoparticles	Melanoma	STAT3 and PD-L1 siRNA	Preclinical	Dual PD-L1 and STAT3 siRNA delivery demonstrated enhanced effects compared to siRNA monotherapy in reducing tumor progression	[380]
Mannosylated chitosan-based ethosomes	Melanoma	mRNA ethosomes and PD-L1 siRNA	Preclinical	Significantly higher antitumor immune response with the combination therapy	[386]
Chitosan-shelled nanobubbles	Melanoma	Nrf2 siRNA	Preclinical	Overcoming cisplatin resistance in melanoma through ultrasound-mediated delivery	[387]
Phenylboronic acid-modified chito-oligosaccharide	Melanoma	Survivin-targeted siRNA	Preclinical	Significantly inhibited melanoma cell proliferation and tumor growth	[388]
Liposome-protamine-hyaluronic acid nanoparticles	Melanoma	Anti-CD47 siRNA	Preclinical	CD47 silencing in tumors and tumor growth inhibition	[393]

Highlights.

Disorders manifest as melanocytic malignancies, hyperpigmentation or hypopigmentation.

Gene delivery is a promising strategy for developing treatments for these conditions.

Lipid and polymer particles have been widely studied for delivering nucleic acids.

Abbreviations

UVR

ultraviolet radiation

FDA

Food and Drug Administration

DNA

deoxyribonucleic acid

RNAi

RNA interference

ASO

antisense oligonucleotides

miRNA/miR

microRNA

siRNAs

short interfering RNAs

shRNAs

short hairpin RNAs

dsiRNA

dicer substrate RNAs

CRISPR

clustered regularly interspaced short palindromic repeats

ZFN

zinc-finger nucleases

MSC

melanocyte stem cells

TYR

Tyrosinase

MC1R

melanocortin-1 receptor

ASIP

agouti signaling protein

TYRP1

tyrosinase-related protein 1

DCT

dopachrome tautomerase

KITLG

KIT Ligand

GPR143

G protein-coupled receptor 143

OCA

Oculocutaneous albinism

AP3B1

adaptor related protein complex 3 subunit beta 1

MLPH

melanophilin

MYO5A

Myosin VA

BRAF

B-Raf proto-oncogene, serine/threonine kinase

NF-1

Neurofibromin 1

NRAS

Neuroblastoma RAS viral oncogene homolog

RAS

rat sarcoma

RAF

Rapidly Accelerated Fibrosarcoma

MEK

Mitogen-activated protein kinase

CDKN2A

Cyclin-Dependent Kinase Inhibitor 2A

PTEN

Phosphatase and Tensin Homolog

TERT

Telomerase Reverse Transcriptase

FGFR3

Fibroblast Growth Factor Receptor 3

PIK3CA

Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha

PRAME

Preferentially Expressed Antigen in Melanoma

AXL

AXL receptor tyrosine kinase

IRF4

Interferon Regulatory Factor 4

EXOC2

Exocyst Complex Component 2

BNC2

Basonuclin Zinc Finger Protein 2

IL

Interleukin

HMOX1

Heme Oxygenase 1

SOD1

Superoxide Dismutase 1

IFNγ

Interferon-gamma

AAV

adeno-associated virus

JAK1

Janus Kinase 1

α-MSH

alpha-Melanocyte-Stimulating Hormone

bFGF

basic Fibroblast Growth Factor

SNAI2

Snail Family Transcriptional Repressor 2

GNAQ

Guanine Nucleotide-Binding Protein (G protein), q polypeptide

BAP1

BRCA1 Associated Deubiquitinase 1

TP53

Tumor Protein 53

DHA

dihydroxyacetone

YAG

Yttrium Aluminum Garnet

PKC

protein kinase C

UTR

untranslated regions

PEG

poly(ethylene glycol)

LNP

lipid nanoparticles

RISC

RNA-Induced Silencing Complex

DC

dendritic cells

CTLA-4

cytotoxic T lymphocyte-associated protein 4

TLR

Toll-like receptors

GFP

Green Fluorescent Protein

AURKA

Aurora Kinase-A

MOF

metal-organic frameworks

PAR1

Protease-Activated Receptor 1

HIF-1α

hypoxia-inducible factor-1α

UCL

ultradeformable cationic liposomes

CAR T

Chimeric Antigen Receptor T cell

PD-L1

Programmed Death-Ligand 1

EV

extracellular vesicles

PEI

poly(ethylenimine)

CPP

cell-penetrating peptide

GRAS

Generally Recognized as Safe

MAPK

Mitogen-Activated Protein Kinase

ERK

Extracellular signal-regulated kinase

TAT peptides

Trans-Activator of Transcription

HIV-1

Human Immunodeficiency Virus type 1

STAT3

Signal Transducer and Activator of Transcription 3

PLGA

poly(d, l-lactide-co-glycolic acid)

PAMAM

poly(amidoamine)

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase