Recent Progress and Future Directions: The Nano-Drug Delivery System for the Treatment of Vitiligo

Introduction

Vitiligo is an acquired idiopathic dermatological disorder characterized by the appearance and development of white macules related to the apoptosis or selective damage of melanocytes. Approximately 0.5–1% of the individuals are diagnosed with vitiligo.¹ The highest reported prevalence has been recorded in India (up to 8.8%), followed by Mexico (2.6–4%) and then Japan (1.68%²). Depigmentation usually occurs on exposed areas of the body including the face, neck and arms. The extreme effects of vitiligo often bring dramatic psychological burden to afflicted patients. Although males and females are equally affected by this disease, women more often openly express and address vitiligo for cosmetic purposes and are more likely to seek treatment.³ The incidence of vitiligo often presents through distinct familial clustering with reports that 20% of the vitiligo patients also have relatives diagnosed with the disorder.⁴ Individuals with a positive family history usually have an earlier age of onset as well as a longer duration⁵ compared to individuals that do not have a family history of the disease.

Thus far, the etiology of vitiligo has been found to result from multiple factors and has not yet fully been elucidated. In the 1950s, Lerner investigated 600 vitiligo patients and found that most patients with segmental vitiligo suffered from emotional imbalances or hyperhidrosis, eventually giving rise to the neural theory.⁶ As research progressed, factors including stress, autoimmune diseases, melanocyterrhagy and autoinflammation have been identified as important factors contributing to vitiligo.⁷ Of all theories, autoimmune disease or autoinflammation^8–11 and oxidative stress¹² as well as interactions among and between them have been accepted as some of the most important factors contributing to the disease.¹³ In the disease, antigen-presenting cells activate T cells through the presentation of melanocyte antigens where T cells then directly kill the melanocytes. It has been reported that endogenous killer and inflammatory dendritic cells are in a hyperactive state in patients with vitiligo.^9,10 Various cytokines including INF-γ,^14–16 CXCL10,^14,17,18 TNF-α, IL-6 and IL-17^19–22 are also secreted by innate cells through an autoimmune response.

Separate from the autoinflammation theory, oxidative stress is also an important risk factor for vitiligo. Melanin synthesized by melanocytes is toxic which stimulates the cell stress signaling pathway in these cells. Moreover, active energy metabolism in the mitochondria leads to an excessive accumulation of reactive oxygen species (ROS). This also gives rise to the development of vitiligo.^23,24

Briefly, when tiny lesions generated through a sunburn, viral infection or physical trauma occur, epidermal damage-associated molecules are released into the body and oxidative stress levels are increased. The adhesion of melanocytes is lost and inflammasomes are activated by the release and induction of molecular and oxidative stress products. After a series of immunoreactions, specific cytotoxic T cells are accumulated on the skin, regulatory T cell activity is down-regulated and inflammatory cytokines as well as autoantibodies are produced, ultimately leading to immune-based melanocyte destruction (Figure 1).²⁵ Likewise, oxidative stress contributes to the onset of depigmentation, and subsequent undesired autoimmune responses lead to the progression of vitiligo.^26–30 Here, we review all nano-drug delivery systems that can be used for the treatment of vitiligo, hoping to offer useful resources and spark inspiration for future research focused on novel vitiligo treatments (Figure 2).

Figure 1 Illustration of the pathogenesis of vitiligo.

Figure 2 A scheme of the nano-drug delivery systems for vitiligo therapy.

Therapeutic Approaches

Since the mechanics and dynamics of vitiligo are not fully understood, it has been challenging to develop an effective treatment for vitiligo. The ultimate goals for vitiligo treatment are to prevent the expansion of existing white spots on the skin, promote repigmentation and reduce psychological burden. Currently, strategies that are typically used for vitiligo treatment mainly include drug therapy and phototherapy (Table 1). Corticosteroids, such as betamethasone dipropionate, clobetasol dipropionate and mometasone furoate, have been first-line options for vitiligo treatment due to their anti-inflammatory and immunosuppressive effects.³¹ In recently published guidelines for vitiligo treatment, calcineurin inhibitors such as tacrolimus and pimecrolimus were also recommended as first-line approaches for the management of vitiligo.^32–34 Some clinical trials have shown that calcineurin inhibitors have a similar effect as glucocorticoids, especially for facial leukomas.^35,36 In addition, the accumulation of oxidative products in the skin is also an important factor contributing to melanocyte dysfunction. Hence, various antioxidants that remove excess ROS and hydrogen peroxide from the epidermis of vitiliginous skin have been used to restore the oxidation-antioxidant system of the skin.³⁷ Oral antioxidants such as polypodium leucotomos, vitamin E, vitamin C and minocycline are used for an antioxidation treatment strategy against vitiligo. Separate from drug therapy, phototherapy including narrow-band ultraviolet (NB-UVA) and psoralen ultraviolet A (PUVA) are relatively safe physical-based approaches.^38–40 Since phototherapy alone and in combination with the drugs mentioned are highly successful in inducing repigmentation, these strategies have been extensively adopted in clinical practice.⁴¹

The onset, progression and management of vitiligo is usually a long process ranging from months to years. Thus, long-term use of certain drugs or treatment approaches will inevitably result in a variety of side effects. For example, the side effects of the long-term use of glucocorticoids include skin atrophy, acne and folliculitis.⁴² The large-scale use of corticosteroids is not feasible and may lead to a high number of side effects. Thus, corticosteroids are not considered appropriate for the treatment of generalized vitiligo. Although glucocorticoids and calcineurin are clinically complementary and have similar therapeutic effects, a recent study emphasized that tacrolimus showed no apparent therapeutic effects in mitigating segmental vitiligo.³² The most common side effect of calcineurin inhibitors is a burning sensation during the first two weeks of treatment. In addition, the financial costs of calcineurin inhibitors are much higher than the costs of corticosteroids, which can be a significant financial burden for patients.^37,43,44 Comparing these different treatments, antioxidant drugs are milder with no obvious side effects. Nonetheless, accepted consensus guidelines do not recommend the use of topical antioxidants as a single therapy for vitiligo since most studies were restricted by a limited number of patients.⁴⁵ In terms of a non-drug treatment, phototherapy for vitiligo requires frequent treatment (two or three times weekly), which brings inconvenience to patients. Despite the higher success rates that these strategies have for repigmentation, recurrence rates still remain high. Over 50% of the patients are prone to recurrent white spots on regimented skin within the first year after therapy is discontinued.^25,46 In addition, side effects such as headaches, nausea and ocular as well as renal toxicity may occur as a result of phototherapy treatment.^47,48 In addition, these therapies are also associated with a risk of skin cancer. Thus, phototherapy is recommended only when major treatments are found to be ineffective by the NHS.⁴⁹

With the development of nanotechnology, voluminous nano-drug delivery systems have emerged and been applied to enhance drug penetration through the skin. These systems include microemulsions, nanoemulsions, nanoparticles, lipid carriers and many other nanovesicles and exhibit prominent advantages over conventional methods. In the field of nanomedicine, the number of existing literature focused on the treatment of vitiligo is relatively small.

Table 1 Treatment Approaches for Vitiligo

Nano-Drug Delivery System in the Treatment of Vitiligo

Human skin is the largest of the body's organ and helps to carry out many essential functions including such as acting as a physical barrier, aiding in immune defense, temperature maintenance, UV protection, and moisture retention.⁵⁰ Three layers comprise the skin: stratum including the corneum, dermis, and hypodermis. The exposed layer and the stratum corneum contain 70–90% protein and 5–15% lipids, and aid in the process of influencing the role of protecting the body from external and environmental stressors and pollutants.⁵¹ One of the most significant challenges of transdermal delivery such as to treat afflictions such as vitiligo is that only a few therapeutic compounds possess ideal penetration behaviors and characteristics.⁵² The best existing technology merely allows drugs with a molecular mass of 100 Da and good lipophilicity to permeate the skin successfully.^53–56 In addition, another obstacle in achieving the desired therapeutic effects is the incompetence of the current dosage forms including creams, ointments, lotions, gels, and other vectors.⁵⁷ Generally, all the problems associated with conventional topical preparations suggest that reform and innovation of transdermal drug delivery for the treatment of vitiligo are urgently needed to improve the understanding of this affliction and to increase the possibility of better patient outcomes.

Recent advancements in cutting-edge nanotechnology provide an unprecedented opportunity to overcome shortcomings related to conventional methodologies (Figure 3). In particular, by taking advantage of some features of the stratum corneum, there is potential for the use of a nano-drug delivery system that can enhance the transdermal penetration of drugs and increase therapeutic effects.⁵⁸ A large number of novel nano-drug delivery systems carrying therapeutic agents are being developed, such as liposomes, polymeric nanoparticles, microspheres, solid lipid nanoparticles and nanofibrous structures (Table 2)

Figure 3 Existing nano-drug delivery system for vitiligo therapy.

Table 2 Nano-Drug Delivery System for Vitiligo Therapy

Liposomes and Niosomes

Liposomes are particles formed by lipids that consist of a double layer similar to the natural cell membrane enclosing an aqueous core. Containing non-toxic, biodegradable properties, liposomes are regarded as the most promising nanocarrier for topical drug delivery.⁶⁹ The apparent success of liposomes may be the result of lipid vesicles fusing into the stratum corneum, allowing the therapeutic to permeate into deeper skin layers.⁷⁰ Liposomes also increase the dissolution of the applied drugs and augment drug solubility on skin surfaces. Furthermore, liposomes are seen as drug repositories that allow for sustained rates of drug release and help reduce the frequency of treatments.⁷¹ One group⁵⁹ prepared an 8-methoxypsoralen-loaded liposomal treatment through a film hydration method. After a series of purges, it was found that the diameter of the final vehicle ranged from 100 to 500 nm and showed excellent penetration behavior when compared to controls treated with hydroalcoholic solution. All nano vehicles displayed high capacities for sustaining the levels of 8-MOP release and its permeation––accumulation into skin layers.

Another group⁶¹ reported the effects of both psoralen and resveratrol that were co-loaded with ultradeformable liposomes (UDL) for the treatment of vitiligo. Psoralen is a natural furanocoumarin derivative used in combination with UV or PUVA for the treatment of various skin diseases.^72,73 Due to the presence of a surfactant, the vesicle changes its shape to respond to conditions from the external environment.⁷⁴ Resveratrol stimulates mitogen-activated protein kinase signaling and has been demonstrated to have antioxidant activity.⁷⁵ Other groups⁷⁶ have conducted optimization studies and the most appropriate formula was selected based on particle size, PDI and zeta potential. Co-loaded liposomes were prepared through modified film hydration and liposome particle size was 120 nm with drug loading efficiencies between 2.5% and 5%. In vitro studies and kinetics showed that the carrier can sustain the release of both drugs over prolonged periods of time. In vitro cell studies showed that the carrier significantly stimulated melanin and tyrosinase activity without affecting the antioxidant capacity of psoralen and resveratrol.

One group⁶⁰ prepared baicalin and berberine samples that were co-loaded with ultradeformable vesicles and demonstrated their potential as adjuvants for the treatment of vitiligo. Baicalin and berberine were selected as curative agents due to their antioxidant, anti-inflammatory and proliferative effects.^76–78 In vitro studies showed that preparation enhanced permeation of drugs and antioxidants. Photoprotective effect evaluations showed that co-loaded vesicles increased melanin and tyrosinase activities.

Others⁶³ constructed an elastic cationic noisome, loading the human tyrosinase plasmid pMEL34. The maximum loading amount of pMEL34 in the elastic vehicle was 150 mg/16 mg of niosomal determined by gel electrophoresis and documentation. Due to the enhancement effects of ethanol on vesicle elasticity, cumulative amounts and flux at 6 h post-treatment with pMEL34 were significantly increased in comparison with pMEL34 loaded into nonelastic cationic niosomes. The level of tyrosinase-associated activities were found to be four times higher than the respective levels for free plasmid and plasmid loaded into nonelastic niosomes, indicating that elastic cationic niosomes have potential as efficient gene topical delivery systems for vitiligo treatment. In 2012, a human tyrosinase plasmid pMEL34 by Tat peptide was described and found to enhance melanin production. The vesicular size and zeta potential were proved that the carrier is still in the range of stable dispersion. In vitro studies have indicated that this type of preparation promotes both tyrosinase gene expression and melanin production with little to no cytotoxic side effects.⁶²

Another group⁶⁶ prepared 8-MOP ethosomes through a central composite design (CCD). Conventional liposomes deliver drugs to superficial layers of the skin, while ethosomes improve the permeation and retention in skin layers of the drugs.⁷⁹ The in vitro skin permeation study showed that this preparation induced enhanced transdermal efficacy, optimized ethosomal formulation and produced significant accumulation of 8-MOP, likely as a result of ethosome deformation. In vivo studies were consistent with in vitro results. Among all the elaborated topical transdermal delivery approaches, lipid nanocarrier is the most optimal one for its high biocompatibility and drug encapsulation efficiency. An increasing number of researchers in this field are paying attention to various novel vesicle delivery systems that possess percutaneous penetration and better therapeutic effects.

Microemulsions

Microemulsions are thermodynamically stable colloidal systems with a transparent appearance. The conventional formula of microemulsions consists of water and oil stabilized by surfactant and sometimes also contains a cosurfactant.⁸⁰ Based on the physical and chemical properties of surfactants and ingredients of microemulsions, microstructures of microemulsions vary. The advantages of microemulsions used as transdermally delivered drugs show them as being an attractive technological platform for novel pharmaceutical formulations.^81–84 One group⁸⁵ reported a clobetasol propionate-loaded microemulsion-based gel that successfully overcame the poor solubility of clobetasol propionate. The effectiveness of the preparation indicated that the stratum corneum swelled due to water retention caused by gel formulation thereby assisting clobetasol propionate penetration into the skin. A clinical study indicated that the extent of repigmentation in patients treated with a clobetasol propionate-loaded microemulsion-based gel was faster and more profound when compared to control groups. Numerous studies and literatures have shown that microemulsion can significantly enhance the percutaneous permeability of drugs though the exact mechanisms that have not been fully understood. It is worth noting that apart from the internal microstructure, the composition or internal phase structure largely determines the permeation of microemulsion. Microemulsions have been a promising approach in the transdermal delivery of anti-inflammatory drugs (NSAIDs) with a fairly obvious improvement in the permeation rate.

Nanoparticles

The emerging use of nanoparticles is one of the most impactful components of nanomedicines. Due to large surface-to-volume ratios, nanoparticles not only enhance the contact area between a drug and target tissue but also permits drug release in a controlled manner.⁸⁶ In recent years, nanoparticles have been incorporated into common and daily goods. Nanoparticles penetrate the skin based on its size, charge and overall structure.⁸⁷ Metal nanoparticles such as palladium and platinum boost enzyme activity and exhibit anti-inflammatory capacity in UV-treated HaCaT keratinocytes. In addition, the rate of apoptosis in cells pretreated with nano-Pt was significantly reduced compared to controls.⁸⁸ A mix of Pd and Pt nanoparticles termed PAPLAL has a history as a treatment for chronic diseases such as burns, gastric ulcers and rheumatoid arthritis.⁸⁹ PAPLAL also exhibited SOD catalase activity and significantly lowered O₂- generation levels in mouse skin tissue. Both in vivo and in vitro results showed that PAPLAL effectively suppressed endogenous superoxide levels through SOD and catalase activity via the AHR and NRF2 pathways.⁶⁷ Further investigation using palladium and platinum nanoparticles in the treatment of vitiligo should be performed with caution since metals may possess allergens.⁹⁰ By coating or other means of modification, nanoparticles can overcome their inherent shortcomings to achieve efficient transdermal delivery and even release drugs with targeting behavior.

Principles of Nano-Drug Delivery Systems Designed for Vitiligo Therapy

Enhancing the Penetrate Capacity of Therapeutic Agents

Similarities between lipid particles and the stratum corneum make liposomes excellent vectors for delivery of therapeutics.⁹¹ Conventional liposomes are thought to be inadequate for transdermal delivery, but more advanced liposomes such as invasomes, transferosomes, ethosomes and niosomes can overcome disadvantages seen with original methods (Table 3).

Invasomes were first proposed as novel modified liposomes in 2003.⁹² The presence of soy-phosphatidylcholine (SPC), ethanol and terpenes make significant improvements in percutaneous permeability compared to traditional liposomes.^93,94 One group investigated the transdermal ability of invasomes loaded with ferulic acid. Skin permeation experiments revealed that invasomes possess good permeation capacity and are an ideal carrier for the transdermal delivery of ferulic acid.⁹⁵

Transfersomes were first reported for transdermal delivery in 1992. Surfactants such as sodium cholate, spans and tweens make these carriers more elastic in comparison to traditional liposomes. Another group prepared a transferosome-encapsulating dexamethasone and evaluated its in vivo performance in a carrageenan-induced rat paw edema model. After a series of formula optimization studies, Span-80 was selected as the optimum edge activator that offered maximum deformability. These results demonstrated that drug-loaded transferosomes have better antiedema activity when compared to liposomes, indicating that transferosomes possess better penetration ability.⁸⁷

Table 3 Brief Introduction of Novel Liposomal Systems

Microneedles (MN) are micron-sized needle projections with a height of 10–2000 μm and a width of 10–50 μm that painlessly penetrates the skin.⁹⁸ In recent years, transdermal drug delivery using microneedles has been widely studied.^99–101 A combination of microneedles and nanocarriers has also been considered for the treatment of diabetes,¹⁰² cancer therapy¹⁰³ and immunotherapy.¹⁰⁴ Various nanoparticles with antioxidant or anti-inflammatory properties can be used as therapeutic agents to remove excess ROS and control vitiligo progression. However, poor water dispersibility and weak percutaneous permeability restrict application.¹⁰⁵ The combination of microneedles and nanoparticles brings hope for vitiligo therapy. Kim et al reported gold nanoparticles that were successfully able to be delivered into the hamster cheek at a depth of 100–200μm.¹⁰⁶ Nevertheless, the main drawback of treating vitiligo with microneedle is that the frequent use of microneedle will inevitably lead to stubborn skin damage and even inflammation. A comprehensive microneedle drug delivery strategy deserves attention and development.

By applying a mild electrical current, iontophoresis allows ionized substances to pass through the adjacent skin or tissue without invasion.^107,108 The combination of nano-drug delivery systems and iontophoresis was first reported in 1996. However, unsatisfactory results were obtained using this method.¹⁰⁹ Another group further demonstrated the mechanisms behind the combination strategy. This group observed connexin43 phosphorylation and filamentous actin depolymerization when charged liposomes crossed the intercellular space. The Ca²⁺ inflow was also stimulated by the electrical stimulus. All evidence suggested that the electric current changed the physiological properties of the skin and enhanced the permeation of liposomes.¹¹⁰

Increasing Therapeutic Agent Accumulation: The Receptor Ligand Binding Mechanism

Melanocytes are dendritic cells that synthesize and secrete melanin and are located in the basal cell layer of the epidermis. There are plenty of ways therapeutic agents can overcome the stratum corneum barrier and penetrate deeper into the skin. However, driving and accumulating the drug to the target site still remains a challenge. The affinity of the receptor and ligand that encompasses active targeting is widely utilized to exert specific retention. Many receptors on the surface of melanocytes enable the construction of such active targeting vectors.

Melanocortin-1 receptor (MC1R) is a G protein-coupled receptor specifically distributed on the surface of melanocytes (Figure 4). Endogenous ligands of this receptor include α-MSH, β-MSH, γ-MSH and adrenocorticotropic hormone (ACTH). Among these ligands, α-MSH shows the strongest affinity with MC1R.¹¹¹ The amino acid sequence of α-MSH is simple, allowing it to serve as a target molecule enabling nano-drug delivery systems to aggregate in the vicinity of melanocytes.

The endothelin receptor is another receptor located on melanocytes that participates in a variety of physiological activities. During the transformation of melanocytes into melanoma cells, endothelin B receptor expression gradually increases.¹¹² One group reported that Endothelin-1 (ET-1) promotes melanin synthesis and the formation of dendrites in normal human epidermal melanocytes.¹¹³ Similarly, a seven-transmembrane receptor on keratinocytes termed protease-activated receptor-2 (PAR-2) plays a central role in the transportation of melanosomes.¹¹⁴ SLIGRL and SLIGKV are two PAR-2-activating peptides that have the same activation capacity as PAR-2 without receptor cleavage.^115,116

Figure 4 Schematic diagram of the role of MC1R in melanin synthesis and secretion.

Promoting Melanin Regeneration

Therapeutic goals for vitiligo treatment consist of inhibiting the inflammatory response and promoting melanin regeneration. Many therapeutic regimens only focus on inhibiting the inflammatory response rather than promoting repigmentation. Thus, there has been a growing demand for drug delivery systems that inhibit the inflammatory response while promoting melanin regeneration.

Tacrolimus is a calcineurin inhibitor that weakens T cell activity and the secretion of proinflammatory cytokines, thus promoting melanocyte migration and repigmentation.^117,118 However, its application is restricted due to the risk of lymphoproliferative disease, narrow therapeutic index and low solubility.^119,120 One group prepared a nanolipid carrier containing lipophilic solubilizers to improve drug solubility. This carrier exhibited a high entrapment efficiency of 96.66% and a significantly higher drug release in vitro and in vivo compared to the commercial ointment. Meanwhile, a skin irritation study revealed less irritation using this carrier when compared to the control group.¹²¹

As described, many receptors specifically located on the surface of melanocytes and keratinocytes play important roles in controlling melanocyte proliferation, melanin synthesis and melanin migration. On the other hand, their endogenous ligands or the analogues can be used as peptide drugs to activate biochemical reactions associated with melanocytes. α-Melanocyte-stimulating hormone (a-MSH), a tridecanoic peptide derived from pro-opiomelanocortin (POMC), plays a significant role in stimulating melanogenic pigmentation in skin tissues.¹²² After binding to MC1R, the receptor typically sends signals by increasing cyclic adenosine monophosphate (cAMP) and exhibits powerful anti-inflammatory effects.¹²³ The short fragments of α-MSH or its derivatives Lys-Pro-Val (KPV) and (CKPV)₂ are reported to possess similar or even stronger biological activity.¹²⁴ ET-1 can also regulate the synthesis of melanin, promote melanocyte proliferation and help form dendrites. Accordingly, these endogenous molecules or their analogues can be anchored on transdermal delivery systems to ameliorate transdermal efficiency as well as promote melanin regeneration.¹²⁵

Conclusion

Vitiligo is a common depigmenting disease affecting 1% of the population worldwide. In some regions, the white spotting caused by vitiligo is confounded with leprosies and has devastating effects on patient psychological health and social activities.¹²⁶ Treatment and management of vitiligo still shows intractable challenges for both scientists as well as dermatologists. More evidence suggests that the dynamics and pathogenesis of vitiligo are closely related to the immune response of skin and melanocyte response to the immune response. Thus, vitiligo should be treated as a chronic immune disease rather than a problem mainly regarding an approach from a regenerative medicine perspective.¹²⁷ Nevertheless, the rapid development of nano-drug delivery systems has brought new insights and ideas for the treatment of vitiligo. These novel methods facilitate the ability for developed drugs to possess sustained or controlled release behavior, enhances the therapeutic efficiency and reduces side effects.

Despite the great potential of the available nano-drug delivery systems, certain limitations exist. Most research regarding novel drug delivery systems for the treatment of vitiligo settle for the minimum control of symptoms rather than optimal outcome to completely cure the disease. Despite these challenges, it is inevitable that scientists will perform and complete the research needed to further optimize nano-drug delivery system efficiency. Various receptors on the surfaces of melanocytes or surrounding keratinocytes including granulocyte colony-stimulating factor (G-CSFR) and melanocortin receptors 1~5 (MC1R~MC5R) increase the feasibility of developing targeted agents. Their endogenous ligands or analogues exhibit great potential for the treatment of vitiligo with minimum side effects. One group proposed ultrashort peptides containing cysteine that spontaneously self-assembled into hydrogels. In vivo studies revealed that one of the formulas exhibits great biocompatibility and limited allergenic potential.¹²⁸ In the following studies, self-assembly behavior, nanostructure formation, hydrogelation and phase transition of peptide nanostructures of two selected tripeptides were investigated by X-ray crystalline diffraction and scanning electron microscopy.¹²⁹ A consistent hydrophobic backbone of acetylated Leu-Ile-Val-Ala-Gly plays the role of self-assembly and the C-terminal residue has an obvious impact on the rate and extent in which the peptide fibril interacts with an SMD.¹³⁰ By elucidating the crosslink and polymerization mechanisms of small molecule peptide drugs, various peptide-based topical drug delivery systems may be an inevitable trend for vitiligo treatment.

Here, we reviewed the pathogenesis, present therapeutic methods and existing nano-drug delivery systems that have been used and that will be used in the future for the treatment of vitiligo. Vitiligo can eventually be better controlled, managed and even cured considering the significant progress that has been made and the ongoing development of strategies to tackle this malady.