Drug Repurposing and Nanotechnology for Topical Skin Cancer Treatment: Redirecting toward Targeted and Synergistic Antitumor Effects

Abstract

Skin cancer represents a major health concern due to its rising incidence and limited treatment options. Current treatments (surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy) often entail high costs, patient inconvenience, significant adverse effects, and limited therapeutic efficacy. The search for novel treatment options is also marked by the high capital investment and extensive development involved in the drug discovery process. In response to these challenges, repurposing existing drugs for topical application and optimizing their delivery through nanotechnology could be the answer. This innovative strategy aims to combine the advantages of the known pharmacological background of commonly used drugs to expedite therapeutic development, with nanosystem-based formulations, which among other advantages allow for improved skin permeation and retention and overall higher therapeutic efficacy and safety. The present review provides a critical analysis of repurposed drugs such as doxycycline, itraconazole, niclosamide, simvastatin, leflunomide, metformin, and celecoxib, formulated into different nanosystems, namely, nanoemulsions and nanoemulgels, nanodispersions, solid lipid nanoparticles, nanostructured lipid carriers, polymeric nanoparticles, hybrid lipid–polymer nanoparticles, hybrid electrospun nanofibrous scaffolds, liposomes and liposomal gels, ethosomes and ethosomal gels, and aspasomes, for improved outcomes in the battle against skin cancer. Enhanced antitumor effects on melanoma and nonmelanoma research models are highlighted, with some nanoparticles even showing intrinsic anticancer properties, leading to synergistic effects. The explored research findings highly evidence the potential of these approaches to complement the currently available therapeutic strategies in the hope that these treatments might one day reach the pharmaceutical market.

Key Concepts

• Skin cancer is a major health concern with rising incidence and limited treatment.

• The search for novel therapeutics is marked by high cost and a likely failure.

• Drug repurposing allows more cost-effective accelerated pharmaceutical development.

• Nanosystem-based formulations lead to improved therapeutic efficacy and safety.

• The combination of drug repurposing and nanotechnology leads to improved outcomes.

1. Introduction

The field of oncology is focused on understanding cancer and on the pursuit of novel therapeutic agents to fight against it, with a considerable amount of research focusing on skin cancer due to its rising prevalence and severe consequences.¹⁻³ Despite recent advancements, the cost and time associated with bringing new drugs to the market are considerably high. Moreover, conventional treatment options are often associated with complex procedures and systemic toxicity and unwanted side effects. Drug molecules with proven anticancer effectiveness have failed to follow through due to leading to life-threatening adverse events, and even several approved treatments are only tolerable up to certain doses, at which they may not have the highest efficacy.⁴⁻⁶ These limitations have prompted a reevaluation of existing drugs, setting off the concept of drug repurposing.⁷⁻⁹

Drug repurposing offers a unique advantage by capitalizing on pre-existing knowledge of drug safety profiles, pharmacokinetics, and mechanisms of action. This strategy not only accelerates the development process of novel therapeutics into clinical applications but also addresses the financial burden associated with novel drug discovery. Repurposed drugs can target pathways involved in skin cancer, offering a potential solution to complement existing therapies.¹⁰⁻¹²

Additionally, a pivotal development in drug delivery that complements the repurposing approach involves the utilization of nanocarrier systems, which can facilitate drug encapsulation and controlled release, enabling targeted and localized delivery and enhancing drug stability, permeation through the skin, and cellular uptake, contributing to an improved therapeutic effect. In the context of skin cancer, nanocarrier-based topical formulations offer several distinct advantages over traditional systemic approaches. Additionally, the topical route of administration allows for direct application to the affected area, minimizing systemic exposure.¹³⁻¹⁵

This Perspective aims to explore the potential of combining drug repurposing with nanocarrier-mediated topical delivery in the context of skin cancer therapy. By examining and providing a comprehensive overview of the current landscape, we aim to highlight the potential of topical formulations featuring repurposed drugs in nanocarrier systems as a promising approach to advancing the battle against skin cancer.

1.1. Using the Skin for Drug Administration: Characteristics and Challenges

The skin is the largest organ in the human body covering about 15% of the body mass and all of the body’s external surface. This continuous self-renewing tissue serves as the barrier between the “inside” and the “outside” of the living organism, blocking the entry of pathogens, protecting the body from chemical assaults, heat, infections, and radiation, and regulating the loss of water and solutes that are essential for human body function.^16,17 Due to the many drawbacks associated with the intravenous (IV) route, alternative options have emerged in order to avoid unwanted secondary effects as well as to increase targeted therapeutic effects. Here the skin has been of great use, namely through topical and transdermal administration (Figure 1), which have been proposed as promising alternative administration routes for the treatment of several diseases, including skin cancer.¹⁸⁻²⁰

Figure 1.

Illustrated difference between topical and transdermal administration and schematic representation of the main skin permeation routes (produced with BioRender).

Although in both drug molecules can be transported through the stratum corneum (SC) by passive diffusion via paracellular (intercellular), transcellular, or appendageal (transfollicular) route (Figure 1), topical delivery systems are typically formulated to target the precise local tissue, whereas transdermal delivery systems are specifically designed to achieve systemic absorption of the drug.²¹⁻²³ Benefits associated with using these administration routes, especially over IV or oral administration, include: providing effectiveness in low and continuous doses, allowing to reduce the dosing frequency; self-administration being a possibility; avoiding drug degradation due to first pass metabolism and other variables that could affect drug absorption, such as pH changes, presence of degradation enzymes, and drug-food interaction in gastrointestinal tract; good patient compliance, being noninvasive and convenient to use (no needle phobia or need to swallow pills); and the fact that the formulation can be easily removed in the event of acute toxicity.²⁴⁻²⁶ Yet, as happens with all administration routes, drug administration on or through the skin does not exist without its limitations, including the already demonstrated therapeutic effectiveness variability that comes with naturally occurring interindividual differences (arising from gender, ethnicity, age), administration site influence, and even disease states, such as skin conditions which may alter the skin’s barrier properties.²⁷⁻³⁰ Additionally, the most important obstacle when using the skin for drug delivery purposes is its potentially low permeability to a large variety of drug molecules.^19,31,32

Permeation through the SC is considered the rate-determining step for drug diffusion in topical and transdermal administration with this layer’s lipidic composition and distinctive structural organization resulting in low permeability for most drug molecules.^31,32 Therefore, there are some requirements for drugs to be successfully applied through the skin, including: high potency (dose <10 mg/day); moderate lipophilicity (log P = 1–5); low molecular weight (less than 500 Da); a low melting point (<250 °C); and being uncharged molecules.^24,33,34 In topical formulations, the great majority exist in the form of semisolids, such as creams, ointments, gels, and pastes, although some liquid dispersions (solutions, suspensions, and emulsions) and solid powders can also be found. Transdermal products are mainly formulated as transdermal patches, which allow for the controlled release of the drugs.³⁴⁻³⁶ Nevertheless, most of these conventional topical and transdermal formulations face relevant issues including low drug skin permeation and/or retention, limited capacity for controlled release, and drug degradation. To tackle these issues, novel nanometric formulation strategies have emerged.³⁷⁻³⁹

1.2. Skin Cancer: Incidence, Types and Current Conventional Treatments

In recent decades skin cancer cases have been increasing across the world, representing the most common type of malignancy in humans.^1,40,41 Although genetic factors have a significant influence on skin cancer risk, nearly all skin cancer types are related to UV exposure, being the most common (yet modifiable) risk factor (Figure 2), with its mutagenic and carcinogenic effects being related to the induction of deoxyribonucleic acid (DNA) damage and errors in its repair and replication.⁴²⁻⁴⁵ Skin cancers are classified into two main categories: nonmelanoma skin cancer (NMSC) and cutaneous melanoma (CM) (Figure 2). While CM has its origin in the transformation of melanocytes, NMSC occurs from other epidermal cells, being subdivided into two main types, depending on the originating epidermal layer: basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) (Figure 2). BCC and SCC are the most common types of skin cancer, but despite being less common, CM is the most dangerous of the three, as it tends to more easily and often spread to other parts of the body, being responsible for the large majority of skin cancer-related mortality.^41,46−50

Figure 2.

Schematic representation of skin cancer risk factors, types, and common conventional treatments (produced with BioRender).

As for common skin cancer treatments (Figure 2), surgery is usually the first option in the early stages. Nevertheless, while effective, it is many times aesthetically undesired by patients, as it compromises the skin’s integrity, and it is no longer effective at advanced stages.⁵¹⁻⁵³ When the cancer grows into deeper parts of the skin and/or metastasizes, other more intensive treatments are required, such as chemotherapy, immunotherapy, radiation therapy, and targeted therapy.⁵⁴⁻⁵⁶ Albeit having been proven to be effective and of significant value to current medicine, these treatments require careful consideration of the risks and challenges involved (Figure 2). Adverse side effects such as diarrhea, nausea and vomiting, alopecia, anemia, thrombocytopenia, fertility issues, delirium, sleep problems, infections, pain, organ inflammation, urinary and bladder problems, among others, can completely disrupt the patients’ daily life.⁵⁷⁻⁵⁹ Furthermore, multidrug resistance, which can be frequently developed, is another obstacle in tumor management, since it often leads to therapeutical ineffectiveness.⁶⁰⁻⁶² In this context, the development of novel, more effective, and safer therapeutic options is urgently needed.

1.3. Nanotechnology for Therapeutic Purposes: General Nanosystem Characteristics and Main Types

Nanotechnology harnesses the potential of manipulating matter at a size range of 1 to 1000 nm. At this scale, the structures, materials, and particles employed in nanotechnology possess distinctive physical, chemical, and biological attributes. These unique characteristics serve as invaluable resources in the realm of nanomedicine, enabling the development of novel components for engineering systems used in diagnosing and treating diseases such as skin cancer.^13,63,64

Although several different innovative strategies have been developed in order to maximize drug permeation through the skin barrier, one of the most studied and effective is without a doubt the use of nanocarriers.^18,19,22,65 Nanosystems offer significant advantages over conventional drug delivery systems, namely: enhanced skin barrier penetration and permeation; continuous, direct, and controlled drug release to the target cells, with facilitated cellular uptake; improved drug bioavailability and reduced off-target toxicity; increased solubility of highly hydrophobic drugs; and improved formulation drug stability.^37,65,66 A nanosystem’s formulation composition and fabrication process must be optimized in order to achieve particles with ideal properties. Particle size (PS), polydispersity index (PDI), zeta potential (ZP) and encapsulation efficiency (EE%) are usually deemed as crucial parameters, being determined during the nanometric formulation’s development process.^67,68 Relevant to this review, it is worth focusing on the three main groups of nanosystems that have distinguishing characteristics for pharmaceutical applications: lipid-based NPs, polymer-based NPs, and inorganic NPs (Figure 3).

Figure 3.

Schematic representation of the main different types of nanoparticle systems with representative examples (produced with BioRender).

Lipid-based NPs exist in many forms. Nanoemulsions (NEs) are a type of lipid-based NP system involving finely dispersed droplets, usually falling between 20 and 200 nm. While high-energy production methods are often necessary in order to obtain nanoscale dimensions, such as high-pressure homogenization or ultrasonication, with the right components in the right proportions some compositions can generate nanoemulsions spontaneously, without requiring high energy throughput.⁶⁹⁻⁷² Compared to conventional emulsions, NEs offer a greater interfacial area, quicker absorption, unique rheological characteristics, enhanced drug solubility, increased drug bioavailability, and are kinetically stable systems, with improved stability and delayed phase separation.⁷³⁻⁷⁵ In order to facilitate skin application and retention, a gelling polymer can be added to these formulation’s composition, transforming NEs into an increased viscosity gel-like consistency preparation, resulting in the production of nanoemulgels (NEGs).⁷⁶⁻⁷⁸ NEGs have the added advantages of having a higher drug loading capacity (due to the existence of an extensive 3D gel network), a more pronounced concentration gradient toward the skin which allows for better skin penetration, greater adhesive characteristics, a more sustained and controlled drug release, and overall enhanced pharmacological effects and reduced adverse reactions.^79,79−82

On the other hand, SLNs are spherical lipid-based carrier systems containing solid nonpolar lipids, surfactants, and water. The use of solid lipids drastically reduces the mobility of incorporated drugs within the lipid matrix, thus preventing particle coalescence and minimizing drug migration to the emulsifier film.⁸³⁻⁸⁵ SLN’s advantages include ease of preparation, economical production, capacity for large-scale manufacturing, controlled drug release profile, chemical versatility, and nontoxicity due to being made of biodegradable lipid components.^13,83,86 SLN’s limitations comprise a relatively low drug loading capacity, and the expulsion of the encapsulated drug during long-term storage due to crystallization.^84,87,88 These challenges have prompted the development of NLCs, which are basically modified SLN, with a lipid phase that incorporates solid (fat) and liquid (oil) lipids, forming an imperfect matrix with more spaces available for the drug to exist in, and a less ordered crystalline arrangement, which allows a more efficient drug incorporation and higher drug loading, and a reduced crystallinity which avoids the potential expulsion of the drug during storage.^84,87,89

Conversely, liposomes are lipid-based vesicular spherical structures formed by the self-arrangement of amphiphilic lipid molecules in solution, such as phospholipids, with one or more concentric phospholipid bilayers enclosing a hydrophilic aqueous core, and thus having the capacity to encapsulate drug molecules with different solubilities: hydrophilic molecules in the aqueous core, hydrophobic molecules in the lipid bilayer, and amphiphilic molecules at the interface between them.⁹⁰⁻⁹² Cholesterol can also be incorporated into the liposomal composition, since being a rigid steroid molecule will enhance the vesicle’s stability and drug encapsulation, as well as prevent drug leakage and thus limit its premature release.^93,94 Liposomes as drug delivery systems present a versatile structure, the ability to protect drugs against enzymatic degradation, and high biocompatibility, biodegradability, nontoxicity and a nonimmunogenic nature, being considered to be the safest nanocarriers due to the phospholipids mimicking natural cell membranes. Nevertheless, liposomes require complex and high-cost production methods, can demonstrate low encapsulation efficiency, have challenging upscaling, and usually have a short shelf life due to poor stability, which pose as limitations.^87,91,95 To address these limitations, liposome-derived systems have been developed, such as ethosomes, which are a modification of classical liposomes containing water, phospholipids, and, in addition, an alcohol (ethanol, isopropyl alcohol, or a polyol) in relatively high concentrations (usually 20% to 45%). Ethanol acts as both a permeation enhancer and a stabilizer, contributing to the flexibility of the vesicles, and enhancing drug permeability across the skin due to fluidizing the lipid bilayer of the SC by intercalating into intercellular lipids.^93,96,97

Polymer-based NPs are nanocarrier systems that depict high versatility, since therapeutic agents can be encapsulated within the NPs’ core, entrapped in the polymer matrix, chemically linked to the polymer chains, or attached to the NP’s surface, which enables the transportation of both hydrophobic and hydrophilic drugs. They allow for a good adhesion capacity, sustained release, target action, and protection of the encapsulated molecules, which along with their general biocompatibility, biodegradability, good shelf life, water solubility, and cost-effectiveness, enable polymeric NPs to be relevant drug delivery platforms.⁹⁸⁻¹⁰⁰ Polymeric NPs can be produced by using either natural or synthetic materials. Natural polymers, derived from animals or plants, are more biodegradable, biocompatible, and overall regarded as less toxic than their synthetic counterparts, including protein-based polymers such as gelatin, and polysaccharides such as chitosan.^98,101,102 On the other hand, synthetic polymers are synthesized through chemical reactions, by either transforming natural polymers or from assembling synthetic monomers, and compared to natural polymers have higher purity (uniformity of composition), stability and batch-to-batch reproducibility, with one of the most used examples being poly(lactic-co-glycolic acid) (PLGA).^{37,99,103−106}

As for inorganic NPs, these are derived from diverse sources (metals, metal oxides, carbon, ceramics, silica, etc.) and are very stable and highly functional nanosystems, which has gotten them significant attention in the field of oncology. Their small size, large surface area, relevant biocompatibility, and capacity for functionalization position them as good candidates for addressing skin cancer.^13,107,108 Additionally, the fact that inorganic NPs have intrinsic therapeutic potential makes them capable of fighting the tumors themselves, also having synergistic therapeutic effects with the encapsulated anticancer drugs. Furthermore, their surface can be altered by physical adsorption, covalent bonding, layer-by-layer assembly, ligand exchange, and/or in situ polymerization, which can enhance their potential to penetrate the skin and lead to targeted delivery.^38,109,110

1.4. Drug Repurposing for Decreased Health Costs and Increased Therapeutic Efficacy and Safety

Conventional pharmaceutical development involves several sequential steps that span over many years, including molecular target identification, high-throughput screening, lead optimization, preclinical development, clinical trials, and regulatory approval.^2,111,112 Additionally, novel drug discovery is a highly costly process, with a significant risk of failure, impacting the industry’s ability to bring to market innovative therapies. In this context, a valuable alternative toward a more cost-effective drug discovery is drug repurposing, an unconventional approach that gives new applications to already existing and marketed drugs.^7,113,114

When compared to the traditional way, drug repurposing is more efficient, cost-effective, and less risky, since knowledge on the pharmacokinetics, pharmacodynamics, dose, metabolic profiles, molecular pathways, mechanisms of action, and different target interactions of the repurposed drug molecules is already available, and hence less effort, time and money will be needed to address these points.^{10,11,115,116} Yet, despite the accelerated timeline and available clinical and toxicological data, the process still demands key investigative work for the new application of the drug, focusing on optimizing the formulation for a new dosing regimen, route of administration, and drug molecule stability, and reprofiling pharmacodynamic, pharmacokinetics and toxicity profiles in its novel indication.^7,117,118

A relevant example of a well-known success case of drug repurposing is minoxidil, which was originally developed for the treatment of arterial hypertension in 1979. In result of leading to unwanted hair growth during clinical trials, it was approved in 1988 for the topical treatment of androgenetic alopecia.^119−121 Aside from the field of dermatology, drugs have been successfully repurposed to treat pain, infections, cardiovascular diseases, psychiatric diseases, and cancer.^121−124

Nevertheless, no review work has been able to summarize drug repurposing specifically for the treatment of melanoma, especially using the many advantages of nanotechnology. Hence, the purpose of this review is to summarize and provide a critical analysis of recent literature evaluating repurposed drugs incorporated in nanosystems for topical delivery as skin cancer therapy alternatives.

2. Topical and Transdermal Repurposed Drug-Loaded Nanosystems for Skin Cancer Treatment

Several topical and transdermal nanoformulations featuring repurposed drugs have been recently developed, with proven antitumor effects in various types of skin cancer, including different therapeutic drug classes such as antibacterial (doxycycline), antifungal (itraconazole), anthelmintic (niclosamide), antidyslipidemic (simvastatin), antirheumatic (leflunomide), antidiabetic (metformin), and nonsteroidal anti-inflammatory drugs (NSAID) (celecoxib). These drugs have been encapsulated within nanosystems with the goal of enhancing their delivery and, consequently, therapeutic effect in skin cancer tissues. The following analysis provides an overview of the developed nanosystems, including relevant formulation parameters such as PS, PDI, ZP, EE%, stability, in vitro drug release, ex vivo skin permeation and/or retention, in vitro cytotoxicity in skin cancer cells, in vitro and/or in vivo safety, and in vivo antitumor activity in skin cancer-induced animal models. A summary of the most relevant parameters is present in Table 1.

Table 1. Summarization of the Most Relevant Analyzed Parameters Regarding Repurposed Drug-Loaded Nanosystems for the Treatment of Skin Cancer, Including the Name of the Repurposed Drug, Its Classification According to Therapeutic Class, the Developed Nanosystem Type, the Nanosystem’s Main Composition, Particle Size, Polydispersity Index, Zeta Potential, and Encapsulation Efficiency, and the Studies’ Other Main Findings^a.

Repurposed drug	Classification according to therapeutic class	Nanosystem type	Main composition	PS (nm)	PDI	ZP (mV)	EE (%)	Other main findings	ref.
Doxycycline	Antibacterial	Hybrid electrospun nanofibrous scaffolds	• PCL	27 ± 7	NR	NR	NR	• Biphasic drug release, with a 60% burst release, followed by controlled release for 55 h	(125)
			• Gelatin
			• Hydroxyapatite					• Enhanced in vitro antitumoral efficacy in A-431 cells (SCC)
Itraconazole	Antifungal	Solid lipid nanoparticles	• Suppocire NB	59.20	0.290	+30.2	98.4	• Biphasic drug release, with a 30% burst release, followed by controlled release for 24 h	(126)
			• DDAB					• Enhanced in vitro antitumoral efficacy in A-431 cells (SCC) and in SK-MEL-5 cells (CM)
								• Adequate safety in healthy skin cells (HaCaT cell line)
								• Selective and synergistic anticancer effects
		Ethosomal gel	• Ethanol	169.0 ± 49.0	0.384 ± 0.037	NR	82.00 ± 1.78	• Gel matrix with adequate viscosity for topical application (1600 to 1740 cP)	(127)
			• Phospholipon 90 G (phosphatidylcholine)					• No skin sensitization on rabbit skin, thus proving to be safe for topical application
			• Carbopol 934 (carbomer 934)					• Enhanced skin permeation
								• Enhanced in vitro antitumoral efficacy in BCC1/KMC cells (BCC)
		Aspasomal cream	• Ascorbyl palmitate	67.83 ± 6.16	0.321 ± 0.14 to 0.493 ± 0.52	–79.4 ± 2.23	>95	• Increased skin deposition	(128)
			• Stearic acid					• Enhanced in vitro antitumoral efficacy in A-431 cells (SCC)
			• Glycerin					• Relevant antitumor effects in an in vivo Ehrlich Carcinoma Model, with significant reduction in tumor weight and volume
			• Propylene glycol
Niclosamide	Anthelmintic	Liposomal gel	• Cholesterol	131	≈ 0.2	–13 ± 9.71	89	• Sustained drug release, 83.26 ± 4.55% after 48 h	(129)
			• Egg lecithin					• Drug release more prolonged at 37 °C than 25 °C (thermogel)
			• Poloxamer 407					• Increased localized skin deposition
			• Poloxamer 188					• Enhanced in vitro antitumoral efficacy in SK-MEL 28 cells (CM)
Simvastatin	Antidyslipidemic	Hybrid lipid-polymer nanoparticles-loaded bioadhesive film	• Chitosan	108 ± 1	0.226	+17 ± 0.6	99.86 ± 0.08	• Sustained release throughout 48 h (99 ± 7% for the optimized film)	(130)
			• Squalene					• Increased skin permeation due to the presence of squalene
			• Glyceryl palmitostearate					• High skin tolerability (human volunteers, 72 h)
			• Polysorbate 80					• No relevant cytotoxicity on healthy skin cells (HaCaT and COS-7 cell lines)
			• Sorbitol					• Enhanced in vitro antitumoral efficacy in COLO-38 and SK-MEL-28 cells (CM)
			• PEG 400					• Synergistic effect between simvastatin and squalene
			• HPMC
			• Limonene
		Polymeric nanoparticles-loaded gel	• PLGA	177	0.147	–28.1 ± 5.64	≈ 90	• Sustained release, 50.79 ± 5.56% after 72 h	(131)
			• Poloxamer 407					• Drug release more prolonged at 37 °C than 25 °C (thermogel)
			• Poloxamer 188					• Enhanced in vitro antitumoral efficacy in SK-MEL-28 cells (CM)
								• High formulation biocompatibility
Leflunomide	Antirheumatic	Nanoemulgel	• Capryol 90	123.7	≈ 0.3	≈ +8	NR	• Increased skin permeation and retention	(132)
			• Cremophor EL					• Enhanced in vitro antitumoral efficacy in A375 and SK-MEL-2 cells (CM)
			• Transcutol HP					• Adequate skin biocompatibility (histopathology studies)
			• Poloxamer 407
Metformin	Antidiabetic	Ethosomal gel	• Ethanol	124.01 ± 14.27	NR	–60.08 ± 1.44	98.40 ± 0.35	• Gel with desired viscosity and adequate bioadhesion	(133)
			• Isopropyl alcohol					• Sustained in vitro drug release, reaching 55.04 ± 0.98% after 8 h
			• Lecithin					• Increased skin permeation
			• Cholesterol					• In vivo safety (animals’ body weight and histopathological studies)
			• Carbopol 974 P (carbomer 974)					• Increased in vivo antitumor efficacy
		Nanodispersions	• Monoolein	<150	<0.2	<−14	NR	• Enhanced skin permeation and retention	(134)
			• Methylene blue					• Enhanced in vitro antitumoral efficacy in A-431 cells (SCC)
			• Poloxamer 407					• Effective synergy between metformin and methylene blue
			• Sodium cholate					• Use of photodynamic therapy led to overall improved outcomes
Celecoxib	Anti-inflammatory	Liposomal gel	• Doxorubicin	142.37 ± 0.78	0.27 ± 0.026	–5.04 ± 0.51	>98	• In vitro drug sustained release	(135)
			• Cholesterol					• Ex vivo skin permeation and retention increase
			• Hydrogenated soya bean phosphatidylcholine					• Enhanced in vitro antitumoral efficacy in B16 cells (CM)
			• Carbopol					• Celecoxib and doxorubicin revealed synergistic behavior
								• Increased in vivo antitumor efficacy
								• Pretreatment with microneedles led to overall improved effects

^aBCC – basal cell carcinoma; CM – cutaneous melanoma; DDAB – didodecyldimethylammonium bromide; EE – encapsulation efficiency; HPMC – hydroxypropyl methylcellulose; NR – not reported; PCL – poly-ε-caprolactone; PDI – polydispersity index; PEG – polyethylene glycol; PLGA – poly(lactic-co-glycolic acid); PS – particle size; Ref. – reference; SCC – squamous cell carcinoma; ZP – zeta potential.

2.1. Antibacterial Drug Repurposing—Doxycycline

Doxycycline (DOX), a Biopharmaceutical Classification System (BCS) class I drug, is a second-generation tetracycline widely recognized as an antibiotic with broad-spectrum efficacy. The bacteriostatic effect of tetracyclines leads to the inhibition of bacterial protein synthesis, preventing the binding of the aminoacyl group of t-RNA to the 30S ribosomal subunit of both Gram-negative and Gram-positive bacteria.^136−138 Moreover, studies have shown that DOX has many significant nonantibiotic properties, such as anti-inflammatory, antiangiogenic, anticollagenolytic, osteoclast inhibitory, fibroblast stimulatory, immunosuppressive, and anticancer effects. These discovered “extra” biological actions have prompted substantial research into alternative applications of DOX.^139−143 The antiproliferative activity of DOX is mainly attributed to its ability to inhibit matrix-metalloproteinases (MMPs), enzymes involved in the degradation and remodulation of the extracellular matrix. Current studies have demonstrated that DOX has the ability to control invasive and metastatic cancer cells, inhibiting their adhesion and migration, affecting their growth and proliferation, and inducing apoptosis. Results of in vitro and in vivo experiments have supported the anticancer properties of this drug against different tumor cells, including melanoma.^139,144,145

Taking this into account, Ramírez-Agudelo et al.¹²⁵ developed a multifunctional system in which hybrid electrospun nanofibrous scaffolds, composed of poly-ε-caprolactone (PCL) and gelatin (Gel) were meant to allow the controlled release of DOX and hydroxyapatite NPs (nHA). The purpose of this study was to investigate the DOX/nHA/PCL-Gel composite nanofiber system’s potential antitumor activity in skin cancer, more specifically in SCC. Hydroxyapatite (HA) is known for being one of the most widely accepted inorganic nanomaterials in clinical use due to its exceptional biocompatibility and acceptable biodegradation. Leveraging its high surface area to volume ratio, high surface activity, and strong ability to absorb diverse chemical species, nHA have proven to be a potential candidate to serve as delivery carriers for antibiotics, nucleic acids, proteins, and anticancer drugs.^146−149 Additionally, nHA themselves have demonstrated anticancer properties in multiple cancer cell types, including melanoma, through intracellular accumulation of reactive oxygen species (ROS), which are associated with apoptosis regulation by elevating oxidative stress.^150−152 Hence, a DOX/nHA combination therapy induces a possible breakthrough, yielding synergistic agents with potential antitumoral effects through different mechanisms of action for fighting cancer. On the other hand, PCL is a hydrophobic, bioresorbable, and biocompatible synthetic polyester, that holds approval from the FDA for its utilization in biomedical devices, in applications related to drug delivery and tissue repair.^153−155 One of the greatest disadvantages of synthetic polymers is the lack of cell-recognition signals, so the combination of natural and synthetic polymers can provide the advantage of fine-tuning the desired properties of the electrospun scaffolds. In this context, Gel was selected, since it is a natural biopolymer derived from the partial hydrolysis of collagen, with several different advantageous attributes, such as cost-effectiveness, natural abundance, and notable biodegradability and biocompatibility in physiological environments.^156−158 PCL/Gel composite scaffolds represent a versatile synergistic synthetic-natural polymer blend for tissue engineering applications, having been proven to allow substantial cell attachment, biocompatibility, and intrinsic bioactivity.^159−161 The polymeric blend nanofibers were also meant to provide the opportunity to design and adjust the DOX release kinetics as an antitumor agent. The synthesized nHA demonstrated excellent dispersion, with uniform morphology, exhibiting NPs with a rod-like shape, showing an average diameter of 27 ± 7 nm and a length of 40 ± 17 nm (Figure 4A). The incorporation of the NPs into the polymeric PCL/Gel nanofibers via electrospinning resulted in their selective entrapment preferentially in the Gel phase, associated with the hydrophilic nature of nHA and DOX. In vitro drug release assays (Figure 4B), using a phosphate-buffered saline (PBS) buffer medium, showed that the DOX release profile exhibited a two-stage pattern. In the first phase, all scaffolds showed a burst release effect, releasing approximately 60% of the loaded drug within the first hour. In the second phase, the remaining drug content was released progressively, over a prolonged period of 55 h. It is worth mentioning that the release rate was not increased with increasing the content of Gel in the fibers. As mentioned, the drug has the tendency to localize primarily within the Gel phase, regardless of the ratio of this biopolymer that is present in the blend. Thus, the phenomenon of the initial burst release can be attributed not only to the high solubility of Gel in the PBS release medium, but also due to the phase segregation between Gel and PCL that occurred during the electrospinning process, that causes the accumulation of Gel on the nanofibers’ surface. The antitumor activity of the optimized nanofibers was also assessed, in vitro, using three distinct tumor cell lines, corresponding to colorectal, breast, and skin cancers. The selected model for skin cancer, A-431 cells, a SCC cell line, was the one where the developed system had the best performance (Figure 4C), with the study showing insignificant cytotoxicity with free nHA treatment and, similarly, with the application of free DOX, which led to a small decrease of cancer cell viability, but in comparison the developed multifunctional system led to a significantly enhanced in vitro antitumoral efficacy, thus proving the high potential of the DOX/nHA/PCL-Gel composite nanofiber system for the treatment of skin cancer, more specifically in SCC. In summary, owing to the notable properties of nHA as a drug carrier and with a repurposed drug such as DOX with marked antiproliferative effects, this topical system stands as an efficient approach that addresses challenges linked to ordinary anticancer treatments. It holds promise for targeted and localized drug delivery applications in the biomedical field, namely as a treatment strategy for skin cancer.

Figure 4.

(A) SEM micrographs depicting the morphology, with additional diameter distribution data, of several variations of the developed formulations, namely PCL/Gel blend nanofibers with a 70:30 w/w % of PCL/Gel (a), PCL/Gel blend nanofibers with a 60:40 w/w % of PCL/Gel (b), PCL/Gel blend nanofibers with a 50:50 w/w % of PCL/Gel (c), nHA/PCL-Gel composite nanofibers with a 70:30 w/w % of PCL/Gel (d), nHA/PCL-Gel composite nanofibers with a 60:40 w/w % of PCL/Gel (e), nHA/PCL-Gel composite nanofibers with a 50:50 w/w % of PCL/Gel (f), DOX/nHA/PCL-Gel composite nanofibers with a 70:30 w/w % of PCL/Gel (g), DOX/nHA/PCL-Gel composite nanofibers with a 60:40 w/w % of PCL/Gel (h), and DOX/nHA/PCL-Gel composite nanofibers with a 50:50 w/w % of PCL/Gel (i); TEM images of the optimized DOX/nHA/PCL-Gel composite nanofibers (j–l). (B) In vitro drug release assays of the developed DOX-loaded HA/PCL-Gel nanofibers (PGHAD1 with a PCL-Gel ratio of 70:30, PGHAD2 with a PCL-Gel ratio of 60:40, and PGHAD3 with a PCL-Gel ratio of 50:50). (C) SCC cell viability after exposure to free nHA, free DOX, and Dox-nHA, at different concentrations; reproduced with permission from Ramírez-Agudelo et al.¹²⁵ Copyright 2018 Elsevier.

2.2. Antifungal Drug Repurposing—Itraconazole

Itraconazole (ITZ), a BCS class II drug, is a broad-spectrum antifungal, chemically being a triazole, and has been clinically used for over 30 years. It inhibits lanosterol 14-demethylase, the enzyme that converts lanosterol to ergosterol, which leads to a compromised ergosterol synthesis, thereby resulting in the inhibition of fungal growth and fungal membrane disruption, which will increase its permeability and change membrane-bound enzymatic activity, hence leading to fungal cell death.^162−164 In recent years, evidence has surfaced demonstrating a plethora of in vitro, in vivo, and even clinical evidence supporting ITZ as a molecule with antitumor activity, mostly by acting on the Hedgehog (Hh) pathway.^165,166 The Hh pathway stands out as one of the primary signaling pathways frequently used during cellular development for intercellular communication, holding an important role in orchestrating the organogenesis of almost all organs in mammals as well as in regeneration and homeostasis, both of which are frequently recurrent processes in a dynamic organ such as the skin. The disruption of the Hh pathway has been observed in various types of cancer, and mutations in the Smoothened protein (SMO) gene can result in abnormal activation of the Hh pathway, leading to uncontrolled cell proliferation and tumor formation. Notably, its crucial involvement in the development of BCC has been convincingly demonstrated by genetic mutations, mouse models of BCCs, and successful clinical trials of BCCs using Hh signaling inhibitors.^167−170 Furthermore, the Hh pathway activity has also been reported to be involved in the pathogenesis of other skin cancers, such as SCC and CM. Additionally, regardless of the emergence of drug-resistant SMO mutations, ITZ has the capability to target this essential component and act as an Hh inhibitor.^168,171 Moreover, ITZ has been demonstrated to lead to a potent suppression of tumor neovascularization and angiogenesis, leading to reduced proliferation of endothelial cells and tumor vascularity. It has also demonstrated to be capable of inducing tumor cell autophagocytosis. Together, these effects may grant ITZ the potential of being repurposed to prompt targeted cutaneous apoptosis and tumor necrosis.^172−174

Nevertheless, despite its potential as an anticancer agent, ITZ formulation development and effective administration have important limitations, due to its poor physicochemical attributes, such as its high molecular weight (705.64 g/mol) and low aqueous solubility (1 ng/mL, at pH 7). These characteristics can hinder not only its formulation at high strength but also its effective delivery and distribution in the body when administered within conventional pharmaceutical preparations. To overcome these challenges and enhance its efficacy in skin cancer therapy, topical formulations incorporating ITZ in nanocarriers have demonstrated favorable results. Through local application and nanoencapsulation of the drug, ITZ concentration has been maximized in the affected areas, hence increasing therapeutic efficacy and reducing the propensity for systemic side effects. In this context, ITZ lipophilicity (log P = 5.66) is an important factor, since it has allowed for easier incorporation within lipid carriers, namely SLN, ethosomes and aspasomes, hence facilitating penetration through cell membranes, potentially enhancing its ability to reach tumor cells effectively, as described in further detail bellow.^126−128

Carbone et al.¹²⁶ developed ITZ SLN, for topical application, using a Suppocire NB (hard fat, consisting of mono-, di- and triglyceride esters of C10 to C18 fatty acids) lipid matrix, featuring a coating layer of didodecyldimethylammonium bromide (DDAB), a cationic lipid known for its pharmaceutical applications as coating material. DDAB has been recently reported to be effective against cancer cell lines, thus enhancing the potential anticancer ITZ activity by synergistic effect. The coating layer of DDAB also contributed to increasing the stability without compromising the homogeneity and size of the NPs, while altering the NPs surface charge to being high and positive (ZP = +30.20 mV), hence enhancing their interaction with the cell membrane, especially with their negatively charged phospholipids, thus facilitating the fusion of NPs with the cancer cells and consequent penetration. The choice to encapsulate ITZ within SLN was driven by the challenge that its poor water solubility represents, since by using a solid biocompatible lipid matrix with a unique chemical structure, characterized by the presence of long chain fatty alcohols (C10–C18), a substantial drug solubilization was obtained. Moreover, matching the ideal parameters for an adequate topical formulation, the developed SLN achieved a small particle size (59.20 nm), homogeneous particle size distribution (PDI = 0.290), and very high EE% (98.4%), also exhibiting a clear visual appearance, indicative of a stable dispersion with no aggregation. The in vitro drug release assay (dialysis membrane) revealed a sustained and prolonged release profile, lasting for oven 24 h, from both uncoated and coated SLN. However, the presence of the coating DDAB layer led to a higher initial drug release (30%), thus confirming the important role that the coating layer plays on the nanoparticle’s features, due to generating an irregular layer on the surface of the SLN, with the formation of protrusions able to capture the drug, thereby promoting its prompt release within the first hours. The in vitro cytotoxicity study on skin cancer cell cultures, namely, SCC (A-431 cell line) and CM (SK-MEL-5 cell line), demonstrated a significant reduction in the viability of these cancer cells when the developed formulations were applied, with the coated NP showing a better performance when compared to the uncoated SLN. Particularly, the coated SLN achieved this effect at a lower concentration of ITZ, indicating enhanced anticancer activity through the synergistic effect of the coating layer and the encapsulated drug. Moreover, the coated SLN not only exhibited a remarkable ability to reduce the viability of tumoral cells, but also preserved the viability of normal skin cells (HaCaT cell line). This suggests a selective cytotoxic effect on cancer cells, potentially minimizing adverse effects. Hence, the researchers successfully designed an innovative topical formulation displaying enhanced cytotoxic effect particular to skin tumor cells, with the coated SLN system offering an improvement to the drug’s delivery profile, allowing the drug to have a greater effect at a lower dose, and with an evident synergistic action between ITZ and the NP system encapsulating it, which highlights the potential of this strategy as a novel therapy against skin cancer.

Saraf et al.¹²⁷ also developed an ITZ nanosystem formulation, but instead of SLN, ethosomes were produced, for topical administration, for the treatment of BCC. The ethosomes were composed of Phospholipon 90 G (phosphatidylcholine) and ethanol, and after preparation were incorporated into a gel matrix, using Carbopol 934 (carbomer 934) as a gelling agent, and hence giving origin to an ethosomal gel. Carbomers are high molecular weight entities derived from the polymerization of acrylic acid and cross-linking with alkenyl ethers of sugars or polyalcohols. Their hydrophilic nature exhibits an important characteristic—when they are exposed to water in a pH range of 4.0 to 6.0, their volume expands significantly due to the large amount of carboxyl groups on the polymer’s backbone, which ionize, resulting in repulsion between the negative charges, which leads to polymer swelling. Carbomers undergo a configuration change, thus increasing the viscosity of the liquid in the presence of water and forming a gel. This unique property makes them versatile excipients in various pharmaceutical formulations to achieve desired product textures, controlled drug release, and stability. Moreover, these gelling agents have low potential for skin irritation and sensitization, being highly suitable for aqueous-based formulations for topical application.^175−177 Thus, in this study a carbomer-based gel was prepared, with a recorded pH of 6.8, and achieved viscosity of 1600 to 1740 cP. Within it, drug-loaded ethosomes were dispersed, and these ethosomal vesicles featured a small PS of 169.0 ± 49.0 nm, a PDI of 0.384 ± 0.037, and good EE% of 82.00 ± 1.78%. Safety studies included an in vivo skin sensitization experiment, using rabbits, and results showed that the ethosomal formulations did not lead to significant erythema, when compared with a control saline solution (0.9% w/w NaCl), indicating that the high concentration of ethanol that was part of the ethosomes’ composition did not induce skin inflammation and that the developed formulation was biocompatible. Additionally, from an ex vivo drug permeation evaluation, in Franz diffusion cells, it was possible to conclude that the formulated ITZ-loaded ethosomes exhibited a greater penetration depth when compared to the free drug. Furthermore, compared with an ITZ suspension, the ITZ-loaded ethosomal gel permeated faster through the membranes. This was justified as the result of the ethanol present in the ethosomal system, which acts as both permeation enhancer and stabilizer. As a result, the ethosomal vesicles became soft and malleable, enabling them to efficiently penetrate the SC. Once within the skin’s layers, ethanol potentially fused with the skin lipids and released ITZ into the deeper layers, which could be beneficial to reach the skin tumor sites, further emphasizing the potential of this type of formulations as a highly efficient drug delivery systems, capable of reaching deeper skin layers and delivering therapeutic agents more effectively. Furthermore, an in vitro study was conducted to assess the efficacy of the ethosomal formulation against the BCC1/KMC cell line (BCC), with results revealing a 34% greater reduction of cell viability when compared to the free drug formulation. Moreover, the cytotoxicity of the developed formulation on tumor cells was approximately 4.6 times higher than that of the free drug form. Hence, in this study, the designed formulation for topical application demonstrated significant cytotoxic effect against BCC. The approach to encapsulate ITZ in ethosomes dispersed in a gel also resulted in better drug permeation when compared to the free drug, thus demonstrating the promise of the developed strategy in achieving an enhanced therapeutic effect for ITZ against BCC.

Lamie et al.¹²⁸ explored the nanovehiculation of repositioned ITZ as well, by harnessing the potential of ascorbyl palmitate (AP) to form aspasomes (Figure 5A), which were in turn incorporated into a cream, thereby creating a topical formulation for ITZ and offering a localized approach to manage cutaneous malignancies. At the core of this approach is AP, a hydrophobic derivative of ascorbic acid (AA), a well-explored vesicle bilayer-forming component that holds FDA approval and is widely popular in skin care products as an inactive ingredient. AP’s capacity to form multilamellar vesicles (aspasomal dispersions) were the root to design the developed ITZ-loaded aspasomes, together with their biocompatibility due to a lipophilic nature.^178−180 Moreover, given the intrinsic antioxidant capacity that has been proven to exist for AA, AP possesses it as well, thus having the ability to reduce cellular levels of ROS after in contact with UV irradiation, making it a potent active oxygen scavenger against dermal oxidative damage. AP is also well-documented to retain the anticancer potential of AA, acting as an inhibitor of DNA synthesis and proliferation in various cancer cells, including those found in the skin.^181−184 This synergistic strategy of employing ITZ and aspasomes was the foundation of the novel and potentially promising approach developed for skin cancer therapy. Additionally, in order to extend the retention of the ITZ-loaded aspasomes on the skin, for improved topical application, an O/W cream (stearic acid, glycerin, and propylene glycol) was chosen as the final product. The formulated cream featured aspasomes with a small PS of 67.83 ± 6.16 nm (Figure 5B), potential for high colloidal stability (evidenced by reasonably low PDI values, from 0.321 ± 0.14 to 0.493 ± 0.52, suggesting moderate monodispersity), negative surface charge with high absolute value (ZP of −79.40 ± 2.23 mV, indicating strong potential for stabilization through electrostatic repulsion and good dispersibility), and quite relevant ITZ EE% (>95%). Ex vivo quantitative evaluation of skin drug deposition, utilizing mice skin layers, demonstrated the superior performance of the ITZ aspasomal cream, achieving maximum deposition in the SC and epidermis up to 3.33- and 2.24-fold greater than an ITZ aspasomal dispersion, respectively. Similarly, in comparison to an ITZ conventional cream, the ITZ aspasomal cream exhibited a 2.19- and 4-fold increase of deposition in the SC and in the epidermis, respectively. The quantitative findings were confirmed via qualitative assessment of skin deposition of fluorescently labeled aspasomal dispersion and aspasomal cream. The in vitro cytotoxicity assessment of both pure ITZ and the optimized ITZ aspasomes on BCC cells (A431 cell line) was conducted, as well. The comparison of IC₅₀ values revealed a clear distinction, indicating that ITZ-loaded aspasomes exhibited a toxicity against A431 cells approximately 2.5-fold higher than that of the free drug. These results indicated that ITZ was able to induce a greater cytotoxicity when loaded within the aspasomal system. The researchers also further explored the anticancer potential of the developed ITZ aspasomes using the Ehrlich Carcinoma Model. Results showed that the ITZ aspasomal cream showed a significant reduction in tumor weight and tumor volume, making it a more effective therapeutic option than all other treatment groups (Figures 5C and 5D). Therefore, according to the findings of this research, encapsulating ITZ within aspasomes allowed for a synergistic effect capable of achieving greater cytotoxicity against BCC, also demonstrating a relevant skin deposition profile, and thus being a highly promising approach for the topical treatment of skin cancer.

Figure 5.

(A) Depiction of the structure and composition of the developed ITZ-loaded aspasomes, with the most relevant performed characterization studies being represented bellow; (B) TEM micrograph of the developed ITZ-loaded aspasomes; (C) tumor volume variation after treatment with the developed formulations (Ehrlich ascites carcinoma mice model); (D) tumor weight variation after treatment with the developed formulations (Ehrlich ascites carcinoma mice model); reproduced with permission from Lamie et al.¹²⁸ Copyright 2022 Elsevier.

In summary, these three works have demonstrated the value and applicability of nanosystem integration for the enhancement of the therapeutic effect of ITZ against skin cancer. The researchers were able to achieve promising topical formulations with encapsulation of ITZ in three distinct types of nanosystems (SLN, ethosomes, and aspasomes) that delivered exceptional performance in enhanced drug delivery. Compared to the free drug, the nanovehiculated ITZ demonstrated improved cytotoxic effect on skin carcinoma cell lines and improved outcomes in in vivo skin cancer models. These promising preliminary findings truly evidence the potential of the developed therapeutic strategies.

2.3. Anthelmintic Drug Repurposing—Niclosamide

Niclosamide (NIC), a BCS class II drug, is an FDA-approved anthelmintic drug used to treat parasitic infections. Despite its widespread usage, NIC’s therapeutic mechanism of action remains only partially comprehended, with early studies associating NIC’s activity to the uncoupling of oxidative phosphorylation.^185−187 Over the past few years, studies have shown that NIC possesses a wide range of clinical applications besides the treatment of parasitic infections, by targeting the Wnt/β-catenin, mTOR, AKT-PI3K, Hh and JAK/STAT3 signaling pathways, which have inevitably linked NIC’s therapeutic potential to many diseases involving these critical signaling cascades.^188−190 In melanoma, signal transducer and activator of transcription 3 (STAT3), a transcription factor, plays a crucial role in various cellular processes, such as tumor cell growth and proliferation, survival, migration, invasion, immune evasion, and metastasis. NIC has been proven to be a potent inhibitor of STAT3, having demonstrated remarkable promise in inducing apoptosis not only in melanoma, but also in several other cancer types, such as breast, lung, and colon cancers.^{188,191−193}

Nonetheless, NIC’s poor water solubility (log P = 4.48) and poor penetration across the skin layers are discouraging factors when considering topical formulations. To address these issues, Shah et al.¹²⁹ developed a thermogel with poloxamers that could incorporate NIC-loaded liposomes for melanoma treatment. This liposome-loaded thermogel was intended to form a local depot for targeted topical administration, specifically to the outermost layers of skin, in order to control the drug’s delivery to the bioactive site and minimize its entry into the systemic circulation. Poloxamer 407 and poloxamer 188 were used, and the ratio between them was 9:1, since it originated the desired gelation temperature (33 °C), thus being optimum for thermogel synthesis. The optimized liposomes exhibited a PS of 131 nm (Figure 6A), an EE% of 89%, and a ZP measuring of −13 ± 9.71 mV, suggesting an effective electrostatic stabilization, contributing to improved vesicle stability. The investigation into the stability of the developed liposomes and liposomal thermogel, over a 1-month period, at temperatures of 2 to 8 °C, and room temperature, revealed that refrigeration was the recommended storage method, a conclusion based on the observation of a slight rise in PS and a reduction in EE% at room temperature. The dialysis bag method was used to evaluate the formulations’ in vitro drug release (Figure 6B), comparing a NIC plain drug suspension and the drug-loaded liposomes. These studies revealed a sustained release profile for the NIC-loaded liposomes with a cumulative drug release of 83.26 ± 4.55% after 48 h. In contrast, the free drug exhibited fast and complete release within 4 h. Furthermore, using Franz diffusion cells, at a higher temperature of 37 °C, the liposomal thermogel effectively prolonged the drug release when compared to the temperature of 25 °C. This effect can be attributed to the gel transition responsible for a heightened formulation viscosity. Ex vivo drug permeation studies were conducted as well on rat skin, using Franz diffusion cells, to evaluate the depth of skin penetration of the NIC-loaded liposomal thermoresponsive gel. It was found that the liposomes exhibited penetration into the skin’s deeper layers, whereas the liposomal thermogel exhibited a different behavior, by localizing the liposomes on the uppermost skin layers, forming a visible band, and thus leading to a depot formation at the bioactive site. In vitro studies were also performed (Figure 6C), to estimate the cell viability of melanoma cells exposed to free NIC, blank liposomes (BLIP) and NIC-loaded liposomes (NLIP). There was an evident accumulation of NIC in SK-MEL 28 (CM cell line) with liposomal encapsulation, when compared to the free drug. The entrapment of NIC within the liposomes elevated its cytotoxicity by 1.756-fold. In contrast, BLIP demonstrated low cytotoxicity with a cell viability of 91.83 ± 2.23%. Based on the data from this study, the NIC-loaded liposomal thermogel was demonstrated to be a safe formulation approach that enhanced the cytotoxicity of NIC against melanoma. The deposition behavior that can offer a localized delivery with prolonged exposure was also evidenced. This research highlights the potential for the liposomal encapsulation of NIC in a topical formulation as a possible therapeutic strategy for helping fight against melanoma.

Figure 6.

(A) TEM image of the developed optimized NIC-loaded liposomes; (B) In vitro drug release profile of the developed NIC-loaded liposomes, compared to a free drug formulation; (C) In vitro cell uptake and viability studies of the developed formulations, using C-6 loaded liposomes, or NIC-loaded liposomes compared to a free drug formulation, in SK-MEL-28 cells; reproduced with permission from Shah et al.¹²⁹ Copyright 2022 Elsevier.

2.4. Antidyslipidemic Drug Repurposing—Simvastatin

Simvastatin (SIM), classified as a BCS class II drug, is a well-recognized cholesterol synthesis inhibitor utilized for dyslipidemia management. It has potent activity in reducing circulating lipids, specifically the cholesterol present in low-density lipoprotein (LDL) The mechanism behind this process stems from the competitive inhibition of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, an essential enzyme to the production of cholesterol.^194−196 HMG-CoA reductase inhibitors, more commonly known as statins, have been found to possess antitumor properties in various types of cancer, including melanoma. These effects are observed when statins are used alone or in combination with conventional treatments. Several studies have shown how these drugs can effectively block the G1-S phase of melanoma cell cycle.^197−200 SIM, more specifically, has demonstrated antitumor potential by prompting apoptosis due to the induction of caspases 3, 8 and 9, and leading to the DNA fragmentation of human melanoma cells, when exposed to the drug’s action for at least 48 h.^{194,201−203}

Nevertheless, SIM is a water-insoluble hydrophobic drug, as evidenced by its high log P value of 4.4659. This water insolubility poses a significant challenge in its delivery, pharmacokinetics, and pharmacological activities. In order to overcome these limitations, Barone et al.¹³⁰ developed a topical bioadhesive film containing SIM-loaded chitosan-coated nanostructured lipid carriers (Ch-NLC), which were used as the drug vehicle to achieve SIM incorporation and controlled release. Ch-NLC, a particular type of hybrid lipid–polymer (HLP) nanoparticle, is composed of a lipid matrix formed by the combination of solid and liquid lipids, which remain solid at room and body temperatures. This lipid matrix is then electrostatically surrounded by a polymer, in this case chitosan. This hybrid nanosystem has the advantages of both lipidic and polymeric materials, improving the developed system’s general interaction with the skin, which is largely attributed to the cationic nature and intrinsic bioadhesive properties of chitosan. Squalene, a natural lipid, was employed as the nanostructuring agent of the lipid NP matrix as well as a permeation enhancer, facilitating skin permeation and uptake into melanoma cells. The formulation approach for this system included a predetermined ideal concentration of chitosan of 1% w/v, with increased chitosan concentrations leading to improved system stability due to the formation of complexes with the coating mechanism, but only until a certain point, with higher concentrations of chitosan resulting in a significant increase in the average PS of the NPs. Further design of the NLC matrix encompassed optimizing critical formulation attributes, squalene concentration, solid:liquid (glyceryl palmitostearate:squalene) lipid ratio, and polysorbate 80 (PS80) concentration to achieve an optimal PS, PDI and ZP. The analysis suggested that an increase in lipid concentration led to an increase in PS. Additionally, when the surfactant (PS80) concentration was increased, a reduction in the PS was observed. This decrease in the PS is a direct consequence of the surfactant’s inherent role as an interface component. Ultimately, a 25:75 solid:liquid lipid ratio was selected, which resulted in a smaller PS compared to a 50:50 ratio. This behavior can be attributed to the increased viscosity of the dispersion when the solid lipid content is present at a higher proportion, leading to an elevated surface tension and consequently larger PS. With the selection of the optimal attributes of 5% w/w lipid concentration, 2.5% w/w PS80 concentration, and 25:75 solid:liquid lipid ratio, the NLC system had a small PS of 108 ± 1 nm and a PDI of 0.226, indicating a narrow size distribution, and the ZP was recorded to be 17.0 ± 0.6 mV, suggesting an electrostatically stable formulation. The EE% was high, being 99.86 ± 0.08%, indicating a very large percentage of SIM incorporated into the NPs. Additional formulation development studies covered the assessment of the effect of chemical permeation enhancers (CPEs) on the drug release, skin permeation, skin irritation, and cytotoxicity profile of the optimized SIM-loaded and unloaded Ch-NLC systems, embedded in a mixture of sorbitol, polyethylene glycol (PEG) 400, and hydroxypropyl methylcellulose (HPMC), to form a adhesive topical films (Figures 7A, 7C and 7E). Propylene glycol (PG) and limonene (L) were the selected CPEs incorporated into the films and compared with the Ch-NLC (reference) film. In vitro release and permeation studies were performed using Franz diffusion cells to comprehend the release behavior of the drug from the formulated nanofilms. This release was proven to be dependent on the partitioning of the drug within the Ch-NLC lipid matrix, followed by the rate of diffusion through the adhesive layer. The results indicated a sustained release throughout the 48 h of the test. The lowest release rate was observed for the PG-film (54 ± 2%), whereas for the reference-film and L-film 98 ± 4% and 99 ± 7% of SIM was released, respectively. Thus, it appeared that the presence of PG in the formulation had an inhibiting effect on the release of SIM. As for skin permeation profiling, newborn pig epidermis (resembles the human skin anatomically, physiologically, and biochemically) was implemented as a barrier model. The study demonstrated that the presence of permeation enhancers (PG and L) did not promote a significantly higher flux, when compared to the reference film. In particular, the film without CPEs presented the highest permeation through the SC, highlighting the permeation enhancing properties of squalene in the NP matrix, originating from its ability to disrupt the ordered lipid bilayer structure of cells. A skin tolerability study of the drug-free PG-, L- and reference-films was also conducted, on human subjects, to evaluate the potential for provoking skin irritation of the developed topical nanofilm formulations (Figure 7B). The films did not cause any skin irritation or alterations in the SC, and they showed no signs of structural damage after 72 h, indicating their suitability for topical use. In vitro evaluation of the cytotoxic activity of SIM, both in free form and encapsulated within the proposed Ch-NLC film, was also determined, and no relevant cytotoxic effects were observed for both free SIM and the reference film (cell viability around 100% after 48 h) on HaCaT and COS-7 cell lines, representing healthy/normal human keratinocytes and fibroblasts, respectively. The cytotoxic effect of the developed formulations on melanoma cell lines (COLO-38 and SK-MEL-28, Figures 7D and 7F) was subsequently investigated, and a dose- and time-dependent response was observed. In general, a similar performance was reported for the proposed hybrid system and free drug. Nevertheless, after a 48 h exposure, the SIM-loaded nanocarriers demonstrated a higher cytotoxic effect, in comparison with the free drug, in the COLO-38 cell line. Additionally, a synergistic effect was observed between SIM and squalene’s intrinsic capacity for destabilizing the cellular membrane, promoting the delivery of drug inside the cell. Further evaluation of the cytotoxicity of the formulated films spiked with CPEs on the COLO-38 cell confirmed that films including SIM encapsulated into Ch-NLC provided an increased cytotoxic activity. Distinct cytotoxicity levels were observed depending on the enhancer that was used, and films containing L demonstrated the most potent cytotoxic effect among the tested enhancers. This publication successfully demonstrated the application of a polymeric-NLC hybrid system for topical administration via an adhesive film featuring repurposed SIM, with reportable cytotoxic effect against melanoma cell lines. The hybrid polymer–lipid (chitosan-glyceryl palmitostearate:squalene) NP matrix featured ideal properties for topical application and revealed an adequate permeation profile, while also demonstrating no decrease of cell viability for healthy cell lines, implying a safe toxicological profile with high tolerability and biocompatibility.

Figure 7.

(A) SEM image of one of the developed nanofilms, containing Ch-NLC, sorbitol, PEG and HPMC; (B) irritation index variation after skin application of the developed formulations; (C) SEM image of one of the developed nanofilms, containing Ch-NLC, sorbitol, PEG, HPMC, limonene and ethanol; (D) In vitro cytotoxicity results in melanoma cells, after exposure of the cells to the developed formulations for 24 h; (E) SEM image of one of the developed nanofilms, containing Ch-NLC, sorbitol, PEG, HPMC, PG and ethanol; (F) In vitro cytotoxicity results in melanoma cells, after exposure of the cells to the developed formulations for 48 h; reproduced with permission from Barone et al.¹³⁰ Copyright 2019 Elsevier.

In another study, Famta et al.¹³¹ also focused on repurposing SIM, loading it into PLGA NPs, which were subsequently integrated in poloxamer-based thermogels, for topical application and targeted therapeutic effect against skin cancer. This strategy was meant to demonstrate that the incorporation of these NPs into a thermogel facilitated the formation of a depot in the upper dermal layers, achieving targeted drug delivery and thus reducing systemic exposure to the drug, increasing both therapeutic efficacy and safety. For this formulation approach, the desired in situ gelation temperature, that would impart a thermoresponsive depot behavior to the formulation, was achieved by adjusting the balance ratio of poloxamers in the preparation. Poloxamers are a class of water-soluble nonionic and synthetic triblock copolymers, formed by central hydrophobic chains of poly(propylene oxide) sandwiched between two hydrophilic chains of poly(ethylene oxide), which give them their amphiphilic character. They show an important inherent thermoreversible behavior and can possess sol-to-gel phase transition near body temperature when at adequate concentrations, being excellent candidates to be a part of thermogels’ composition. Poloxamer 407 and poloxamer 188 stand out as the most widely used grades of poloxamers in the development of drug delivery systems. When concentrated aqueous solutions of these poloxamers are exposed to body temperature (near 37 °C), they form a reversible gel that returns to a solution state at room temperature (25 °C). While Poloxamer 407 has the ability to toughen the gel matrix and enhance bioadhesion, its phase transition temperature is higher than the required gelation temperature. Poloxamer 188 is more hydrophilic and has a lower molecular weight than Poloxamer 407, which helps it regulate the sol–gel transition temperature and shear-thinning behavior of Poloxamer 407, when used in combination. Therefore, the addition of Poloxamer 188 to Poloxamer 407 causes a reduction of the phase transition temperature, leading to thermogels with ideal characteristics.^204−206 In this study, it was observed that the optimal ratio of Poloxamer 407 and Poloxamer 188 was 9:1, since it showed the optimal gelation temperature (34 °C) for thermogel synthesis (Figure 8A). Using a Quality by Design (QbD) approach, the formulation was optimized, taking into consideration the NPs’ PS, PDI and EE% as Critical Quality Attributes (CQAs). The optimized formulation achieved a ZP of −28.1 ± 5.64 mV, a small PS (177 nm), and a low PDI (0.147), and the EE% was confidently assumed to be around 90%, based on preliminary model screening. In vitro drug release studies of free drug and SIM-loaded NPs were performed by using the dialysis bag method. The results revealed a sustained release pattern for SIM-loaded NPs, with approximately 50.79 ± 5.56% of the drug being released over a period of 72 h. In contrast, the free drug showed a fast release, where it was completely released within 6 h. To evaluate the thermoresponsiveness of the thermogel, additional in vitro diffusion studies were performed, using Franz cells, maintaining two different temperatures in the receiver compartment, 25 ± 2 and 37 ± 2 °C (Figure 8B). Notably, at 37 °C the nanoparticulate thermogel demonstrated a more sustained drug release, which was significantly different when compared to its performance at room temperature. This can be attributed to the sol-to-gel transition that occurred at the higher temperature, where the observed increase in viscosity restricted the loaded NPs diffusion within the formulation, creating the desired drug depot. Furthermore, in vitro studies were performed to measure the uptake and cell viability of melanoma cells (SK-MEL-28 cell line, a CM cell line) upon exposure to various treatments: free SIM, blank PLGA (BPLGA) NPs, and SIM-loaded PLGA (SPLGA) NPs (Figures 8C and 8D). It was observed that SPLGA NPs’ cytotoxic activity was 9.38-fold higher than free SIM. Thus, the entrapment of SIM in PLGA NPs appeared to increase its accumulation in the SK-MEL-28 cells and reduced its precipitation in the aqueous cell culture media, which is a common obstacle with the administration of the free drug. As a result, the increased intracellular accumulation of SIM led to an enhanced anticancer activity. Additionally, the BPLGA NPs exhibited low cytotoxicity, as evidenced by the observed high cell viability of 97.55 ± 1.29%, indicating that the NPs themselves are a biocompatible option for drug delivery. Furthermore, the sustained release of SIM from the PLGA NPs exposed the cancer cells to the drug over a longer period of time, leading to an increased cytotoxicity. Therefore, in this study, the researchers successfully repurposed SIM in polymeric PLGA NPs for topical application and formulated a thermogel to achieve drug depot formation, for enhanced cytotoxicity in melanoma cell lines by localized prolonged drug exposure.

Figure 8.

(A) Evident sol-to-gel transition behavior of the developed PLGA NPs-loaded thermogel, at room temperature (25 °C) and 34 °C, and thermoresponsive viscosity increase behavior assessment results; (B) In vitro drug diffusion profiles (Franz cells) of the developed NP-loaded thermogel, at 37 ± 2 °C and 25 ± 2 °C; (C) cellular uptake of the developed NPs in SK-MEL 28 cells; (D) SK-MEL 28 cell viability and drug uptake after exposure to the different developed formulations; reproduced with permission from Famta et al.¹³¹ Copyright 2022 Elsevier.

In summary, the analyzed works have highlighted the benefit of encapsulating SIM for enhanced drug delivery in topical applications and increased antitumor activity against skin cancer. Incorporation of nanosystems (hybrid polymer-NLC and polymeric NPs) in these formulations has substantially aided in unlocking the potential of SIM as a repurposed drug targeting skin cancer therapy by topical delivery.

2.5. Antirheumatic Drug Repurposing—Leflunomide

Leflunomide (LFD) is an isoxazole-derived BCS class II drug. It functions as a disease-modifying antirheumatic drug (DMARD) and has demonstrated effectiveness in managing and treating rheumatoid arthritis, and it is a prodrug involved in multiple signaling pathways and cellular processes. LFD undergoes swift conversion into an active metabolite, known as teriflunomide (A771726), via isoxazole ring cleavage. A771726 inhibits dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis in lymphocytes, thus modulating DNA synthesis.^207−209 Over the years, LFD has been used in numerous studies as a potential treatment against several types of cancer, including lung cancer, pancreatic cancer, thyroid cancer, oral cancer, and myeloma, among others, with multiple antitumor action mechanisms having been identified.^210−215 In melanoma, the proliferation of human melanoma cells has been proven to be dose-dependent and mainly due to the activity of the metabolite A77172657. Additionally, the LFD, acting as an agonist, strongly activates the transcriptional activity of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that regulates a wide range of biological activities. These findings imply the potential clinical application of LFD for addressing melanoma.^{113,210,216,217}

Nevertheless, despite its proven beneficial pharmacological effects in the treatment of melanoma, the oral administration of LFD has been linked to moderate to severe side effects such as nausea, vomiting, diarrhea, alopecia, elevated liver enzymes, and hepatic dysfunction. To address these issues, the topical administration of a LFD could be pertinent, especially for skin diseases. Furthermore, due to its poor aqueous solubility (less than 40 μg/mL) and lipophilicity (log P = 2.52), it is worth exploring its incorporation into nanosystems for topical application. In this context, Pund et al.¹³² developed a thermodynamically stable high viscosity nanoemulsion-based nanocarrier system for LFD, to evaluate its anticancer effect against melanoma. The thermodynamic stability derives from the use of self-emulsification, utilizing Capryol 90, Cremophor EL, and Transcutol HP as the nanoemulsifying components and incorporating Pluronic F-127 (Poloxamer 407) as a gelling agent, to achieve the desired final formulation, a nanoemulgel (Figure 9A). Capryol 90 is a propylene glycol monocaprylate with an HLB of 5, denoting its lipophilicity. Due to its low polar surface area, LFD exhibits enhanced solubility in modified oils with small/medium molar volume oil, like medium chain glycerides. In this study, from a screening of oils and modified oils, Capryol 90 showed the highest solubility for LFD. Cremophor EL, a nonionic surfactant which has an HLB value higher than 12, is considered as a comparatively safe, biocompatible, less toxic surfactant, and has the ability to form micelles at a lower concentration than the other surfactants screened in this study. Cremophor EL also showed the highest LFD solubility among the tested surfactants, making it the chosen candidate for subsequent developmental stages. Additionally, it was the surfactant that had a better emulsification ability together with Capryol 90. As for Transcutol HP, it is a diethylene glycol monoethyl ether that has an additional effect of enhancing the skin penetration of lipophilic drugs. Moreover, this cosolvent also showed maximum solubility of LFD. Thus, these were the selected excipients, which led to the formation of a preliminary optimized nanoemulsion with a droplet size of 102.3 nm, PDI of 0.278, and ZP of −7.8 mV. This low ZP absolute value was expected, since the excipients used in the formulation were nonionic. The observed slight negative charge may be attributed to the ionization of trace amounts of free fatty acids present in the chosen excipients. Nonetheless, in the current scenario, the magnitude of the ZP is of lesser significance because the formulation was intended to undergo gelation. Subsequently, the preliminary nanoemulsion was transformed into a nanoemulgel, which was achieved by the incorporation of Poloxamer 407 as the gelling agent in the nanoemulsion matrix, and this formulation exhibited a slight but nonsignificant increase in droplet size, transitioning from 102.3 to 123.7 nm. This expansion in the hydrodynamic size of the formulation droplets can be attributed to the absorption of the gelling polymer onto the emulsion’s droplets. Remarkably, the PDI remained unchanged, thus increasing the probability of maintaining the overall stability of the system. Ex vivo permeation studies, performed in Franz diffusion cells, showed that a conventional gel formulation showed slow, inefficient, and incomplete LFD delivery across rat abdominal skin. As previously noted, the limited aqueous solubility of the drug acts as the first limiting step before the permeation can effectively occur. Nevertheless, the nanoemulgel showed not only a significant improvement of LFD skin permeation, but also a significant increase in the accumulation of LFD in the skin tissues, thus leading to a potentially more effective and prolonged therapeutic effect at the target site. In vitro cytotoxicity studies (Figures 9B and 9C) were performed as well on two melanoma cell lines (A375 and SK-MEL-2), to evaluate the antiproliferative potential of the formulated LFD nanoemulgel. A significant reduction in cell viability and an accelerated cell mortality rate was observed, providing compelling evidence for the heightened efficacy of the nanosized LFD emulsion in gel form, when compared to the free drug. This improved therapeutic outcome was mostly justified by the authors as being due to the enhanced drug skin permeability provided by the nanoemulgel. Furthermore, the formulation appeared to be potentially safe, since histopathology studies, performed on rat abdominal skin after formulation administration, showed no signs of hemorrhage, necrosis, and/or ulceration, thus confirming the nontoxicity of the developed nanoemulgel (Figure 9D). Overall, this research marks another great application of a NP system enhancing the cytotoxic effect of a repurposed drug with therapeutic potential for melanoma. The applied approach was able to evidence LFD’s antimelanoma activity with a targeted delivery topical nanoformulation system (nanoemulgel). This activity was demonstrated to have an enhanced cytotoxic effect against melanoma, that can be attributed to the impact of the nanoemulsion-based formulation.

Figure 9.

(A) Schematic representation of the developed preliminary LFD-loaded nanoemulsion and final nanoemulgel formulations; (B) microscopic images of A375 melanoma cells after no treatment (a), treatment with the free drug (b), or treatment with the developed LFD-loaded nanoemulgel (c), and images of the SK-MEL-2 melanoma cells after no treatment (d), treatment with the free drug (e), or treatment with the developed LFD-loaded nanoemulgel (f); (C) cell viability of A375 and SK-MEL-2 melanoma cells after treatment with the developed formulation, and controls; (D) histopathological images of rat abdominal skin after treatment with the developed formulation, and controls; reproduced with permission from Pund et al.¹³² Copyright 2015 Elsevier.

2.6. Antidiabetic Drug Repurposing—Metformin

Metformin (MET) is a BCS class III biguanide drug used as a first-line therapy for the treatment of type II diabetes. It has been used for over 60 years due to its outstanding ability to decrease plasma glucose levels, well-established safety profile, and relatively low cost. Studies have placed energy metabolism at the center of MET’s mechanism of action in diabetes.^218−220 However, other therapeutic action mechanisms have been identified, making MET relevant for other diseases including cancer. Adenosine 5′ monophosphate-activated protein kinase (AMPK) functions as an energy central regulator, being actively involved in the restoration of energy balance within numerous metabolic pathways. MET directly triggers the activation of AMPK, which leads to the inhibition of the mammalian target of rapamycin (mTOR), a signaling that contributes to the inhibition of the proliferation of cancer stem cells. mTOR is responsible for the regulation of many important physiological functions such as cell growth, proliferation, metabolism, protein synthesis, and autophagy. Thus, by inhibiting this protein kinase, all of these important cellular functions will not occur. MET has also been proven to inhibit skin cancer progression through alternate pathways: stimulation of the immune system, enhancing autophagy and cell apoptosis by p53 and p21 activation.^221−225

Given this context, Mousa et al.¹³³ focused on developing an ethosomal gel to incorporate MET and evaluating its potential as a topical treatment against skin cancer. The design of the drug loaded ethosomal gel centered on achieving optimal PS, EE%, drug release percentage, and ZP, properties which are significantly impacted by the formulation’s composition. For the preparation of the ethosomes, the defined critical parameters were the amounts of lecithin, ethanol, and cholesterol in the formulation. Lecithin was responsible for the assembly of the multilamellar phospholipid membrane of the ethosomes. Higher lecithin concentration led to a reduction of the EE% because of the hydrophilic nature of MET, and also led to an increase in the ethosomal vesicle size due to increasing the tendency of the molecules to coalesce and aggregate. Cholesterol had a relevant influence on both vesicle stability and MET EE%. Increasing the cholesterol concentration in the formulation resulted in an increase in the vesicle size of the ethosomes. Additionally, the mixture of isopropyl alcohol with ethanol notably reduced vesicle size and caused a high negative surface charge and a high EE%, as MET is a cationic drug. Furthermore, isopropyl alcohol was added due to its reported capacity to enhance drug release. Moreover, when Carbopol was mixed with the ethosomes, the desired viscosity and adequate formulation bioadhesion were achieved. The optimal formulation exhibited a remarkable EE% of 98.40 ± 0.35%, a small average PS of 124.01 ± 14.27 nm (Figure 10A), and a measured ZP of −60.08 ± 1.44 mV. From a dialysis-mediated in vitro assessment of the drug’s release into Sorensen phosphate buffer medium, the drug release percentage was recorded as 55.04 ± 0.98% for the optimized ethosomal formulation, revealing a sustained drug release profile (Figure 10B). Skin permeation studies were also performed (Figure 10C) using diffusible membranes that were collected from abdominal rat skin. The results showed that the ideal permeability behavior was observed for the formulation that had the highest ethanol and isopropyl alcohol concentration, the lower lecithin concentration, and a moderate concentration of cholesterol. This was likely related to the fact that when the concentrations of lecithin and cholesterol are increased, the rigidity of the ethosomal vesicle bilayer is also increased. Consequently, a reduction in the concentration of these two components led to an increase in MET permeation rate. In vivo studies were conducted as well, in a mice model, to evaluate the antitumor activity and toxicity of the developed MET-loaded ethosomal gel (Figures 10D, 10E and 10F). The developed formulations did not lead to significant variations in the animals’ body weight, thus implying their safety upon administration. Additionally, to assess the advancement of induced skin cancer, the dimensions of the lesions were measured. It was noticed that the MET-loaded ethosomal gel produced a significant decrease in the lesions’ diameters, when compared with the empty-ethosomal gel, empty gel, and free-MET gel, over 28 days. Hence, the curative effect was significantly enhanced in the MET-loaded ethosomal gel. Furthermore, histopathological studies of the animals’ skin tissues after formulation application revealed only minimal inflammatory cell infiltrates, apparently intact keratinocytes, abundant collagen formation, and a mildly thick dermal layer, thus resulting in improved histologic features, when compared to the other treatment groups (Figure 10G). Thus, the designed ethosomal gel demonstrated effective MET repurposing for an increased antitumor effect in murine skin cancer. These results highlight the potential of the developed approach as a localized application therapeutic option to fight skin cancer.

Figure 10.

(A) TEM photomicrograph of the developed optimized MET-loaded ethosomes; (B) cumulative drug release from the developed ethosomal formulations, with different compositions; (C) cumulative drug permeation from the developed ethosomal formulations, with different compositions; (D) animal body weight variation after administration with the developed ethosomal formulations, and controls; (E) skin cancer lesion length variation after administration with the developed ethosomal formulations, and controls; (F) skin cancer lesion width variation after administration with the developed ethosomal formulations, and controls; (G) histopathological pictures of the animals’ skin tissues after formulation application, stained with hematoxylin and eosin, with (i) and (ii) corresponding to no treatment, (iii) and (iv) to empty gel application, (v) and (vi) to free drug formulation administration, (vii) and (viii) to empty ethosome application, and (ix) and (x) to the optimized MET-loaded ethosomal gel group; reproduced with permission from Mousa et al.¹³³ Copyright 2022 MDPI.

Exploring an alternative treatment strategy that combined photodynamic therapy (PDT) and MET repurposing on a topical formulation, Donadon et al.¹³⁴ evaluated the capability of monoolein (MO)-based nanodispersions to enhance the codelivery of MET and methylene blue (MB) for the treatment of nonmelanoma skin cancer. PDT is a modern and noninvasive clinically approved form of therapy that can have a selective cytotoxic activity against malignant cells. It involves the interaction between a photosensitizer (PhS), the wavelength that corresponds to an absorbance band of the sensitizer, and the presence of oxygen. This reaction triggers the activation of the whole process, leading to selective destruction of tumor cells by necrosis or apoptosis. The photocytotoxic reactions occur only within the pathological tissues located in the region where PhS is distributed, enabling precise and targeted destruction. Initially, PDT relied on administering PhS systemically, but its introduction through topical administration brought a revolutionary shift in the medical field. Moreover, the combination of PhS with nanomaterials has also been an important advancement, due to the potential to enhance the efficiency of PDT and eliminating some of its adverse effects.^226−228 In this context, MB is a PhS with favorable photophysical and photochemical properties, having demonstrated suitable photobiological qualities for PDT, inducing cell death of various types of cancerous cells, encompassing skin malignancies like BCC and melanoma.^229−231 In addition, MET may also induce a mild and specific inhibition of mitochondrial respiratory chain complex I, resulting in a reduction in oxygen consumption by tumor cells and reversing tumor hypoxia. Tumor hypoxia can be a barrier to PDT in cancer treatment because the production of ROS depends on the availability of oxygen during PhS excitation.^232−234 Hence, in this research, the combination of MET and MB was introduced to enhance the effectiveness of PDT as a potential new strategy for treating skin cancer. However, neither MET (log P = −1.43) nor MB (log P = −0.9) possesses suitable physicochemical characteristics for efficient delivery through the skin. Thus, to address this limitation, the creation and evaluation of nanodispersions based on MO for codelivery and colocalization of MET and MB within the skin was performed, meant to represent an enhanced delivery system capable of surpassing the SC barrier and facilitating the simultaneous delivery of MB and MET into the viable skin layers (Figure 11A). The formulation development and screening process focused on achieving optimal drug-loaded nanocarrier properties that met a PS under 150 nm, a PDI under 0.2, and a ZP less than −14 mV, as well as the drug release, skin permeation, and retention profiles of MET and MB components that demonstrated a more pronounced improvement over free-MET and free-MB suspensions (controls). The dispersions with low MO content and stabilized with Kolliphor P407 (Poloxamer 407) and sodium cholate were the most promising in maximizing cutaneous delivery and minimizing transdermal delivery of MB and MET (Figure 11B). Additionally, in vitro viability studies on A-431 cells (SCC cell line) demonstrated that the combination therapy of MET and MB in MO nanodispersions displayed significant cytotoxicity, which could be maximized by the photoactivation of MB. Thus, the MO-based nanoencapsulation of MET and MB allowed for improved antitumor activity. The integration of nanotechnology, drug synergy, and PDT for enhanced cutaneous delivery and cancer cell cytotoxic effects appears to be an innovative approach that holds potential for enhancing treatment outcomes for nonmelanoma skin cancer.

Figure 11.

(A) Summary of the most relevant assays performed in the characterization of the developed nanodispersions; (B) light microscopy image of porcine ear skin after a 12-h treatment with water (i), and confocal microscopy images of porcine ear skin after a 12-h treatment with water (ii), MB and MET aqueous solution (iii), and MB and MET nanodispersions (iv, v, and vi), where the seen fluorescence is related to MB skin penetration; reproduced with permission from Donadon et al.¹³⁴ Copyright 2023 Elsevier.

In summary, the two analyzed studies describe two different lipid-based nanosystems capable of enhancing skin permeation and lead to the targeted delivery of MET, therefore optimizing the antitumor effect of the repurposed drug molecule. Moreover, the research suggests that MET has the potential to augment the desired effect of PDT when treating nonmelanoma skin cancer. These two proposed strategies have successfully highlighted the value of drug delivery systems featuring nanosized configurations, as well as the potential of MET repurposing to target skin cancer.

2.7. Anti-inflammatory Drug Repurposing—Celecoxib

Celecoxib (CEL), a BCS class II drug, is an NSAID that selectively inhibits cyclooxygenase-2 (COX-2). COX-2 is responsible for the prostaglandin synthesis. Prostaglandins are inflammation-promoting molecules. Hence, their inhibition promotes an anti-inflammatory environment. The analgesic, anti-inflammatory, and antipyretic properties of CEL enable it to be commonly used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and acute pain. Additionally, CEL has also been proven to be effective in the inhibition of the progression of skin cancer as well as to enhance the effectiveness of other chemotherapeutic drugs. In melanoma, cell growth is influenced by COX-2 activity, and blocking this enzyme with CEL could offer a reasonable and clinically feasible approach to impede tumor advancement and induce tumor cell death.^235−239

In this context, Ahmed et al.¹³⁵ investigated the combination of a coloaded liposomal gel, containing both doxorubicin (DOXO) and CEL, and the use of Derma roller microneedles (MNs), as a promising therapeutic strategy for the management of melanoma. DOXO stands as the most popular and widely used anthracycline antibiotic turned antitumor agent, finding frequent application in the treatment of both hematological and solid tumors, and also having been proven effective in a few studies against melanoma.^240−244 However, the use of a single chemotherapeutic agent in cancer management has revealed several limitations, such as a lack of sustained efficacy, development of drug resistance, and severe adverse side effects. For these reasons, this study attempted a novel strategy of codelivery of DOXO and CEL, aiming for synergistic effects, with reduced drug doses, thus minimizing undesirable side effects. The developed approach, with skin pretreatment using physical penetration enhancers, aimed to improve the permeation of the drugs through the skin, thus increasing their anticancer efficacy. The utilization of MNs as a means of increasing the physical skin penetration has been widely used in transdermal drug delivery. This approach is valued for its painless properties, no need for specialized medical proficiency (unlike intramuscular and intravenous injections), and for presenting minimal risk of infection at the application site.^245−247 The developed nanogel formulation featured a versatile and widely used gelling agent, Carbopol. The obtained coloaded liposomes displayed a small PS (142.37 ± 0.78 nm) and a homogeneous particle size distribution (PDI = 0.27 ± 0.026), and a ZP of −5.04 ± 0.51. In vitro drug release studies were conducted, in Franz diffusion cells equipped with a semipermeable dialysis membrane, to evaluate DOXO and CEL release from the developed liposomes and liposomal gel, in PBS. When compared with the release profile observed in liposomal drug suspensions, all liposomal gel formulations exhibited similar and sustained release behaviors, devoid of any initial burst release, which was an adequate result for obtaining a potentially prolonged therapeutic effect. Ex vivo skin permeation studies were also conducted, using abdominal skin from mice, in modified Franz diffusion cells, and results showed a notable increase in the penetration of DOXO and CEL through the skin’s layers into the receptor compartment in the groups that underwent derma roller pretreatment, as opposed to the untreated group. Moreover, the permeation and retention of DOXO improved when CEL was incorporated. The in vitro cytotoxicity of the developed formulations was tested as well on B16 murine melanoma cells. A synergistic effect was observed when CEL and DOXO were combined in solution. However, liposomal formulations coloaded with DOXO and CEL exhibited greater cytotoxicity than the DOXO/CEL solution. Furthermore, the inhibitory rates of the DOXO/CEL coloaded liposomes were superior in comparison to liposomes with a single drug. Moreover, the formulations’ in vivo anticancer effect was evaluated on the skin area where B16 cells had been implanted in a mice model. Mice that were treated topically with the coloaded liposomal gel denoted significantly enhanced antitumor effect, which can be attributed to an effective accumulation within the tumor tissue. The pretreatment with the MNs also helped to increase the tumor inhibition rate, by improving skin permeability from the microsized holes induced in the SC. Additionally, although mice that received topical treatment with a DOXO liposomal gel displayed visibly smaller tumor sizes compared to untreated mice, however, the tumor size in this group was still significantly larger than in the animals treated with the DOXO/CEL coloaded liposomal gel formulation. To summarize, the developed coloaded DOXO/CEL liposomal gel, combined with the utilization of MNs, was able to significantly elevate the tumor growth inhibitory rate in melanoma-grafted mice. These results demonstrated the benefit in opting for a combination therapy approach, rather than single-drug administration, as well as selecting topical application by nanocarrier-facilitated delivery for an increased localized effect, further enhanced by the influence of MN application.

3. Final Remarks

Drug repurposing has had a tremendous impact on the discovery of new therapeutic options in the fight against skin cancer. It has allowed researchers to confidently unlock the anticancer potential of various marketed drugs with less effort and less cost, backing on their known pharmacological profile from their original applications. As highlighted in the analyzed research, various drugs, namely, doxycycline, itraconazole, niclosamide, simvastatin, leflunomide, metformin, and celecoxib, were successfully repurposed for skin cancer treatment. Thus, several different pharmacological classes were included, comprising antibacterial, antifungal, anthelmintic, antidyslipidemic, antirheumatic, antidiabetic, and anti-inflammatory drugs.

Unlike most cancers, the location of cutaneous carcinoma allows for targeted treatment options that rely on simple technologies, such as topical formulations directly applied on the affected area, that shy away from complex procedures, prioritize the localized therapeutic effect, offer convenience to the patient, and minimize systemic exposure and the adverse symptoms often associated with it. However, topical drug delivery is often focused on overcoming the greatest challenge: penetrating the skin barrier. Here, nanotechnology has been a great help. Nanotechnology has revolutionized the field of drug delivery, especially in oncology. In the context of topical administration of repurposed drugs targeting skin cancer, nanoparticle-based therapeutics have made remarkable improvements to skin permeation, therapeutic efficacy, toxicity, and stability. The repurposed drug molecules studied in the analyzed literature were encapsulated into different nanosystem types, namely, nanoemulsions and nanoemulgels, nanodispersions, solid lipid nanoparticles, nanostructured lipid carriers, polymeric nanoparticles, hybrid lipid–polymer nanoparticles, hybrid electrospun nanofibrous scaffolds, liposomes and liposomal gels, ethosomes and ethosomal gels, and aspasomes.

In the present review, all studied approaches presented unique NP systems successfully integrated in topical formulations for the encapsulation of repurposed drugs, with the goal of improving these drugs’ antitumor activity toward melanoma and nonmelanoma skin cancers. The proposed repurposed drugs have been evidenced to have potential anticancer activity, which was evaluated in a topical delivery scenario. Most strategies focused on maximizing the localized delivery and enhancing the drugs’ skin permeation and upper layer retention profiles for better treatment efficacy. In all cases, this goal was remarkably achieved by formulation optimization, namely, regarding the nanosystem. The results when evaluating the cytotoxicity of the drugs incorporated in the NP-based formulations, compared to free drug formulations, evidence that the designed approaches significantly outperformed the conventional formulations. In some cases, an intrinsic antitumor effect from the NPs themselves was registered, providing great evidence for the benefit of nanotechnology in skin cancer therapy.

It was also observed that in general, the NPs that achieved the best performance had a PS smaller than 200 nm. As for the system’s homogeneity, a PDI under 0.3 is recommended and should be targeted to be as close to 0 as possible, which happened for most of the optimized formulations. When the NP’s surface (evaluated by ZP) was described as positively charged, an interactive behavior of the particles fusing with the negatively charged cellular membranes was responsible for improved drug permeation and retention profiles. The effect of the ZP in formulation stability was more pronounced in liquid formulations (nanodispersions and nanoemulsions), where the repulsive behavior of the suspended particles or droplets is very important to prevent the aggregation phenomenon. A high absolute value was targeted, with most designed systems achieving at least ±10 mV. In the case of semisolid products, due to their higher viscosity, the ZP does not have such a strong relation to formulation stability.

Furthermore, most of the designed formulations showcased a favorable toxicological profile, highlighted by negligible cytotoxicity in healthy cells and a high skin tolerability. The inclusion of biocompatible materials was certainly a priority for the formulation design process to ensure that the proposed systems can offer little to no harm to future patients upon administration.

Despite no current commercialization of nanobased topical formulations for cancer treatment, the explored research findings highly evidence the potential of these approaches to complement the currently available therapeutic strategies. Hopefully these treatment options will soon be available in the pharmaceutical market, as their inexpensive and convenient nature as well as their proven high anticancer efficacy drives their applicability.

4. Conclusions

Several repurposed drugs, namely, doxycycline, itraconazole, niclosamide, simvastatin, leflunomide, metformin, and celecoxib, formulated for topical application, had their effects successfully enhanced by incorporation into different nanosystems, namely, nanoemulsions and nanoemulgels, nanodispersions, solid lipid nanoparticles, nanostructured lipid carriers, polymeric nanoparticles, hybrid lipid–polymer nanoparticles, hybrid electrospun nanofibrous scaffolds, liposomes and liposomal gels, ethosomes and ethosomal gels, and aspasomes. The skin permeability of these nanosystems allowed for enhanced antitumor effect on melanoma and nonmelanoma research models. In some instances, the nanoparticles acted not only as drug carriers but also as anticancer agents. Hence, despite no current commercialization of nanobased topical formulations for cancer treatment, the explored research findings highly evidence the potential of these approaches to complement the currently available therapeutic strategies. Hopefully these treatment options will soon be available in the pharmaceutical market to help in the fight against skin cancer.

Glossary

Abbreviation list

AA

Ascorbic acid

AhR

Aryl hydrocarbon receptor

AMPK

Adenosine 5′ monophosphate-activated protein kinase

AP

Ascorbyl palmitate

API

Active pharmaceutical ingredient

BCC

Basal cell carcinoma

BCS

Biopharmaceutical Classification System

BLIP

Blank liposomes

BPLGA

Blank PLGA

CEL

Celecoxib

Ch-NLC

Chitosan-coated nanostructured lipid carrier

CM

Cutaneous melanoma

COX-2

Cyclooxygenase-2

CPE

Chemical permeation enhancers

CQA

Critical quality attribute

DDAB

Didodecyldimethylammonium bromide

DHODH

Dihydroorotate dehydrogenase

DMARD

Disease-modifying antirheumatic drug

DNA

Deoxyribonucleic acid

DOX

Doxycycline

DOXO

Doxorubicin

EE%

Encapsulation efficiency

FDA

Food and Drug Administration

Gel

Gelatin

HA

Hydroxyapatite

Hh

Hedgehog

HLB

Hydrophilic–lipophilic balance

HMG-CoA

Hydroxymethylglutaryl-coenzyme A

HPMC

Hydroxypropyl methylcellulose

ITZ

Itraconazole

IV

Intravenous

L

Limonene

LDL

Low-density lipoprotein

LFD

Leflunomide

MB

Methylene blue

MET

Metformin

MMPs

Matrix-metalloproteinases

MNs

Microneedles

MO

Monoolein

mTOR

Mammalian target of rapamycin

NEGs

Nanoemulgels

NEs

Nanoemulsions

nHA

Hydroxyapatite nanoparticles

NIC

Niclosamide

NLCs

Nanostructured lipid carriers

NLIP

Niclosamide-loaded liposomes

NMSC

Nonmelanoma skin cancer

NP

Nanoparticle

NSAID

Nonsteroidal anti-inflammatory drug

O/W

oil-in-water

PBS

Phosphate-buffered saline

PCL

Poly-ε-caprolactone

PDI

Polydispersity index

PDT

Photodynamic therapy

PEG

Polyethylene glycol

PG

Propylene glycol

PhS

Photosensitizer

PLGA

poly(lactic-co-glycolic acid)

PS

Particle size

PS80

Polysorbate 80

QbD

Quality by Design

ROS

Reactive oxygen species

SC

Stratum corneum

SCC

Squamous cell carcinoma

SIM

Simvastatin

SLNs

Solid lipid nanoparticles

SMO

Smoothened protein

SPLGA

Simvastatin-loaded PLGA

STAT3

Signal transducer and activator of transcription 3

UV

Ultraviolet

W/O

Water-in-oil

ZP

Zeta potential

The authors declare no competing financial interest.