Nail disorders are beyond cosmetic concern; besides discomfort in performance of daily chores, they disturb patients psychologically and affect their quality of life. Fungal nail infection (onychomycosis) is most prevalent nail related disorder affecting major population worldwide. Overcoming the impenetrable nail barrier is the toughest challenge for development of efficacious topical ungual formulation. Sophisticated techniques such as Iontophoresis, Photodynamic therapy have been proved to improve transungual permeation. This article provides updated and concise discussion regarding conventional approach and upcoming novel enhancers/research approaches focused to alter nail barrier. A comprehensive description regarding pre-formulation screening techniques for identification of potential ungual enhancers is described in this review. An attempt has been made to elaborately describe the characterization techniques for pre-screening of ungual enhancers and to highlight the current pitfalls for development of ungual delivery.
Skin and its auxiliary appendages such as hair and nails represent an area of great importance in dermatology or any cosmetic field because disorder in any of these parts have a direct impact on external appearance, psychological and normal daily routine. Nails disorders are not life threating but if untreated can transform from a non-specific to an exasperating problem, which consumes lot of time to restore into its normal condition. A synergistic combination of systemic with topical delivery is preferred approach for efficient treatment of onychomycosis. In spite of availability of several treatment options for ungual infection, none of the remedies give absolute fungal eradication. Psoriatic nail dystrophy is another common nail disorder, compared to skin psoriasis; treatment options for nail psoriasis are limited and often give disappointing result. Numerous strategies have been employed and succeeded to improve drug diffusion through the dense ungual keratin layers. A brief research based on novel approaches to treat ungual disorders has been described in this review. These agents have shown marked improved transungual diffusion of drug into the nail and compared to conventional ungual enhancers cause minimal damage to nail keratin. Major emphasis is given to biophysical and bioengineering techniques to utilize their potential to understand and characterize nail barrier for screening ungual enhancers.
1.1. The human nail and its anatomy
A nail is a horn-like envelope covering the dorsal portion of the terminal phalanges of fingers and toes in humans, primates, and a few other mammals.
Figure 1a. External nail antaomy
Figure 1b. Layers of Nail plate
The human nail apparatus comprises of nail plate, nail bed, nail folds, and the nail matrix. As shown in figure 1-the nail plate is the actual fingernail, consisting of translucent keratin covering the entire nail bed. The nail plate is a thick, elastic, convex structure composed of approximately 25 layers of tightly bound dead keratinised cells. The nail plate is divided into three layers, upper dorsal layer, intermediate layer followed by the inner ventral layer (figure 1b). The thickness of each layer is in the ratio 3:5:2 respectively .The dorsal layer is most resistant barrier for penetration of molecules.The cutaneous wedge shaped skin folds overlapping the sides and proximal end of the nail are the nail folds. The visible part of the nail matrix or the edge of the germinal matrix is called as lunula. It is white cresent moon shaped and is located at the base of nail (prominently visible on thumb nail) .The junction between the free edge and the skin of the fingertip is known as the hyponchium. It is an epithelium tissue and its function is to protect the nail bed. The seal between the nail plate and the hyponchium is known as the onchodermal band. A small band of epithelium extending between the posterior nail wall onto the base of the nail is known as the eponychium. The paronychium is the border tissue around the nail. It is also known as the paronychial edge and is the site of the infection of the nail disorder known as paronychia. 
Figure 2. Internal nail anatomy
Figure 2. Diagrammatically represents the interior structure of fingernail -The nail bed is the immediate living tissue present beneath the nail plate. The nail matrix (keratogenous membrane or onychostroma) is a living tissue located exactly below the lunula which protects the nail extending several millimetres into the finger. according to its Based on their function nail matrix is classified into subtypes namely sterile matrix and germinal matrix. The sterile matrix is responsible for the production of the nail bed. The germinal matrix produces the cells which subsequently become the nail plate. The edge of germinal matrix is visible, called as lunula of nail plate. The nail root (radix unguis) is the base of nail formed from the tissue growing below the matrix. The nail sinus (sinus unguis) is a deep furrow into which the nail root is inserted. 
1.2. Nail Disorders
The two most frequent ungual disorders are onychomycosis and nail psoriasis. Onychomycosis is responsible for 50% of the nail disorders. It affects approximately 10% of the general population .It is more prevalent in diabetic and elderly population. Use of excessive immunosuppressant’s can also lead to onchomycosis. Nail psoriasis is reported in 80-90% of the patients suffering from skin psoriasis. It affects 1-3% of the total population .
A comprehensive description of nail disorders and their characteristics symptoms observed are listed briefly in table 1
Table 1. Nail disorders and their characteristics symptoms
Disorder Characteristics observed
Onychomycosis ‘ Fungal infection of nail plate caused by dermatophytes such as Trichophyton, epidermophyton and microsporum species(responsible for 80-90% of the cases), and seldom by non dermatophyte fungi such as Aspergillus, Fusarium spp, and yeasts such as Candida spp.
‘ Fungii digests the nail keratin causing discolouration, thickening and splitting of nails.
‘ Irritation of the nails and pain is observed
Nail Psoriasis ‘ Presence of scales pits on the nails, red and yellow discolouration of the nails. The skin under the nail gets thickened.
‘ Crumbling of nail is also observed.
‘ The nail plate gets separated from the nail bed.
‘ Pain, redness and swelling of the nail fold and formation of pus filled blisters.
‘ The nail plate becomes thickened with prominent transverse ridges.
Tinea unguis ‘ Thickening of the nails due to presence of ringworm infection. If left untreated can lead to complete loss of nail plate.
Onychogryposis: ‘ Thickening of the nail plate and the nail plate is observed to curve inwards the nails, with a characteristics claw-type appearance .
Onychatrophia: ‘ Nail plate gets atrophised, loses its lustre, reduces in size and sometimes sheds entirely .
Koilonychia ‘ The nails become thin and concave in shape like a spoon and show raised ridges .
Melanonychia ‘ Black or brown pigmentation of the nail plate .
1.3. Nail growth and regeneration
The growth rate of normal fingernails varies from <1.8mm to '4.5mm per month . The average growth of nail per day is 0.1mm. Toenails grow at a rate one-half to one-third of the growth of the fingernails. A normal fingernail generally grows fully in about 6 months whereas a toenail takes about 12 to 18 months for complete growth .In a dominant hand the nail growth is faster. The rate of nail growth is higher in males than females. Age and environment also play a major role for growth of nails. The rate of growth in nails is slow in the old age and high in cold climate. Environmental factors such as exposure to chemicals, strong detergents, reaction to adhesives used in artificial nails can lead to nail abnormalities. It is observed that after nail avulsion nails grow at faster rate . Treatment with drugs such as benoxaprofen, biotin, cysteine, methionine, levodopa, itraconazole accelerates the nail growth. The nail growth is retarded in presence of infections and in conditions like fever, malnutrition, decreased circulation and lactation. Administration of antimycotic drugs also decreases the rate of nail growth . 2. STRATEGIES TO ENHANCE TRANSUNGUAL PERMEATION OF DRUGS The nail keratin cells are tightly bound, arranged in form of compact blocks with no interstitial space in between. The thickness of the nail plate, its high sulphur content and the marked differences between the nail plate and the stratum corneum (Table 2) makes the nail plate an impenetrable barrier for transport of the drug across the nail plate. To overcome the tough nail barrier and enhance transungual drug delivery, several methods and techniques have been adapted by researchers, which are briefly described in this review. Table 2: Comparison between the nail plate and the stratum corneum . Composition Nail Stratum corneum Thickness 500-1000??m 10-40??m Disulphide linkage 10.6% 1.2% % swelling in water 25% 200-300% Lipid content 0.1-1% 10-20% Table 3: Amino acid composition of the human nail plate and the stratum corneum . Amino acid Stratum corneum Nail Lys 4.2% 3.1% S 1.4% 3.2% Glu 12.6% 13.6% Gly 24.5% 7.9% ?? Cys 1.2% 10.6% 2.1. Conventional approach Due to lack of basic understanding of nail anatomy and its permeability, initially mechanical methods such as partial removal of nail plate/complete nail avulsion followed by subsequent application of drug were used for treatment of onychomycosis [12, 13].These methods are non-patient compliant and are practically infeasible solution as a complete cure for onychomycosis. Disrupting basic nail keratin backbone, using disulphide reducing agents (sodium sulphite, dithioreitol) and keratolytic agents (urea, lactic acid, salicylic acid) are one of the common approaches for enhancing permeation of antifungals into the nails. Chemical agents such as urea, thioglycollic acid, and enzyme like papain interact with the disulphide bond of the nail keratin and facilitate their breakage, aiding improved transport of drug across the nail plate.  Recently sequential application of oxidising and reducing agents for improved transungual delivery was reported by M.D. brown et al. Two penetration enhancers (PEs), thioglycolic acid (TA) and urea hydrogen peroxide (urea H2O2) and their sequential pre-treatment onpermeation of three model permeants (caffeine, terbinafine and methyl paraben) were studied. The diffusion flux of all permeates were significantlyincreased in presence of the penetration enhancers. The sequential application of TA followed by urea H2O2 increased flux of terbinafine and caffeine but reversing their application order mild increase in flux of methyl paraben was observed . 2.2. Sophisticated technologies Sophisticated technologies employing iontophoresis, ultrasound, and ultraviolet energy could alter nail keratin physically, with minimum damage, enhancing penetration of drug into the nail. a) Iontophoresis Iontophoresis is most effective technique for driving higher amount drug into the nail through the dense keratin layer [16, 17, 18, 19, 20]. With aid of iontophoresis drug depot can be formed into layers of nail keratin which gradually releases drug with time. Hao and li examined the effect of iontophoresis on permeation of antifungal drug ciclopirox . A small portable, disposable, user friendly device was developed which significantly delivered high amount of ciclopirox iontophoretically from its lacquer formulation compared to its passive delivery from same formulation and marketed lacquer penlac. Similar study was performed by Nair et al on human nail for enhancing delivery of terbinafinehcl . Ionotophoresis could successfully drive ionic terbinafine molecules into the nail. Light microscopy study using methylene blue was performed and uptake of methylene blue was found to be highest using iontophoresis into the three layers of nail when compared with control. Manda et al studied iontophoretic delivery of terbinafine through proximal nail fold using cadaver toe nail model . A custom designed polyurethane foam pad was employed as iontophoreic device which significantly delivered high amount of terbinafine into the nail matrix and deeper ungual layers compared to its passive delivery. b) Co2 lasers Lim et al used combination of fractional Co2 laser therapy with topical antifungal treatment for treating 24 patients suffering from onychomycosis . Nail plate were punctured using ablative co2 laser followed by topical application of amolorofine cream. At the end of study, it was observed that the fungi resided area of the infected toenail of patients wassignificantly decreased with improved visual appearance. Out of 24, total of 22 patients (92%) showed a clinical response, and 12 patients (50%) showed a complete cure with a negative microscopic result and no adverse effect. The authors postulated that ablative fractional Co2 laser exerted direct fungicidal effect and created multiple porosities into nail plate enhancing the penetration of antifungal agent into the nail bed or matrix . c) Etching/mesoscissing Etching involves production of minuscule micropores on surface of nail plate. Certain surface modifying agents such as phosphoric acid, tartaric acid, or devices such as (Path Former) creates microporosites on the nail surfaces, decreasing the contact angle providing a better surface for the drug to bind . Path Former(Path Scientific, Carlisle, USA) is an FDA approved etching device, which creates miniature pin holes into the nails without affecting the nail bed and helps in draining the subungual hematomas . The device uses electrical resistance of the nail as the feedback and eliminates the need for anaesthesia. The drilling of the nail plate is done by using a 400 micrometer tissue cutter and is retracted when it has penetrated into the nail plate. After the nail is etched a nail lacquer can be applied on the nails promoting sustained release of the drug. d) Ultrasound An ultrasound-mediated drug delivery system was developed by Abadi and Zderic, 2011 for treating onychomycosis. The slip-in device consists two compartments namely ultrasound transducers and drug delivery compartments above each toenail. The device is connected to a computer, where a software interface allows users to select their preferred course of treatment. Using an ultrasound-mediated drug delivery system, thrice amount of drug was delivered into the nail . e) Laser/UV Photodynamic therapy Laser wavelength in near infra- red region (780 nm -3000 nm) has capacity to directly heat the target tissues. Laser therapy has been reported in articles for curing onychomycosis [28, 29, 30, 31]. A pulsed laser technology has been employed for eradication of Trichophyton rubrum. Direct thermal killing of fungal mycelia on nail clippings was observed when the temperature exceeded 50 degrees centigrade. Photodynamic therapies have shown remarkable results in treatment of skin related disorders [30, 31, 32]. Same technology was utilized Ryan et al, the authors treated infected fungal nail using a combination of a light sensitive drug (5-Aminolevulinic acid- ALA) and visible light which causes destruction of selected cells. Incubation of dermatophytes such as Candida albicans and Trichophyton interdigitale in presence of ALA (10 mM), followed by irradiation with light caused reductions in viability of organisms by 87% and 42%, respectively. ALA was applied in form of bioadhesive patch on the human nails, ALA induced accumulation of photosensitizer called protoporphyrin IX which subsequently lead to photodynamic destruction of fungi.  2.3. NOVEL UNGUAL PERMEATION ENHANCERS a. Water- Primary ungual permeation enhancer Water diffuses into the nail more rapidly compared to stratum corneum, also the rate of transonchial water loss from nail keratin is higher than tewl of stratum corneum [34, 35, 36]. Kelly et al compared the effect of plain organic and binary mixture of aqueous organic solvents systems on nail hydration and permeability .Ungual uptake and transport was correlated to concentration of organic solvent employed in study. It was observed that substituting water with a non-polar solvent decreases drug penetration across the nail plate. Higher the concentration of organic solvent, slower was ungual uptake and transport of radioactive probes across the nail. Water miscible solvents such as polyethylene/polypropylene glycol could hydrate the nail to higher extent compared to plain organic solvents. Nail keratin swells in presence of water and becomes more flexible. The hydrated keratin cells moves apart and the dense keratinized nail behaves like a hydrophilic gel matrix. Drug moieties can therefore diffuse through the hydrated keratin network with less resistance . This principle was utilized by gunt et al to increase the permeation of ketoconazole through human nail. The permeability of antifungal ketoconazole was tested at different relative humidity (RH) to study effect of hydration on permeation of ketoconazole. Radiolabelled [3H] ketoconazole was employed to study the permeability of ketoconazole solution through human cadaver nails over a period of 40 days. The permeability of ketoconazole increased in order of three fold as the ambient RH was increased from 15 to 100%. . Hui et al compared the penetration profile of ciclopirox between marketed organic solvent based lacquer (penlac), aqueous marketed gel and experimental gel . The rate of permeation of ciclopirox in aqueous gel formulations were higher compared to penlac. Water itself acted as permeation enhancer which hydrated the nail and ultimately lead to an increased permeation of drug into nail. Similar results were obtained by D. Monti et al, permeation of two antifungal drugs ciclopirox and amolorfine in novel chitosan based water soluble nail lacquer were studied and compared with marketed amolorolfine lacquer (Loceryl) using bovine hoof slices . The hydro soluble lacquers showed enhanced permeation and invitro antifungal activity into hoof keratin compared Loceryl. It was found that application of chitosan based ciclopirox nail lacquer on hoof keratin, resulted in rapid penetration of ciclopirox compared to marketed non aqueous lacquer. The growth of the fungus Candida parapsilosis was inhibited up to 30 hours after the application of hydrosoluble nail lacquer. The reason postulated by authors was presence of aqueous vehicle along with adhesion promoters like chitosan could lead effective transport of active across the nail keratin.  General conventional nail lacquers are based on water-insoluble resins and have limited potential to enhance the transungual drug delivery. On the contrary, aqueous-based lacquers can promote the nail hydration and drug diffusion across the nail plate, but suffer limitation of being easily wiped off or washed off the nail surface. Hence, to incorporate the properties of both water soluble and water insoluble nail lacquer Shivakumar et al. proposed a bilayered nail lacquer for onchomycosis treatment . The lacquer consisted of two layers, underlying hydrophilic layer containing the drug terbinafine hydrochloride and an upper hydrophobic vinyl layer. The hydrophilic layer was based on HPMC and adhered well to the surface of the nails. The vinyl layer was applied to protect the underlying drug containing layer getting washed off during daily chores. It was found that the bilayered lacquer was resistant to drug loss on multiple washings and a significant high amount of terbinafinehcl was retain into nail layers compared to hydrophilic monolayer lacquer and control. In-vitro efficacy demonstrated an enhanced activity with bilayered lacquer.  b. SEPA: Hui et al used SEPA (2-n-Nonyl-1,3-dioxolane) for improving penetration of econazole from a nail lacquer formulation (Econail) . It was found that addition of SEPA could increase permeation of econozole 6 times higher than control. Econail could deliver significantly higher amount of econozole in all three layers of nail and nail bed as compared to control. Dioxalones are generally skin permeation enhancers acting by altering lipid diffusion pathway of skin. The exact mechanism of dioxalone promoting econozole influx into the nail was not clearly understood but it was reported by author that SEPA acted as adhesion promoter and plasticizer for nail which facilitated increased diffusion of econozole into the nail. c. Hydrophobins: Hydrophobins are amphiphilic fungal proteins, which were recently proved by Vejnovic et al., 2010 as prospective transungual permeation enhancers .Vejnovic et al, investigated permeation enhancement potential of hydrophobins A-C for transport of terbinafinehcl across the nail. All the hydrophobins successfully increased permeability of terbinafine across the nail, hydrophobin B was superior among all of them showing highest permeability coefficient and 13 fold enhanced permeation of terbinafine. The mechanism of action by which hydrophobins act as permeation enhancers is still under research, some of their modes of action were reported by authors as follows. Structurally hydrophobins are stable having eight cysteine residues and four disulphide linkages, which lead to better protein interactions with keratin fibres and also with fungi proteins. Further, hydrophobins had amphipihilc structure with unique self- assembling and adherent properties and were able to coat terbinafine improving its solubility and physical stability. The coated terbinafine was found to have greater affinity for the hydrophilic gel membrane of the human nail thus increasing its permeation. Results suggested that the addition of hydrophobins improved permeability in the range of 3E'10 to 2E'9 cm/s. As of interest hydrophobins are new emerging ungual enhancers with unique features, more research is still required for investigating their complete potential as ungual enhancer and probably will be found. d. Keratolytic enzymes M. Mohorcic et al studied the effect of fungal keratinase produced by P. marquandiion on permeation characteristics of nail plate and bovine hoof . It was found that the enzyme acted on the intercellular matrix which holds nail cells together, which resulted corneocytes separation from one another. SEM images showed that the corneocytes were 'lifted off' the plate and their surface was corroded. Pre-treatment of hoof with keratinase resulted in enhanced transungual permeation of model drug metformin. The permeability co-efficient and drug flux were found to be significantly increased in the presence of the enzyme. It was concluded that the enzyme, via its hydrolytic action on nail plate proteins can improve permeation and ungual uptake of drug. Similar Tiwary and Gupta isolated a combination of novel enzymes termed as Ker N which is chemically subtilisin-??-Glutamyl Transpeptidase from a feather degrading strain of Bacillus licheniformis . The KerN enzyme increased the permeability of nail by loosening nail matrix and corroding the dorsal surface, which was confirmed by SEM images of nail plate treated with KerN. Drug permeation studies revealed that 58% clotrimazole was retained into the nail plates after 24 h exposure with 300 ??g/mL of kern in presence of drug. The enzyme had high potency and was found to be stable in presence of drug even after 72 h. The authors therefore proposed, KerN as a novel ungual enhancer to increase the permeability of drug during topical application on nail plates. e. Inorganic salts Inorganic salts such as sodium phosphate can serve as excellent, non-toxic, cheap ungual permeation enhancers . Optimum concentrations of these salts have ability to increase the nail hydration with increased thermodynamic activity of drug. Nair et al studied effect of different inorganic salts (ammonium carbonate, sodium phosphate, calcium phosphate, potassium phosphate , sodium sulphite) on transungual permeation of terbinafineHCl. All the above salts enhanced transungual permeation of terbinafine in nail plate by 3 ' 5 folds. Among this, sodium phosphate showed highest, 5 fold enhancement of terbinafine permeation as compared to control. A 0.5 M sodium phosphate was employed as permeation enhancer in polaxamer based terbinafine gel and transungual in-vitro diffusion studies was carried using Franz diffusion cell. The cumulative amount of terbinafine permeated after 24 h from the formulated gel was higher than control. Hence, inorganic salts such as sodium phosphate can serve as promising novel ungual penetration enhancers f. Lipid diffusion enhancers Maibach et al incorporated ciclopirox in an oily vehicle consisting of benzyl alcohol, peppermint oil, turpentine and mineral oil, for enhanced transungual permeation. In contrast to hydrogel nature of nail, this lipophilic formulation showed significant rate of penetration of ciclopirox compared to its commercial lacquer penlac after 11 days in vitro diffusion study on human nail plate. Ciclopirox content into all three layers of nail plate and nail bed from the novel lipidic formulation was found to be significantly higher than penlac. The authors hypothesized that though hydrophilic pathway is predominant pathway for molecules to diffuse into the nail, there exist miniature lipidic pathway into the nail through which lipophilic moieties traverses and by passes the impenetrable keratin corneocytes. This new pathway can be studied and further explored to develop more efficacious ungual formulations. Thus an appropriate combination of hydrophilic and lipophilic enhancers is suspected to give optimum and efficacious drug delivery into ungual layers.
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