Effect of penetration enhancers on drug nail permeability from cyclodextrin/poloxamer-soluble polypseudorotaxane-based nail lacquers

pharmaceutics Article Effect of Penetration Enhancers on Drug Nail Permeability from Cyclodextrin/Poloxamer-Soluble Polypseudorotaxane-Based Nail Lacquers Elena Cutrín-Gómez 1, Soledad Anguiano-Igea 1, M. Begoña Delgado-Charro 2, JoséLuis Gómez-Amoza 1and Francisco J. Otero-Espinar 1,* 1Department of Pharmacology, Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Santiago de Compostela 15782, Spain; [email protected] (E.C.-G.); [email protected] (S.A.-I.); [email protected] (J.L.G.-A.) 2Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK; [email protected] (M.B.D.-C.) *Correspondence: [email protected] Tel: +34-609-718-821 Received: 18 November 2018; Accepted: 11 December 2018; Published: 13 December 2018   Abstract: Nail delivery has interest for local treatment of nail diseases. Nevertheless, the low permeability of drugs in the nail plaque precludes the efficacy of local treatments. The use of penetration enhancers can increase drug permeability and improve the efficacy of the treatment of nail pathologies. In this work, different chemical substances have been evaluated as potential penetration enhancers. With this aim, the effect of different substances such as sodium lauryl sulfate (SLS), polyethylene glycol 300 (PEG 300), carbocysteine, N-acetylcysteine, lactic acid, potassium phosphate, Labrasol ® and Labrafil ® in the microstructure, nail surface and drug permeability has been evaluated. The models obtained by mercury intrusion porosimetry and PoreXpert ™ software show a more porous structure in nails treated with different enhancers. Permeation studies with bovine hooves and nails revealed that all the hydroalcoholic lacquers developed, and particularly those prepared with SLS, provide better nail penetration of the drugs ciclopirox olamine and clobetasol propionate. Results have shown that the increase of the drug penetration in the nail is caused by the formation of a porous random microstructure and by the decrease of the contact angle between lacquers and the surface or the nail plaque. The presence of SLS produces an improvement in the spreading of the solution on the nail surface and promotes the penetration of the solution into the nail pores. The hydroalcoholic lacquer, elaborated with cyclodextrin/poloxamer soluble polypseudorotaxane and sodium lauryl sulfate as an enhancer, allowed the rate of diffusion and penetration of the active ingredient within the nail to be significantly higher than obtained with the reference lacquers when using either ciclopirox olamine or clobetasol propionate as the active ingredient. Keywords: transungual drug delivery; nail; medicated nail lacquers; onychomycosis; nail psoriasis; polypseudorotaxanes; methylβ -cyclodextrin; poloxamers; ciclopirox olamine; clobetasol propionate; penetration enhancer 1. Introduction Ungual drug delivery has been receiving increasing attention in the past few years [ 1 , 2 ]. This is caused by the need to have topical formulations available that improve drug penetration in the nail structure and, consequently, the efficacy of treatment of nail pathologies. The objective pursued with an ungual topical therapy is the attainment of, at least, the minimum therapeutic concentration in every layer of the nail relevant to the pathology considered, including Pharmaceutics 2018,10, 273; doi:10.3390/pharmaceutics10040273 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2018,10, 273 2 of 18 the deepest ones. This is a challenging task since the nail layer, besides being much thicker than the stratum corneum, presents completely different physicochemical and structural characteristics [ 3 ]. For this reason, dermal topical formulations present very low or even no efficacy in ungual drug delivery since the methods used to improve drug penetration into the skin are ineffective in this area of administration [4–7]. The nail plate is mainly composed of keratinocytes with different degrees of keratinization depending on the location. The superficial (dorsal) area of the plate consists of dead cells that have lost their nucleus and store a great amount of fibrous proteins, such as keratin, which provides elasticity and resistance. The intermediate area consists of cells that are strongly bound to one another by desmosomes and hemidesmosomes and contains high proportions of keratinized cells, although the cells contain a smaller amount of keratin than those of the superficial area. Lastly, the lower (ventral) area, which rests on the nail bed, consists of two layers of cells stemming from the epidermic bed [ 8 – 11 ]. Finally, nails have a lower proportion of intercellular lipids than skin [ 12 ]. For these reasons, the nail plate represents a more hydrophilic barrier than the skin [12]. Incorporation of certain chemicals into ungual topical formulations might modify the nail plate structure, promoting drug penetration and increasing the efficacy of topical therapies. It is not surprising, therefore, that chemical methods have been explored together with physical and mechanical enhancement approaches to promote drug penetration across the nail plate [ 1 , 3 , 13 ]. Because mechanical methods, including abrasion and avulsion of the nail, have poor patient acceptance since they are traumatic, painful and invasive, most recent research has focused on finding new non-invasive physical methods and chemical procedures [14,15]. Chemical methods involve incorporating permeation enhancers that increase drug penetration into the nail into the formulation [ 16 ]. A priori, permeation enhancers, normally used in dermal and transdermal delivery, are expected to be ineffective on the nail for the reasons above. Thus, the search for chemicals that facilitate drug ungual penetration, the main objective of this study, is especially timely [2,3]. So far, few substances with the capacity of promoting penetration into the nail have been described. For example, N-acetylcysteine [ 17 , 18 ], phosphoric acid [ 19 ] or fungal keratinase [ 20 ] were proven to promote drug penetration through bovine hooves, this being a nail model typically used for preliminary studies [ 21 ]. Likewise, molecular screenings have been carried out in order to identify possible candidates for ungual enhancers [ 19 , 22 – 24 ]. Table 1summarizes the state of the art and collects the most frequent enhancers described in the literature as well as a brief description of their mechanism of action. The aim of this work is to identify better penetration enhancers to promote the drug nail absorption from heat-sensitive water-alcohol hydrogels based on soluble polypseudorotaxanes of Poloxamer 407 and soluble derivatives of the β -cyclodextrin. We have previously shown that these vehicles form a hydrated film coating on the surface of the nail, facilitating the hydration of the nail plate and the release and diffusion of the drug into the nail [25–27]. Based on these preliminary studies, our starting point was the selection of compounds with, in principle, greater potential as ungual penetration enhancers (lactic acid, sodium lauryl sulfate, PEG 300, cysteine, carbocysteine and potassium phosphate), along with other chemicals considered to be efficacious enhancers of transdermal and oral routes of administration such as Labrasol ® [ 28 ] and Labrafil ® [ 29 ]. The candidates have been the subject of several studies (mercury intrusion porosimetry and scanning electron microscopy) that aimed at identifying and characterizing the possible alterations that they produce on the microstructure and composition of nail components (RAMAN spectroscopy). The efficacy of these enhancers in enabling drug penetration into the nail was evaluated through diffusion studies involving bovine hoof membranes and human nail clippings. Two model drugs were selected: clobetasol propionate, a drug of choice in the treatment of nail psoriasis [ 30 ], and ciclopirox olamine, used for the treatment of onychomycosis [31]. Pharmaceutics 2018,10, 273 3 of 18 Table 1. Common nail penetration enhancers described in the bibliography. Enhancer Proposed Mechanism of Action Reference Sodium lauryl sulfate (SLS) Denaturation of proteins because of electrostatic interactions or micelle formation [32] Polyethylene Glycol 300 (PEG) Increase in the hydration and swelling of the nail [33] Carbocysteine (CB) Reduction of keratin’s disulfide bridges [34] N-acetylcysteine (AC) Reduction of keratin’s disulfide bridges [34,35] Lactic Acid (LAC) Formation of micropores [19,34] Potassium Phosphate (PK) Increases hydration of the nail and the thermodynamic activity of the drug [33] Labrasol® Enhancer of absorption for transdermal release [36,37] Labrafil® Enhancer of absorption for transdermal release [38] 2. Materials and Methods 2.1. Materials The enhancers tested were sodium lauryl sulfate (Fagron Iberica, Tarrasa, Spain), polyethylene glycol 300 (Merck Millipore, Darmstadt, Germany), Labrasol ® and Labrafil ® (GattefosséSAS, Madrid, Spain), lactic acid and sodium phosphate (VWR, BDH Prolabo, Barcelona, Spain), carbocysteine and acetylcysteine (Acorfarma, Madrid, Spain). The drugs used were Ciclopirox olamine (Fagron Iberica, Barcelona, Spain), clobetasol-17-propionate (Acorfarma, Spain). The compounds used in the elaboration of the vehicles were Poloxamer 407 (Pluronic ® F127, Sigma-Aldrich-Merck, Darmstadt Germany), partially methylated β -cyclodextrin (Crysmeb ® , Roquette Laisa, Valencia, Spain), ethanol (Merck Millipore) and purified water (Helix®Millipore). The phosphate-buffered saline was prepared, following the 9th edition of the European Pharmacopoeia, from potassium dihydrogen phosphate, sodium chloride, and sodium dihydrogen phosphate dodecahydrate, all of them of analytical quality. Sodium azide was added to the buffer to prevent microbial growth in permeation studies; this was from Panreac Quimica SA (Barcelona, Spain). Finally, methanol from Prolabo (VWR, BDH Prolabo, Barcelona, Spain) was used to extract ciclopirox olamine and clobetasol-17propionate from nails and hooves. Bovine hooves were obtained from the local slaughterhouse (Compostelana de Carnes S.L; Santiago de Compostela, Spain). The hooves were cleaned with water and kept frozen. Before the experiments, they were defrosted and hydrated in order to facilitate the process of cutting them (Ufesa Professional Slicer FS50, Ufesa, Barcelona, Spain) into small slices (0.3–0.7 mm thick), which were dried at room temperature. Nail samples were obtained from volunteers between the ages of 25 and 65. The volunteers signed an informed consent form for the use of the samples, and their use was authorized by the Ethics Committee of Galicia (Project identification code: 2018/099; date of approval: 22 February 2018). The volunteers cut their own fingernails and toenails and provided them to the researchers. The samples were meticulously cleaned and washed with water, dried at room temperature and stored in a glass container also at room temperature. The nail samples used in diffusion studies were approximately 8 mm long, whereas the samples used in the rest of the studies were between 1 and 3 mm. 2.2. Nail Incubation in the Presence of the Enhancers The nail samples were immersed for 24 h at room temperature, in aqueous solutions containing 5% of the enhancers. The one exception was carbocyisteine, which, due to its low solubility, was tested at 0.2% (Table 2). Following the incubation, the nails were cleaned with water and freeze-dried in liquid nitrogen (Telstar LyoQuest Plus, Telstar, Terrassa, Spain). Pharmaceutics 2018,10, 273 4 of 18 Table 2. Chemicals and concentrations tested as enhancers of nail penetration. SLS was tested at 5% in incubation experiments and at three levels in diffusion experiments. Enhancer Concentration (% weight/volume) Sodium lauryl sulfate (SLS) 5/1/0.5 PEG 300 (PEG) 5 Carbocysteine (CB) 0.2 N-acetylcysteine (AC) 5 Lactic Acid (LAC) 5 Potassium Phosphate (PK) 5 Labrasol® 5 Labrafil® 5 The nail samples (both treated and non-treated) were subjected to the following tests: 2.3. Scanning Electron Microscopy (SEM) The samples were placed on adhesive graphite and copper disks and were analyzed using a Zeiss Evo LS 15 SEM microscope. Images from both the dorsal and ventral surface were taken from randomly chosen locations of the surface. The analysis of the microphotographs from the surface of the samples was carried out using Image Pro Plus 6 software (Media Cybernetics Inc., Rockville, MD, USA) from 10 SEM images of the nail surface. 2.4. Mercury Intrusion Porosimetry (MIP) The samples were analyzed in a Micromeritics Autopore IV porosimeter (Norcross, GA, USA) equipped with a penetrometer with 3-mL capacity, and an analysis pressure interval of 0.004 to 172.4 MPa was used. The amount of combined nails used in each study was, approximately 0.6 g, which may amount to 50–60 specimens depending on the size. The porosimeter gives the pore size distribution data and the % of porosity of samples. The data regarding pore size distribution were modeled using PoreXpert ™ software (Environmental and Fluid Modelling Group, University of Plymouth, UK). A detailed description of the methods can be found elsewhere [17]. The permeability to water and the rate of penetration of water into the nail were estimated based on the modeled structures, as previously described [ 17 ]. To calculate the permeability, enhancer solutions were used to first determine the surface contact angle between the solutions and the nail to include this value in the PoreXpert simulation. To identify the layers in PoreXpert nail models that can act as a limiting step in molecular diffusion, each model structure was divided into 20 parallel horizontal planes. Each layer occupies one-tenth of the thickness of the entire model. Thus, the surface occupied by the pores, connections and bottlenecks in each of the 10 layers of the models was calculated from the PoreXpert data. Finally, the percentage of the surface of each layer occupied by the pores, connections and bottlenecks relative to the total layer surface was determined. This value is the critical surface value. 2.5. RAMAN Spectroscopy Three sets of spectra from each nail surface were obtained using a Raman Bruker Fourier transform Scope spectroscope. The spectra were taken from three randomly chosen locations on the ventral and dorsal surfaces of the nail. The ratio between the height of the signal, corresponding to the C–C (900 cm−1) and S–S (500 cm−1) vibrations, was calculated. 2.6. Determining the Contact Angle through Goniometry Solutions of the enhancers at the same concentrations used in the incubation and diffusion studies (Table 2) have been tested in an SEO Phoenix 300 (Surface Elector Optics, Gyeonggi-do, Korea). For this, Pharmaceutics 2018,10, 273 5 of 18 a small drop of solution has been placed on the surface of nail samples and then a series of photographs have been taken, so the evolution of the drop over 10 s can be observed. The contact angle with the nail, for each of the enhancers, has been determined by the analysis of the obtained images. The angle value has been selected based on the moment at which the drop becomes stable on the nail surface. 2.7. Permeation Studies The aqueous lacquers used in the in vitro diffusion studies were prepared according to previous studies [ 27 ]. A 1:1 water-ethanol solution was prepared containing 10% w/vof partially methylated β -cyclodextrin and 5% w/vof poloxamer 407. Next, the enhancer (Table 2) was incorporated and stirred until it was completely dissolved. Lastly, the drug, either ciclopirox olamine or clobetasol-17-propionate, was added in excess and the vehicles were continuously stirred for 24 h. Finally, the formulations were filtered through nylon membranes of 0.45 µ m (Merck Millipore) to eliminate the undissolved drug. The concentration of ciclopirox olamine in the formulations prepared is determined through UV spectroscopy (Hewlett-Packard 8452A, Hewlett-Packard Española, S.A, Madrid, Spain) and the concentration of clobetasol propionate through Ultra Performance Liquid Chromatography –mass spectrometry UPLC-LC/MS (see below). In a first step to assess the efficacy of the enhancers, a model of bovine hooves was used [ 39 , 40 ]. In a second phase, the enhancers showing higher efficacy were tested using human nail samples. Bovine hooves and human nails permeation studies were performed with vertical Franz penetration cells (Vidrafoc, Barcelona, Spain) with ~7 mL receptor solution. For this, prior to the test, hoof slices and human samples were hydrated in water for two hours to provide them with flexibility so they could be clamped between two Teflon cylindrical adapters (polytetra-fluoroethylene, Mecanizados del Noroeste, Santiago de Compostela, Spain), providing an effective diffusional area of 0.196 cm 2 . Then, adapters with samples were clamped between the donor and receptor chambers of Franz diffusion cells. The thickness of the slices was measured with a micrometer (Mitutuyo, Guipúzcoa, Spain). Samples with a similar thickness and without visible faults or fissures were selected for the studies. The average thickness was 0.4-0.7 for nails and 0.4–0.8 mm for hooves. The solutions containing the enhancers (2 mL each) were added to the donor compartment. The receiving solution for ciclopirox olamine was phosphate-buffered saline (pH 7.4; 37 ± 0.5 ◦ C), to which sodium azide (30 mg/L) had been added to prevent algae and microbiological growth. Sink conditions were maintained in all the tests as the drug concentration in the receiver was always less than 10% of the drug’s PBS solubility. In the case of clobetasol propionate, the receiving solution was PBS containing 5% HPB with sodium azide (30 mg/L) to increase drug solubility in the receptor. The diffusion studies lasted 11 days; 1-mL samples were obtained from the receiver every 24 h, and the same volume was replaced with fresh PBS. The receptor samples were filtered (0.45 µ m nylon filter) before analysis. In the case of ciclopirox olamine, samples from tests with human nails were diluted with NaOH 1 M to prevent interferences with protein residues coming from the nail extraction. Once the diffusion studies were finished, the amount of ciclopirox olamine or clobetasol propionate present inside the nail or hoof was determined. In order to do this, and after dismantling the diffusion cells, the samples were cleaned with distilled water and then dried with cellulose paper. Then, the sections of nails or hooves exposed to the lacquer were cut into small pieces, which were weighed and incorporated into 10-mL vials, to which 5 mL phosphate buffer with methanol at 5% were added. Then, the vials were incubated at a temperature of 25 ◦ C and under continuous stirring for six days to facilitate the drug extraction. The concentration of ciclopirox olamine in the samples was spectrophotometrically quantified at 308 nm (Diode-Array Spectrophotometer Hewlett Packard 8452 (Hewlett-Packard Española, S.A, Madrid, Spain). Regarding the clobetasol propionate, quantification was performed on a MS/MS tandem Waters Xevo ® TQD detector linked to an Acquity UPLC ® H-Class system (Waters ® , Czech Republic) using TargetLynxTM Application Manager. An Acquity BEH C18 column (2.1 × 50 mm, 1.7 µ m particle size, Waters ® Czech Republic) at 40 ◦ C was used, using isocratic conditions: Pharmaceutics 2018,10, 273 6 of 18 water-methanol 20:80 at 0.5 mL/min. The autosampler was kept at 10 ◦ C and the volume of injection was 5 µ L. Acquisition of mass spectrometric data was in multiple reaction monitoring mode (MRM) via positive electrospray ionization. Ion transitions of m/z 467.1 > 355.1 (Cone voltage 100 V, collision energy 10 V) a desolvation gas flow of 1100 l/h, cone gas of 80 l/h and capillary voltage of 0.55 kV were used for acquisition. The desolvation and source temperature were 450 ◦ C and 146 ◦ C, respectively. The amounts of accumulated drug diffused (M) to the receiver according to the type (t) were normalized in accordance with the diffusion area (A) (0.196 cm2). 3. Results and Discussion Figure 1shows SEM images of the dorsal and ventral surfaces of healthy nails treated with the different enhancers studied. Pharmaceutics 2018,10,x6of18 methanol20:80at0.5mL/min.Theautosampler waskeptat10°Candthevolumeofinjectionwas5 μL. Acquisitionofmassspectrometricdatawasinmultiplereactionmonitoringmode(MRM) via positiveelectrosprayionization.Iontransitionsofm/z467.1>355.1(Conevoltage 100V,collision energy10V)adesolvation gasflowof1100l/h, conegasof80l/h andcapillaryvoltageof0.55kV wereused foracquisition.Thedesolvationand sourcetemperature were450 °Cand146 °C, respectively.Theamounts ofaccumulateddrugdiffused(M) tothereceiver accordingtothetype(t) werenormalizedinaccordance withthediffusionarea(A)(0.196cm 2 ). 3.Results and Discussion Figure1showsSEM imagesofthedorsal andventralsurfaces ofhealthynailstreatedwiththe Figure1.Scanning electronmicroscopyphotomicrographsatdifferent magnificationsofthehealthynail platesincubatedwiththeenhancers(WT: nailwithouttreatment,AC:acetylcysteine, CB:carbocysteine, SLS:sodiumlauryl sulfate PK: potassiumphosphate, andPEG:polyethylene glycol300)ontheirexternal (dorsal)surface(twofirstleft columns) andontheinternal(ventral)surface(tworightcolumns). Red arrowspointtosome ofthepores. Figure 1. Scanning electron microscopy photomicrographs at different magnifications of the healthy nail plates incubated with the enhancers (WT: nail without treatment, AC: acetylcysteine, CB: carbocysteine, SLS: sodium lauryl sulfate, PK: potassium phosphate, and PEG: polyethylene glycol 300) on their external (dorsal) surface (two first left columns) and on the internal (ventral) surface (two right columns). Red arrows point to some of the pores. Pharmaceutics 2018,10, 273 7 of 18 Photomicrographs were taken at different augmentations in order to accurately determine the size of the pores and identify fractures or alterations on the surface. Healthy nails appear to be compact and present a surface with small pores and fissures. Superficial porosity were estimated based on these SEM photomicrographs (Table 3). In all cases, the appearance of the surface and the size of the surface pores were similar to each other. Table 3. Values of the ratio value between the height of the band signal corresponding to the vibration frequency of the S–S bonds and of the C–C bonds (υss/υcc) for external (dorsal) and internal (ventral) surfaces of the nail exposed to the enhancers’ solutions for a 24-h period. Enhancer Surface Pore Size υss/υcc Geometric Mean (Geometric Standard Deviation) Dorsal Ventral Untreated nail 4.20 (0.41) 1.07 1.04 Water 3.91 (0.39) 1.01 1.05 SLS 5% 4.34 (0.37) 0.87 0.94 PEG 300 5% 4.01 (0.40) 0.97 0.86 CB 0.2% 3.69 (0.37) 0.95 0.98 AC 5% 3.87 (0.37) 0.96 1.02 LAC 5% 0.37 (0.39) 1.04 1.02 PK 5% 4.06 (0.33) 0.98 1.03 Labrasol®5% -0.93 0.83 Labrafil®5% -0.90 0.83 Figures 2and 3show the Raman spectra of the surface of nails treated with the different enhancers. All samples showed the characteristic spectrum of protein structures, similar to the one obtained by Wessel et al. [ 41 ] and by ourselves in previous studies [ 26 , 27 ] for the healthy nail and after being hydrated with water. The spectra were typical of the keratins or main components of the nail plate, that is, the vibrations corresponding to C–C bonds at a wave number slightly above 900 cm −1 , to hydroxyl groups between 1600 and 1700 cm −1 , to disulfide bridges S–S around 500 cm −1 and to amide or amine groups at 1300 and 1600 cm−1. Pharmaceutics2018,10,x7of18 Photomicrographsweretakenatdifferentaugmentationsinordertoaccuratelydeterminethe sizeoftheporesandidentifyfracturesoralterationsonthesurface.Healthynailsappeartobe compactandpresentasurfacewithsmallporesandfissures. SuperficialporositywereestimatedbasedontheseSEMphotomicrographs(Table3).Inallcases, theappearanceofthesurfaceandthesizeofthesurfaceporesweresimilartoeachother. Figures2and3showtheRamanspectraofthesurfaceofnailstreatedwiththedifferent enhancers.Allsamplesshowedthecharacteristicspectrumofproteinstructures,similartotheone obtainedbyWesseletal.[41]andbyourselvesinpreviousstudies[26,27]forthehealthynailand afterbeinghydratedwithwater.Thespectraweretypicalofthekeratinsormaincomponentsofthe nailplate,thatis,thevibrationscorrespondingtoC‒Cbondsatawavenumberslightlyabove900 cm ‐1 ,tohydroxylgroupsbetween1600and1700cm ‐1 ,todisulfidebridgesS‒Saround500cm ‐1 and toamideoraminegroupsat1300and1600cm ‐1 . . Figure2.RAMANspectraoftheexternal(dorsal)surfaceofnailstreatedwiththedifferentenhancers intherangeof200‒1800cm ‐1 .AC:acetylcysteine,CB:carbocysteine,SLS:sodiumlaurylsulfatePK: potassiumphosphate,PEG:polyethyleneglycol300,LAC:lacticacid. . Figure3.RAMANspectraina2400‒2800cm ‐1 intervaloftheexternalnailsurfacetreatedwiththe differentenhancers.AC:acetylcysteine,CB:carbocysteine,SLS:sodiumlaurylsulfatePK:potassium phosphate,PEG:polyethyleneglycol300,LAC:lacticacid. Figure 2. RAMAN spectra of the external (dorsal) surface of nails treated with the different enhancers in the range of 200–1800 cm −1 . AC: acetylcysteine, CB: carbocysteine, SLS: sodium lauryl sulfate, PK: potassium phosphate, PEG: polyethylene glycol 300, LAC: lactic acid. Pharmaceutics 2018,10, 273 8 of 18 Pharmaceutics2018,10,x7of18 Photomicrographsweretakenatdifferentaugmentationsinordertoaccuratelydeterminethe sizeoftheporesandidentifyfracturesoralterationsonthesurface.Healthynailsappeartobe compactandpresentasurfacewithsmallporesandfissures. SuperficialporositywereestimatedbasedontheseSEMphotomicrographs(Table3).Inallcases, theappearanceofthesurfaceandthesizeofthesurfaceporesweresimilartoeachother. Figures2and3showtheRamanspectraofthesurfaceofnailstreatedwiththedifferent enhancers.Allsamplesshowedthecharacteristicspectrumofproteinstructures,similartotheone obtainedbyWesseletal.[41]andbyourselvesinpreviousstudies[26,27]forthehealthynailand afterbeinghydratedwithwater.Thespectraweretypicalofthekeratinsormaincomponentsofthe nailplate,thatis,thevibrationscorrespondingtoC‒Cbondsatawavenumberslightlyabove900 cm ‐1 ,tohydroxylgroupsbetween1600and1700cm ‐1 ,todisulfidebridgesS‒Saround500cm ‐1 and toamideoraminegroupsat1300and1600cm ‐1 . . Figure2.RAMANspectraoftheexternal(dorsal)surfaceofnailstreatedwiththedifferentenhancers intherangeof200‒1800cm ‐1 .AC:acetylcysteine,CB:carbocysteine,SLS:sodiumlaurylsulfatePK: potassiumphosphate,PEG:polyethyleneglycol300,LAC:lacticacid. . Figure3.RAMANspectraina2400‒2800cm ‐1 intervaloftheexternalnailsurfacetreatedwiththe differentenhancers.AC:acetylcysteine,CB:carbocysteine,SLS:sodiumlaurylsulfatePK:potassium phosphate,PEG:polyethyleneglycol300,LAC:lacticacid. Figure 3. RAMAN spectra in a 2400–2800 cm −1 interval of the external nail surface treated with the different enhancers. AC: acetylcysteine, CB: carbocysteine, SLS: sodium lauryl sulfate, PK: potassium phosphate, PEG: polyethylene glycol 300, LAC: lactic acid. Table 3shows the ratio value between the height of the band signal, corresponding to the vibration frequency of the S–S bonds and of the C–C bonds ( υss / υcc ) for the dorsal and ventral surfaces of the nails untreated and treated with different enhancers. No significant differences were observed between treatments for the ratio values (one way Analysis of the Variance, α n.s.) suggesting no significant alteration in the S–S bonds. However, the analysis of the bands corresponding to the group –SH (2600 cm −1 , Figure 3) shows that a significant occurrence of these groups happened when the nail was treated with N-acetylcysteine. The occurrence of the –SH is related to disulfide bridges. As a consequence, the acetylcysteine is the only compound affecting these bridges, whereas the formation of of -SH radicals was not observed in the rest of the enhancers. Mercury intrusion porosimetry was used to investigate potential modifications occurring in the microporous structure of the nails following the enhancers’ treatment. Mercury intrusion porosimetry tests were performed with both treated and untreated nails. To use this technique, it is essential to completely eliminate the water from the pores while keeping the microstructure of the nail intact and allowing the intrusion of the mercury. Therefore, after the 24-h treatment the nails were rapidly frozen by immersion in liquid nitrogen and then freeze-dried. Pore size distributions corresponding to the nails treated with the enhancers are shown in Figure 4and the total porosity in Table 4. Pharmaceutics2018,10,x8of18 Table3showstheratiovaluebetweentheheightofthebandsignal,correspondingtothe vibrationfrequencyoftheS‒SbondsandoftheC‒Cbonds(  ss / cc )forthedorsalandventralsurfaces ofthenailsuntreatedandtreatedwithdifferentenhancers.Nosignificantdifferenceswereobserved betweentreatmentsfortheratiovalues(onewayAnalysisoftheVariance,αn.s.)suggestingno significantalterationintheS‒Sbonds.However,theanalysisofthebandscorrespondingtothegroup –SH(2600cm ‐1 ,Figure3)showsthatasignificantoccurrenceofthesegroupshappenedwhenthenail wastreatedwithN‐acetylcysteine.Theoccurrenceofthe–SHisrelatedtodisulfidebridges.Asa consequence,theacetylcysteineistheonlycompoundaffectingthesebridges,whereastheformation ofof‐SHradicalswasnotobservedintherestoftheenhancers. Table3.Valuesoftheratiovaluebetweentheheightofthebandsignalcorrespondingtothevibration frequencyoftheS‒SbondsandoftheC‒Cbonds( ss / cc )forexternal(dorsal)andinternal(ventral) surfacesofthenailexposedtotheenhancers’solutionsfora24‐hperiod. EnhancerSurfaceporesize ss / cc  Geometricmean(geometricstandarddeviation) DorsalVentral Untreatednail4.20(0.41)1.071.04 Water3.91(0.39)1.011.05 SLS5%4.34(0.37)0.870.94 PEG3005%4.01(0.40)0.970.86 CB0.2%3.69(0.37)0.950.98 AC5%3.87(0.37)0.961.02 LAC5%0.37(0.39)1.041.02 PK5%4.06(0.33)0.981.03 Labrasol®5%‐ 0.930.83 Labrafil®5%‐0.900.83 Mercuryintrusionporosimetrywasusedtoinvestigatepotentialmodificationsoccurringinthe microporousstructureofthenailsfollowingtheenhancers’treatment.Mercuryintrusion porosimetrytestswereperformedwithbothtreatedanduntreatednails.Tousethistechnique,itis essentialtocompletelyeliminatethewaterfromtheporeswhilekeepingthemicrostructureofthe nailintactandallowingtheintrusionofthemercury.Therefore,afterthe24‐htreatmentthenails wererapidlyfrozenbyimmersioninliquidnitrogenandthenfreeze‐dried.Poresizedistributions correspondingtothenailstreatedwiththeenhancersareshowninFigure4andthetotalporosityin Table4.  Figure 4. Cumulative curves of porous volume obtained through mercury intrusion porosimetry for healthy nails treated with the different enhancers. AC: acetylcysteine, CB: carbocysteine, SLS: sodium lauryl sulfate, PK: potassium phosphate, PEG: polyethylene glycol 300, LAC: lactic acid. Pharmaceutics 2018,10, 273 9 of 18 Table 4. Porosity and correlation values for untreated and treated nails. Enhancer Porosity (%) Correlation Untreated nail 6.98 0.692 Hydrated nail 15.24 0.159 SLS 5% 28.20 0.074 PEG 300 5% 13.90 0.194 CB 0.2% 22.75 0.124 AC 5% 27.86 0.053 LAC 5% 19.14 0.052 PK 5% 16.14 0.012 Labrasol®5% 11.27 0.200 Labrafil®5% 7.54 0.816 With the exception of Labrafil, Labrasol and PEG, the porosity of untreated nails and hydrated nails was much lower than that measured for nails treated with enhancers. Likewise, clear differences were observed between the enhancers. The porosity of nails was ranked as: treated with SLS > CB > LAC~AC > PK > water. The cumulative curves for PEG and water-treated nails were very similar. Lastly, nails treated with Labrasol ® had higher porosity than those treated with Labrafil ® ; both groups exhibited lower porosities than hydrated nails. Labrafil®and untreated nails showed similar porosity profiles. Porous structures were modeled based on the cumulative curves of mercury intrusion porosimetry using PoreXpert ™ software. Figure 5shows the single cell structures resulting from this modeling, in which the pores are represented by cubic spaces connected through cylindrical bottlenecks (this being the same representation as in the mercury intrusion analysis). Pharmaceutics2018,10,x9of18 Figure4.Cumulativecurvesofporousvolumeobtainedthroughmercuryintrusionporosimetryfor healthynailstreatedwiththedifferentenhancers.AC:acetylcysteine,CB:carbocysteine,SLS:sodium laurylsulfatePK:potassiumphosphate,PEG:polyethyleneglycol300,LAC:lacticacid. Table4.Porosityandcorrelationvaluesforuntreatedandtreatednails. EnhancerPorosity(%)Correlation Untreatednail6.980.692 Hydratednail15.240.159 SLS5%28.200.074 PEG3005%13.900.194 CB0.2%22.750.124 AC5%27.860.053 LAC5%19.140.052 PK5%16.140.012 Labrasol®5%11.270.200 Labrafil®5%7.540.816 WiththeexceptionofLabrafil,LabrasolandPEG,theporosityofuntreatednailsandhydrated nailswasmuchlowerthanthatmeasuredfornailstreatedwithenhancers.Likewise,cleardifferences wereobservedbetweentheenhancers.Theporosityofnailswasrankedas:treatedwithSLS>CB> LAC~AC>PK>water. ThecumulativecurvesforPEGandwater‐treatednailswereverysimilar.Lastly,nailstreated withLabrasol®hadhigherporositythanthosetreatedwithLabrafil®;bothgroupsexhibitedlower porositiesthanhydratednails.Labrafil®anduntreatednailsshowedsimilarporosityprofiles. Porousstructuresweremodeledbasedonthecumulativecurvesofmercuryintrusion porosimetryusingPoreXpert™software.Figure5showsthesinglecellstructuresresultingfromthis modeling,inwhichtheporesarerepresentedbycubicspacesconnectedthroughcylindrical bottlenecks(thisbeingthesamerepresentationasinthemercuryintrusionanalysis).  Figure5.Modelsforungualstructuresfornailstreatedwithdifferentenhancersandforuntreated controlsobtainedthroughPoreXpert™.AC:acetylcysteine,CB:carbocysteine,SLS:sodiumlauryl sulfatePK:potassiumphosphate,PEG:polyethyleneglycol300,LAC:lacticacid. Figure 5. Models for ungual structures for nails treated with different enhancers and for untreated controls obtained through PoreXpert ™ . AC: acetylcysteine, CB: carbocysteine, SLS: sodium lauryl sulfate, PK: potassium phosphate, PEG: polyethylene glycol 300, LAC: lactic acid. In agreement with our previous findings [ 17 , 27 ] models of untreated nails showed a very compact internal area, with very few and very small pores, throats and bottlenecks that inhibit drug penetration. Pharmaceutics 2018,10, 273 16 of 18 4. Conclusions The chemicals LAC, PK, AC, CB and SLS significantly modified the structure of the nails and hooves, making them more permeable to drug diffusion. This capability identifies them as penetration enhancers for the nail. The models obtained by mercury intrusion porosimetry and PoreXpert ™ show a more porous structure in the nails treated with enhancers than for those chemicals without enhancement effects and control untreated nails. Raman spectroscopy studies suggested that, with the exception of N-acetylcysteine, the modifications of the microporosity could not be attributed to the rupture of disulfide bridges and that other mechanisms were involved in the increase in porosity. Permeation studies with bovine hooves and nails revealed that all the hydroalcoholic lacquers developed, and particularly those prepared with SLS, have superior performance as drug delivery systems compared to the reference lacquers used in current therapies. The hydroalcoholic lacquer elaborated with a sodium lauryl sulfate content of 1% allowed the rates of diffusion and penetration of the active ingredient within the nail to be significantly higher than those obtained with the reference lacquers when using either ciclopirox olamine or clobetasol propionate as the active ingredient. 5. Patents This work is part of the submitted patent WO2015185647A1. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/10/4/273/s1, Figure S1. Diffusion profiles of ciclopirox olamine through the bovine hoof, using lacquers with different penetration enhancers. Data correspond to mean +/−SD (n= 3). Author Contributions: E.C.-G. and S.A.-I. participated in the study design, performed most experiments and wrote the original draft; M.B.D.-C. participated in the data analysis and the review and editing of the manuscript; J.L.G.-A. and F.J.O.-E. participated in the conceptualization, design of experiments, data analysis, supervision, writing and editing. Funding: This research was funded by the Technology Transfer Accelerator of the University of Santiago de Compostela and by Xunta de Galicia grant numbers GRC2013/015 and GPC2017/015. E.C.-G. was supported by a predoctoral grant from Xunta de Galicia. Conflicts of Interest: The authors declare no conflict of interest. References 1. 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