Polymeric Micelle Nanocarriers for the Cutaneous Delivery of

Article pubs.acs.org/molecularpharmaceutics

Polymeric Micelle Nanocarriers for the Cutaneous Delivery of Tacrolimus: A Targeted Approach for the Treatment of Psoriasis Maria Lapteva, Karine Mondon, Michael Möller, Robert Gurny, and Yogeshvar N. Kalia* School of Pharmaceutical Sciences, University of Geneva & University of Lausanne, 30 Quai Ernest Ansermet, 1211 Geneva, Switzerland S Supporting Information *

ABSTRACT: Tacrolimus (TAC) suffers from poor cutaneous bioavailability when administered topically using conventional vehicles with the consequence that although it is indicated for the treatment of atopic dermatitis, it has poor efficacy against psoriasis. The aim of this work was to formulate TAC loaded polymeric micelles using the biodegradable and biocompatible methoxypoly(ethylene glycol)-dihexyl substituted polylactide (MPEG-dihexPLA) diblock copolymer and to investigate their potential for targeted delivery of TAC into the epidermis and upper dermis. Micelle formulations were characterized with respect to drug content, stability, and size. An optimal 0.1% micelle formulation was developed and shown to be stable over a period of 7 months at 4 °C; micelle diameters ranged from 10 to 50 nm. Delivery experiments using human skin and involving quantification by UHPLC-MS/MS demonstrated that this formulation resulted in significantly greater TAC deposition in skin than that with Protopic (0.1% w/w; TAC ointment), (1.50 ± 0.59 and 0.47 ± 0.20 μg/cm2, respectively). The cutaneous biodistribution profile of TAC in the upper 400 μm of tissue (at a resolution of 20 μm) demonstrated that the increase in cutaneous drug levels was due to improved TAC deposition in the stratum corneum, viable epidermis, and upper dermis. Given that there was no increase in the amount of TAC in deeper skin layers or any transdermal permeation, the results suggested that it would be possible to increase TAC levels selectively in the target tissue without increasing systemic absorption and the risk of side effects in vivo. Micelle distribution and molecular penetration pathways were subsequently visualized with confocal laser scanning microscopy (CLSM) using a fluorescently labeled copolymer and fluorescent dyes. The CLSM study indicated that the copolymer was unable to cross the stratum corneum and that release of the micelle “payload” was dependent on the molecular properties of the “cargo” as evidenced by the different behaviors of DiO and fluorescein. A preferential deposition of micelles into the hair follicle was also confirmed by CLSM. Overall, the results indicate that MPEG-dihexPLA micelles are highly efficient nanocarriers for the selective cutaneous delivery of tacrolimus, superior to the marketed formulation (Protopic). Furthermore, they may also have significant potential for targeted delivery to the hair follicle. KEYWORDS: immuno-mediated skin diseases, tacrolimus, skin topical delivery, polymeric micelles, colloids

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treatment of severe psoriasis.5 However, TAC has demonstrated a 10-fold greater immunosuppressive activity than ciclosporin A in vivo.5 TAC binds to the immunophilin FKBP 12 (or FK506 binding protein) inside the activated T-cell. The complex inhibits dephosphorylation of NF-AT (nuclear factor of activated T-cells) by calcineurin and thus prevents migration of NF-AT into the nucleus. This in turn inhibits the expression of IL-2 and other immune response inducing cytokines and suppresses the T-cell response.6 A TAC containing ointment (Protopic; 0.03 and 0.1% (w/ w)) is indicated for the topical treatment of moderate to severe atopic dermatitis. However, its immunosuppressant activity has

INTRODUCTION The management of atopic dermatitis or psoriasis usually requires long-term administration of anti-inflammatory drugs. Treatment involving topical application of corticosteroids, although having the advantage of being targeted, is often associated with skin atrophy due to the inhibition of collagen synthesis.1,2 In severe cases, topical dosage forms are not practical and may also lack efficacy with the result that oral administration is required. In both atopic dermatitis and psoriasis, the cause of inflammation is related to an exacerbated immune response;3 therefore, administration of immunosuppressant drugs is a valid therapeutic strategy. Tacrolimus (TAC; FK506) is a potent “second generation” macrolide immunosuppressant isolated from Streptomyces tsukubaensis.4 Its mechanism of action is similar to that of ciclosporin A, a cyclic undecapeptide immunosuppressant, which is administered orally for the © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2989

October 29, 2013 July 9, 2014 July 24, 2014 July 24, 2014 dx.doi.org/10.1021/mp400639e | Mol. Pharmaceutics 2014, 11, 2989−3001

Molecular Pharmaceutics

Article

loaded micelles and a commercial liposomal cream (Pevaryl).36 The results showed that the micelles displayed high incorporation efficiency and also possessed a much sought after selectivity for drug deposition over permeation. Moreover, in addition to selectivity, the amount of econazole deposited in the skin from the micelle formulation was superior to that using Pevaryl. Given these promising observations, it was decided to determine whether the micelles could increase the cutaneous bioavailability of TAC and, if so, was drug delivered selectively to the stratum corneum/viable epidermis/upper dermis (the target compartments); affirmative responses would have clinical significance by expanding the range of potential indications. Cutaneous bioavailability studies typically involve tape-stripping to determine the amount of drug in the stratum corneum and separation of the viable epidermis from the dermis in order to quantify the amounts of drug retained in these layers. It was decided to use a more sophisticated approach in the present study. This involved snap-freezing the skin samples in isopentane cooled by liquid nitrogen followed by cryotome slicing to produce fine lamellae with a thickness of only 20 μm. The amount of TAC in each individual slice was quantified, enabling a TAC biodistribution profile to be obtained as a function of position in the skin with far superior resolution to conventional tape-stripping/epidermal separation studies. Therefore, in summary, the objectives of the present investigation were (i) to develop an optimized, stable MPEGdihexPLA micelle TAC formulation with drug content equivalent to that of Protopic (0.1% w/w), (ii) to evaluate its potential to deliver the drug selectively to the skin, (iii) to determine the cutaneous biodistribution of TAC, that is, the amounts of drug present as a function of depth in the membrane (at a resolution of 20 μm), hence (iv) to compare the TAC biodistribution profiles following application of the micelle formulation and Protopic, and finally (v) to formulate fluorescent micelles to investigate the localization of the copolymer and the loaded compound in skin using confocal laser scanning microscopy (CLSM) and hence to gain insight into the mechanism of penetration enhancement.

encouraged its off-label use in other inflammatory skin diseases, including psoriasis.7 While systemic administration of TAC was indeed effective in the treatment of severe and recalcitrant psoriasis,8,9 topically applied Protopic failed to promote TAC deposition in hyperkeratotic psoriatic plaques.10 Subsequent studies demonstrated that delivery was only possible under occlusion11 or to particular regions of the body where the skin was thinner and more permeable, e.g., the face 12 or intertriginous areas.13−19 Given its pharmacological properties and clinical effects, it is clear that TAC should have a role in the topical treatment of inflammatory skin diseases such as psoriasis; however, its delivery must obviously be improved for its potential to be fully exploited. An optimal TAC formulation must convey the drug to its target site, (i.e., the epidermis and dermis, where reside infiltrated and skin homing T-cells, respectively) while concomitantly minimizing systemic exposure so as to decrease the risk of side effects.20,21 However, formulation of lipophilic, poorly water-soluble drugs to overcome the skin barrier and selectively target the epidermis/dermis is a considerable challenge. Excipients, such as surfactants, used to improve drug solubility and partitioning may also serve as penetration enhancers, resulting in unnecessary systemic delivery of drugs that are administered to treat local, dermatologic indications. In the case of TAC, colloidal systems including microemulsions,22 liposomes and ethosomes,23,24 liquid crystalline nanoparticles,25 and nanolipid carriers,26,27 have all been investigated. While some of these formulations indeed increased the topical bioavailability of TAC, they also increased its transdermal permeation,22,27 thus potentially increasing the risk of systemic side effects in vivo. Micelles are colloidal systems composed of amphiphiles that self-assemble above the critical micelle concentration (CMC) to form a specific core−corona structure. In the case of polymeric micelles, the amphiphilic compound is a block copolymer containing blocks with different lipophilic/hydrophilic properties. In 2006, Trimaille et al.28 developed novel polymeric micelles using methoxy-poly(ethylene glycol)-poly(hexylsubstituted lactides) (MPEG-hexPLA) diblock copolymers.29 The hydrophilic “head” consists of a methoxypoly(ethylene glycol) chain, whereas the lipophilic “tail” is composed of a hexyl substituted polylactide moiety. The presence of hexyl substituents on the polyester structure increases the lipophilicity of the polymer compared to that of standard PLA, decreasing the CMC to 8 μg/mL (1.6 × 10−6 M)28 and increasing drug loading efficiency. For example, lipophilic drugs including meso-tetra(p-hydroxyphenyl)porphine (THPP, a poorly water-soluble photosensitizer with a log P of 8.4)30 and hypericin (log P of 10.8)31 were successfully incorporated with efficiencies exceeding 90%. The copolymer has been extensively studied with respect to its biocompatibility and toxicity. Its forced degradation resulted in the formation of nontoxic 2-hydroxyoctanoic acid and lactic acid.32 Biocompatibility was demonstrated in vitro in different cell lines and in vivo using the chick embryo chorioallantoic membrane (CAM) model for both the copolymer (below the CMC) and polymeric micelles up to copolymer concentrations of 20 mg/mL.33 Dihexyl substituted micelles (MPEG-dihexPLA) have previously been investigated for systemic34 and ocular35 administration. The first investigation into their potential for dermatologic applications compared the topical delivery of econazole nitrate into porcine and human skin from drug

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EXPERIMENTAL SECTION Materials. TAC was purchased from Hangzhou Dayang Chem (Hangzhou, P.R. China). Acetone, acetonitrile (Chromasolv HPLC grade), sodium and potassium chloride, sodium and potassium phosphate, ammonium acetate, acetic acid, fluorescein (free acid), 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), and isopentane were purchased from SigmaAldrich (Buchs, Switzerland). Bovine serum albumin (BSA) was purchased from Axon Lab (Baden-Dättwil, Switzerland). Ultrapure water (Millipore Milli-Q Gard 1 Purification Pack resistivity >18 MΩcm; Zug, Switzerland) was used to prepare all solutions. All other chemicals were at least of analytical grade. Protopic ointment (0.1% (w/w), Astellas; Wallisellen, Switzerland) was purchased from a local pharmacy. In addition to TAC, it contains, white soft paraffin, liquid paraffin, propylene carbonate, white beeswax, and hard paraffin.37 Analytical Methods. Quantification of TAC by HPLC-UV. The HPLC apparatus consisted of a P680A LPG-4 pump equipped with an ASI-100 autosampler, a thermostated column compartment TCC-100, and a UV170U detector (Dionex; Voisins LeBretonneux, France). TAC was assayed using a detection wavelength of 210 nm. Isocratic separation was performed using a LiChrospher100, RP-18e, 5 μm, 125 × 4 mm column (BGB Analytik AG; Boeckten, Switzerland) which 2990

dx.doi.org/10.1021/mp400639e | Mol. Pharmaceutics 2014, 11, 2989−3001


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monomer (8.9 mmol) was polymerized in bulk at 100 °C under argon with 1.6 g of dried initiator (MPEG 2000 Da, 0.8 mmol) to obtain the required molecular weight. Once the mixture had melted, the catalyst, Sn(Oct)2 (0.35 g/mL in dry THF), was added to the initiator. The ring opening polymerization was stopped after 1.5 h by adding 5 mL of normal THF (traces of water help to stop the reaction), which was then evaporated. The resulting copolymer was precipitated into 100 mL of cold methanol (−20 °C), filtered, and dried under vacuum. The structure of the resulting polymer is shown in Scheme 1a.

was maintained at 35 °C. Chromeleon software was used for integration and data analysis. The mobile phase consisted of a mixture of acetonitrile and water (80:20 v/v). The flow rate and injection volume were 0.6 mL/min and 25 μL, respectively. A peak for TAC was obtained at 4.82 min, and the run time was 7.0 min. The limits of detection (LOD) and quantification (LOQ) were 1.31 and 3.95 μg/mL, respectively. The HPLCUV method was validated (complete details are provided in the Supporting Information, section 1). Quantification of TAC by UHPLC-MS/MS. To increase sensitivity and specificity, UHPLC with tandem mass spectrometry detection was used to quantify the drug deposited in and permeated across the skin during the in vitro transport experiments. UHPLC-MS/MS analysis was carried out using a Waters Acquity UPLC system (Baden-Dättwil, Switzerland) comprising a binary solvent pump and sample manager and a Waters XEVO TQ-MS detector (Baden-Dättwil, Switzerland). Isocratic separation was carried out using a Waters XBridge BEH C18 2.1 × 50 mm column containing 2.5 μm particles. The column was thermostated at 45 °C. The mobile phase consisted of 10 mM ammonium acetate buffer with 0.01% acetic acid and acetonitrile (15:85 v/v). The flow rate was set at 0.7 mL/min and the injection volume was 5 μL. As ion suppression was observed in the samples, presumably due to the presence of endogenous compounds released from the skin, sirolimus (SIR) was used as an internal standard. Each injected sample contained the internal standard at a concentration of 10 ng/mL. Mass spectrometric detection was performed with electrospray ionization in positive ion mode using multiple reaction monitoring (MRM). The detection settings for both TAC and SIR are presented in Table 1. The LOD and LOQ were 1.65 and 5.00 ng/mL, respectively. The UHPLC-MS/MS method was also validated (complete details are provided in the Supporting Information, section 2). Preparation of the Micelle Formulation. Synthesis of MPEG-dihexPLA and NL-MPEG-dihexPLA Copolymers. MPEG-dihexPLA Copolymer. The block copolymer methoxypoly(ethylene glycol)-)-dihexyl-substituted lactide (MPEGdihexPLA) was synthesized in-house as described previously.28,38 Briefly, 2.5 g of the corresponding dried lactide

Scheme 1. (a) Structure of the MPEG-dihexPLA Copolymer and (b) Structure of the NR-MPEG-dihexPLA Copolymer

The copolymer was characterized with respect to its molecular weight (Mn) and polydispersity index (P.I.) using gel permeation chromatography (GPC). The GPC setup consisted of a Waters 717 autosampler, Waters 515 HPLC pump, and a Waters 410 differential refractometer (Waters; Baden-Dättwil, Switzerland) using Waters Styragel HR1-4 columns. The analysis was carried out with polystyrene (PS) of different molecular weights as calibration standards (PSS; Mainz, Germany). The copolymer was also characterized by 1H NMR (Brüker 300 MHz). NR-MPEG-dihexPLA Copolymer. The Nile Red labeled MPEG-dihexPLA copolymer (NR-MPEG-dihexPLA) was synthesized by coupling a Nile Red derivative to the MPEGdihexPLA block copolymer via a mild Mitsunobu reaction.39 The structure of NR-MPEG-dihexPLA is shown in Scheme 1b. The stability of the covalent bond between the Nile Red and the MPEG-dihexPLA copolymer in the presence of skin was checked by mass spectrometry (API 150EX mass spectrometer (AB/MDS Sciex; Zug, Switzerland) prior to the in vitro skin permeation experiments. Preparation of TAC Loaded Micelles. Micelles of different TAC loading (50, 100, 150, 200, 300, and 500 mg of TAC per g of copolymer) were prepared using the solvent evaporation method.31,36 Briefly, a known quantity of drug and copolymer was dissolved in 2 mL of acetone. The mixture was added dropwise under sonication (Branson Digital Sonifier S-450D) to 4 mL of ultrapure water. Acetone was then slowly removed by using a rotary evaporator (Büchi RE 121 Rotavapor). The final copolymer concentration was adjusted with water to 5

Table 1. MS/MS Settings for Detection of TAC and SIR

nature of parent ion parent ion (m/z) daughter ion (m/z) collision energy (V) cone voltage (V) capillary voltage (kV) capillary temperature (°C) desolvation gas flow (L/h) cone gas flow (L/h) collision gas flow (L) LM resolution 1 HM resolution 1 ion energy 1 (V) LM resolution 2 HM resolution 2 ion energy 2 (V)

tacrolimus

sirolimus

ammonium adduct [M + NH4]+ 821.7 768.6 24 28 2.79 350

ammonium adduct [M + NH4]+ 931.5 864.6 24 25 2.79 350

600

600

30 0.15 2.8 15 0.7 2.8 14.78 0.7

30 0.15 2.8 15 0.7 2.8 14.78 0.7 2991



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were harvested using a Zimmer air dermatome (Münsingen, Switzerland). Hair was removed from the skin surface using clippers. Discs corresponding to the permeation area were punched out (Berg & Schmid HK 500; Urdorf, Switzerland). Skin samples were frozen at −20 °C and stored for a maximum period of 3 months. Prior to the experiment, skin samples were thawed at room temperature and placed for 15 min in 0.9% saline solution for rehydration. Human skin samples were collected immediately after surgery from the Department of Plastic, Aesthetic and Reconstructive Surgery, Geneva University Hospital (Geneva, Switzerland). The study was approved by the Central Committee for Ethics in Research (CER: 08-150 (NAC08051); Geneva University Hospital). The hypodermis and fatty tissue were removed, and discs corresponding to the permeation area were punched out (Berg & Schmid HK 500; Urdorf, Switzerland). The skin discs were subsequently horizontally sliced with a Thomas Stadie-Riggs slicer (Thomas Scientific; Swedesboro, NJ, US) to a thickness of ∼0.8 mm. The skin was stored in a biobank at −20 °C for a maximum period of 3 months. Evaluation of TAC Skin Delivery in Vitro. Skin samples (porcine or human skin) were mounted in standard twocompartment vertical (Franz) diffusion cells ((Milian SA; Meyrin, Switzerland) with an area of 2 cm2. The receptor compartment (volume = 10 mL) was filled with sonicated phosphate buffered saline (PBS, pH 7.4) containing 1% bovine serum albumin (BSA). After equilibration, 200 mg of either the optimized micelle TAC formulation or Protopic ointment (0.1% w/w) was placed in the donor compartment and left in contact with skin for the given application time; this corresponded to an application of 100 μg TAC per cm2. The receptor compartment was stirred at 250 rpm and maintained at 33 °C throughout the experiment. Upon completion of the experiment, 1 mL of receptor phase was withdrawn to quantify TAC permeation. Samples were diluted in acetonitrile to precipitate BSA. After centrifugation at 10 000 rpm for 15 min, the permeation samples were analyzed by UHPLC-MS/MS. The diffusion cells were dismantled, and each sample was carefully washed in order to ensure the removal of the residual formulation from the skin surface. Skin samples were dried with a cotton swab. To verify the effectiveness of the wash procedure, the TAC remaining on the skin was quantified by UHPLC-MS/MS and was found to be below the LOQ of the analytical method. The skin was subsequently cut into small pieces, and TAC deposited in the skin was extracted by soaking the pieces in 4 mL of acetonitrile for 4 h with continuous stirring at room temperature. The extraction procedure was validated (Supporting Information, section 3.1). The extraction samples were centrifuged at 10 000 rpm for 15 min and diluted prior to UHPLC-MS/MS analysis. Optimization of the Formulation Application Time. The micelle formulation (0.1%) and Protopic (0.1% w/w) were each applied for a series of application times (1, 4, 8, 12, and 24 h) in order to evaluate TAC delivery kinetics into and through porcine skin. Experiments were performed at each time-point with 6 replicates. Upon completion of the experiment, an aliquot (1 mL) was withdrawn from the receptor compartment to assess TAC permeation, and skin was processed (as described above) to quantify TAC deposition in the tissue. Investigation of the TAC Biodistribution Profile in Human Skin. Protopic (0.1% w/w) and micelle formulation (0.1%) were applied for 12 h (optimal application time selected using

mg/mL. After equilibration overnight, the micelle solution was centrifuged at 10 000 rpm for 15 min (Eppendorf Centrifuge 5804) to remove excess drug, and the supernatant was carefully collected. Preparation of Fluorescent Micelles. In this case, the copolymer solution consisted of a mixture of MPEG-dihexPLA and NR-MPEG-dihexPLA (70:30) in 2 mL of acetone. Then, either fluorescein (1.4 mg) or DiO (0.02 mg) was added to this mixture. The fluorescent micelles were prepared using the solvent evaporation method described above. The final copolymer concentration was adjusted to 5 mg/mL. Characterization of Micelle Formulations. Size Determination. The hydrodynamic diameter (Zav), polydispersity index (P.I.), and volume weighted and number weighted diameters (dv and dn, respectively) of the micelles were measured using dynamic light scattering (DLS) with a Zetasizer HS 3000 (Malvern Instruments Ltd.; Malvern, UK). Measurements were performed at an angle of 90° and at a temperature of 25 °C. All values were obtained after 3 runs of 10 measurements. Morphology Observation. Micelle morphology was characterized with transmission electron microscopy (TEM) (FEI Tecnai G2 Sphera, Eindhoven, Netherlands)) using the negative staining method. Briefly, 5 μL of the micelle solution was dropped onto an ionized carbon-coated copper grid (0.3 Torr, 400 V for 20 s). The grid was then placed for 1 s in a 100 μL drop of a saturated uranyl acetate aqueous solution and then in a second 100 μL drop for 30 s. Excess staining solution was removed, and the grid was dried at room temperature prior to the measurement. TEM images were processed using ImageJ software (ImageJ 1.45s). Determination of TAC Content in the Micelles. TAC loaded into the micelles was quantified by HPLC-UV. To ensure complete micelle destruction and release of incorporated drug, 1:20, 1:50, and 1:100 dilutions in acetonitrile were made for each formulation. The drug content, drug loading, and incorporation efficiency were calculated using eqs 1−3: drug content (mg drug/mL formulation) = [TAC] mass of TAC in the formulation (mg) = volume of the formulation (mL)

(1)

drug loading (mg drug/g copolymer) =

[TAC] in the formulation (mg/mL) [copolymer] in the formulation (g/mL)

(2)

incorporation efficiency (%) =

mass of TAC incorporated into micelles (mg) × 100 mass of TAC introduced (mg) (3)

Evaluation of the Stability of the Micelle Formulations. Formulations with different target drug loadings (50, 100, 200, 300, and 500 mgTAC/gcopolymer) were prepared and stored at 4 °C for 7 months, and the incorporated TAC was quantified by HPLC-UV at a series of time-points (day 1, followed by 1, 2, 3, and 7 months). Skin Preparation. Porcine ears were purchased from a local abattoir (CARRE; Rolle, Switzerland). After washing under running cold water, skin samples with a thickness of ∼0.75 mm 2992



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between Nile Red and MPEG-dihexPLA in the fluorescently labeled copolymer (NR-MPEG-dihexPLA) was shown to be stable in the presence of skin. Development and Characterization of Micelle Formulations. Choice of Optimal Formulation. Drug Content. A series of formulations (A−F) was prepared with constant copolymer content (5 mg/mL) but different target TAC loadings: 50, 100, 150, 200, 300, and 500 mg of TAC per g of copolymer. All formulations appeared as clear limpid liquids. The actual drug loadings achieved and the drug contents and incorporation efficiencies obtained for each formulation are given in Table 3. The micelles incorporated TAC with efficiencies ranging from 34.82 ± 0.05% to 93.51 ± 0.20%. This resulted in high drug content in each formulation (from 0.224 ± 0.001 mg/mL to 1.166 ± 0.003 mg/mL). The highest drug content was provided by formulation E (1.166 ± 0.003 mg/mL). Size Characterization. TAC loaded MPEG-dihexPLA micelles were characterized to determine their size using DLS (Table 3). All TAC loaded micelle formulations presented homogeneous nanometer scale sizes with hydrodynamic diameters (Zav) from 71.7 to 105.7 nm. The number weighted diameter (dn) ranged from 14.6 to 17.7 nm, and the volume weighted diameter (dv) measurements ranged from 24.0 to 25.7 nm. Stability of Micelle Formulations. TAC content in Formulations A−F was quantified over 7 months at different time points (Figure 1). Formulations D, E, and F were considered to be unstable since TAC precipitation was observed 1 month after micelle preparation. Relative to the initial value, TAC content fell by 44.1−63.5% after 1 month of storage and had decreased by 68.1−79.1% at the end of the stability study. In contrast, formulations A, B, and C proved to be more stable, retaining more than 80% of their initial drug content after 7 months. Of these stable formulations, formulation C (target 150 mg/g) had the highest TAC content of 0.539 ± 0.001 mg/mL and was selected for further optimization. Development of a 0.1% Micelle Formulation. In order to make a “head-to-head” comparison between Protopic (0.1% w/ w) and the micelle formulation, it was necessary to achieve the same TAC content, that is, 0.1% TAC per gram of formulation. It had already been demonstrated that increasing drug loading resulted in TAC precipitation out of the micelle. Therefore, in order to increase the TAC content in the formulation, the drug loading was kept constant at its target value of 150 mg (i.e., as in formulation C, henceforth called C5), but the copolymer content was increased. Micelles with 10 and 15 mg/mL of copolymer were formulated (formulations C10 and C15, respectively), and it was observed that TAC content in the formulation increased linearly with the copolymer content (y = 0.1197x + 0.0026; r2 = 0.97). The formulation with the highest copolymer content (15 mg/mL) enabled the highest drug loading (1.736 ± 0.001 mg/mL of TAC). Subsequently, an investigation into the stability of these formulations was performed (Figure 2). For the three tested formulations, more than 80% of the drug remained incorporated after storage for 7 months. Moreover, there was no significant difference between the three formulations (ANOVA single factor, p < 0.05) with respect to the percentage of the residual TAC remaining in the formulation after 7 months (this ranged from 81.2 ± 1.7 to 83.2 ± 2.6%). Thus, increase in copolymer content did not induce

the results of the previous study) to human skin using the conditions described above (n = 5). At the end of the experiment, a small area of 0.8 cm2 was punched out from the 2 cm2 skin samples. These small skin discs were snap-frozen in isopentane cooled to its freezing point (−160 °C) with liquid nitrogen and mounted in a cryotome (Microm HM 560 Cryostat, Walldorf, Germany) so as to obtain horizontal (XYplane) 20 μm slices. A total of 20 slices was taken from each sample, theoretically, going from the skin surface to a depth of 400 μm, providing tissue samples from the stratum corneum, viable epidermis, and upper dermis. Each slice was collected in an individual Eppendorf tube. TAC deposited in each slice was extracted for 30 min with 200 μL of acetonitrile under stirring at room temperature. The extraction procedure was validated (Supporting Information, section 3.2). Each extract was subsequently subjected to UHPLC-MS/MS analysis. Visualization of Micelle Penetration Pathways. Skin samples were mounted in Franz diffusion cells (area = 2 cm2). Two hundred milligrams of fluorescent micelle formulation or control formulation (saturated aqueous solutions of DiO or fluorescein) was added to the donor compartment. The dye content in the micelle formulation for fluorescein and DiO was 350.0 and 5.0 μg/mL, respectively. The concentration of fluorescein and DiO in the saturated solutions was 4.50 and 0.07 μg/mL, respectively. All cells were protected from light. The receptor compartment was stirred at 250 rpm at room temperature during 24 h to ensure sufficient delivery to enable visualization with a microscope (LSM 710, Zeiss, Germany). After completion of the experiment, the skin samples were washed and placed on a glass slide with the stratum corneum side up. The excitation wavelengths, fluorescence emission wavelengths, laser power, pinhole, and master gain for each dye are presented in Table 2. The confocal images were obtained Table 2. Settings Used for CLSM dye Nile Red DiO fluorescein

excitation

emission

laser power (%)

HeNe diode laser at 543 nm Ar laser at 488 nm Ar laser at 488 nm

607−674 nm

10.0

38.5

700

469−511 nm

10.0

38.5

1000

504−530nm

5.0

38.5

600

pinhole (μm)

master gain

with an air Achroplan 20× objective and analyzed using Zen software (Carl Zeiss, Germany). To ensure accurate comparison, controls were visualized using the same parameters as those used for samples exposed to micelles. Data Analysis. Data were expressed as the mean ± SD. Outliers determined using the Dixon test were discarded. Results were evaluated statistically using either analysis of variance (ANOVA) or Student’s t test. The Student− Newman−Keuls test was used when necessary as a posthoc procedure. The level of significance was fixed at p = 0.05.

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RESULTS Synthesis of MPEG-dihexPLA and NR-dihexPLA Copolymers. The MPEG-dihexPLA copolymer was synthesized by bulk ring opening polymerization using Sn(Oct)2 as catalyst and MPEG 2000 as initiator. The copolymer had a Mn of 6080 g/mol and a polydispersity of 1.15. The ester bond 2993



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Table 3. Micelle Formulation Characterization with Respect to Drug Content and Size drug content

formulation unloaded micelles A B C (C5) D E F C5 C10 C15 optimal 0.1% micelle formulation a

copolymer content (mg/mL)

TARGET drug loading (mgTAC/gcopol)

drug loading ± SD (mgTAC/gcopol)

drug content ± SD (mgTAC/mL formulation)

5

n.a.

n.a.

n.a.

5 5 5 5 5 5 5 10 15 7.5a

50 100 150 200 300 500 150 150 150 150

44.72 ± 0.16 93.51 ± 0.20 107.89 ± 0.15 147.34 ± 0.55 233.21 ± 0.64 174.11 ± 0.26 107.89 ± 0.15 132.25 ± 0.31 115.73 ± 0.09 132.25 ± 0.31

0.224 0.468 0.539 0.737 1.166 0.871 0.539 1.322 1.736 1.001

± ± ± ± ± ± ± ± ± ±

0.001 0.001 0.001 0.003 0.003 0.001 0.001 0.003 0.001 0.001

size incorporation efficiency ± SD (%)

Zav (nm)

P.I.

dn (nm) [%]dn

dv (nm) [%]dv

n.a.

57.5

0.580

15.2 [100.0%]

19.7 [100.0%]

105.7 92.1 84.0 77.1 71.7 74.2 84.0 51.1 82.1 52.9

0.619 0.595 0.602 0.625 0.581 0.647 0.602 0.665 0.883 0.596

17.3 15.9 14.6 17.2 17.2 17.7 14.6 17.9 15.7 17.9

25.7 24.0 24.4 25.1 24.7 24.6 24.4 22.7 20.9 19.4

89.44 93.51 71.92 73.67 77.74 34.82 71.92 88.14 77.16 88.14

± ± ± ± ± ± ± ± ± ±

0.32 0.20 0.10 0.27 0.21 0.05 0.10 0.20 0.06 0.20

[100.0%] [100.0%] [100.0%] [100.0%] [100.0%] [100.0%] [100.0%] [100.0%] [100.0%] [100.0%]

[64.7%] [98.3%] [100.0%] [99.1%] [99.3%] [99.5%] [100.0%] [100%] [98.9%] [99.6%]

The optimal 0.1% micelle formulation was prepared by the dilution of formulation C10.

before the experiment, and the formulation was subsequently diluted to adjust the drug content to 1 mg/mL (0.1%). The pH of the final formulation was 6.5, which indicated that the formulation was suitable for application to the skin. The TEM micrograph of the optimized formulation (Figure 3) demonstrates that micelles were spherical in shape with diameters ranging from 10 to 50 nm; these dimensions were confirmed by DLS (Table 3).

Figure 1. Stability of TAC loaded micelles: evolution of drug content as a function of time over 7 months at 4 °C. Formulation A: target 50 mg/g. Formulation B: target 100 mg/g. Formulation C: target 150 mg/g. Formulation D: target 200 mg/g. Formulation E: target 300 mg/g. Formulation F: target 500 mg/g. (Mean ± SD; n = 3.)

Figure 3. TEM micrograph of the optimized 0.1% micelle formulation. (Bar = 100 nm.)

Figure 2. Stability of TAC loaded micelles with different copolymer content: evolution of drug content as a function of time during 7 months at 4 °C. Formulation C5, target 150 mg/g, 5 mg/mL of copolymer; formulation C10, target 150 mg/g, 10 mg/mL of copolymer; formulation C15, target 150 mg/g, 15 mg/mL of copolymer. (Mean ± SD; n = 3.)

Evaluation of TAC Delivery in Vitro. Optimization of the Application Time. The amounts of TAC permeated across porcine skin were below the LOQ of the UHPLC-MS/MS method for application times up to 12 h. However, after application for 24 h, TAC was detected in the receiver compartment: 318 ± 70 ng/cm2 and 210 ± 164 ng/cm2 for the optimized 0.1% micelle formulation and Protopic (0.1% w/w), respectively; permeation from the two formulations was not significantly different at 24 h (t test, p < 0.05). Skin deposition of TAC using the optimized 0.1% micelle formulation after application for 4, 8, 12, and 24 h was significantly greater than

formulation instability during the 7 month study period. Moreover, there was no difference in micelle size among formulations C5, C10, and C15 (Table 3). The optimized formulation with 10 mg/mL of copolymer was selected for the skin delivery experiments since TAC content was increased sufficiently with the least increase in copolymer content. The exact drug content was assessed by HPLC-UV on the day 2994



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that from Protopic (0.1% w/w) at each time-point (t test, p < 0.05). The maximal TAC deposition was achieved after 24 h (11.51 ± 3.05 μg/cm2 and 0.75 ± 0.23 μg/cm2 for micelles and Protopic (0.1% w/w), respectively) (Figure 4). However, given

Figure 4. TAC deposition in porcine skin as a function of formulation application time; 0.1% micelle formulation (●) and Protopic (0.1% w/ w) (○). (Mean ± SD; n = 5.)

that application for 24 h of either formulation led to the detection of TAC in the receiver compartment suggesting a potential risk for undesirable systemic drug absorption, it was decided to perform experiments using a 12 h application time, which also corresponds to the recommended (i.e., twice daily) therapeutic regimen in the treatment of atopic dermatitis using Protopic.40 Comparison of TAC Delivery into Porcine and Human Skin. As with porcine skin, TAC levels in the receptor compartment were below the LOQ following application to human skin for 12 h. TAC deposition from the optimized 0.1% micelle formulation was significantly higher than that from Protopic for both porcine (4.03 ± 0.88 vs 0.47 ± 0.20 μg/cm2) and human (1.50 ± 0.59 vs 0.36 ± 0.05 μg/cm2) skins (Figure 5). In the case of Protopic, TAC deposition in porcine and

Figure 6. Cutaneous biodistribution profile of TAC in the upper layers of human skin (stratum corneum, viable epidermis, and superficial dermis to a total depth of 400 μm and at a resolution of 20 μm) following a 12 h application of the 0.1% micelle formulation (●) and Protopic (0.1% w/w) (○). TAC deposition from the micelle formulation was significantly superior to that from Protopic for delivery to the stratum corneum and viable epidermis (to a depth of 160 μm; 0.0012 < p < 0.0376). (Mean ± SD; n = 5.)

Visualization of Micelle Penetration Pathways. CLSM Investigation of DiO and Fluorescein Skin Delivery Using Fluorescent Micelles. After application of fluorescent micelles loaded with either DiO or fluorescein for 24 h, the porcine skin surface was visualized using CLSM (Figures 7 and 8). Skin penetration of NR-MPEG-dihexPLA (red) and loaded dyes (green) is presented in Figures 7a and 8a for DiO and fluorescein, respectively. Each figure consists of panels showing the signal given by the copolymer (red), the loaded dye (green), and a superimposition of both signals. Respective skin penetration of DiO and fluorescein from the control formulations (saturated aqueous solutions) is shown in Figures 7b and 8b. The use of the covalently bound NR-MPEG-dihexPLA copolymer allowed the localization of the polymeric “carrier” to be visualized, whereas the dyes mimicked the “payload,” that is, the incorporated drug. From the optical sections of skin samples exposed to micelles, it can be seen that the green signal of both dyes was increased in comparison with the control formulations. In each experiment, the NR-MPEG-dihexPLA copolymer was found to be deposited homogeneously at the skin surface. For the DiO loaded micelle formulations, a colocalization of DiO and NR-MPEG-dihexPLA copolymer was observed at the skin surface as evidenced by the superposition of the red + green signals resulting in yellow coloration. This suggested that the dye was not released from the micelles. In contrast, for the fluorescein loaded micelle formulations, although the NR-MPEG-dihexPLA copolymer remained at

Figure 5. Comparison of TAC deposition in porcine and human skin after a 12 h application of the 0.1% micelle formulation (■) and Protopic (0.1% w/w) (□). TAC deposition from the micelle formulation was significantly superior to that from Protopic for both porcine (p = 0.00059) and human (p = 0.0046) skins. (Mean ± SD; n = 6.)

human skin was similar; however, delivery from the micelle formulation was lower in human skin than in porcine skin (see Discussion). Biodistribution of TAC in Human Skin. The biodistribution profile of TAC in human skin (Figure 6) showed that TAC deposition from the optimized 0.1% micelle formulation was significantly greater than that from Protopic (0.1% w/w) in the uppermost 140 μm of skin. In the deeper skin layers, TAC levels achieved using the two formulations were similar. 2995



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accumulation of red signal (NR-MPEG-dihexPLA copolymer) inside the appendage.

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DISCUSSION Development and Characterization of Micelle Formulations. Choice of Optimal Formulation. It is well known that a major problem in the formulation of highly lipophilic drugs is their lack of aqueous solubility.41 Micelle formulations (A to F) overcame this challenge and successfully incorporated large amounts of TAC yielding high drug loadings and incorporation efficiencies (Table 3). The MPEG-dihexPLA copolymer and its micelles can be considered as excellent carriers for TAC allowing up to a ∼540-fold enhancement of its solubility in aqueous media (formulation E, 1.166 ± 0.003 mg/ mL vs 2.15 μg/mL in unbuffered water16). Although formulation F had a nominally higher target drug loading, it showed a lower actual drug loading and incorporation efficiency suggesting that the micelle core had been supersaturated with the drug during the preparation procedure, which resulted in immediate drug precipitation. Indeed, the three formulations with the highest drug loadings (formulations D, E, and F) were all unstable due to drug precipitation. Formulation C was selected as optimal in terms of drug incorporation and stability (enabling a >250-fold increase in aqueous solubility). In general, micelles usually possess diameters below 100 nm,42 and this was indeed the case for the micelles prepared in this study. The size of the micelles is important for several reasons. First, it is inversely proportional to the interface area; thus, small micelles enable an increased contact area with the skin.36 Second, it has been shown that the size of the carrier has an effect on its interaction with skin structures: smaller particles are thought to penetrate deeper into skin appendages.43 Development of a 0.1% Micelle Formulation Equivalent to Protopic (0.1%). In order to develop a 0.1% micelle formulation, the copolymer content of the formulation was increased to 10 and 15 mg/mL (formulations C10 and C15). The linear relationship between TAC loading and copolymer content suggested that increasing the latter caused the number of micelles to be increased without affecting their individual drug loading. The increased micelle population within the formulation did not influence the size suggesting that there was no micelle aggregation. The optimal formulation for head-to-head comparison with Protopic (0.1%) was prepared by dilution of formulation C10 to obtain a TAC content of 0.1%; this did not cause any drug precipitation or modification of micelle size. TEM images confirmed DLS data concerning the nanometer size of the optimized micelles. High Zav and P.I. values determined with DLS can be explained by the presence of both small (∼10 nm) and large micelles (>40 nm). In conclusion, a stable micelle formulation containing 0.1% of TAC was successfully developed (Table 3), and the micelles were found to be spherical with diameters between 10 and 50 nm (Figure 3). TAC Delivery in Vitro. As can be seen from Figure 4, TAC delivered from the micelle formulation accumulated in skin faster and in significantly greater amounts than that from Protopic. Because of the low glass transition temperature of the MPEG-dihexPLA copolymer (Tg of ∼ −42 °C), the micelle core is thought to be in a flexible rubbery state.32 Therefore, TAC diffusivity in the micelle formulation should be higher than that in the ointment leading to faster deposition onto skin. A 12 h application of the formulation was sufficient to enable significant TAC delivery, superior to Protopic (0.1% w/w),

Figure 7. Visualization of skin penetration of NR-MPEG-dihexPLA and DiO after application for 24 h of (a) DiO loaded in NR-labeled fluorescent micelles and (b) DiO water saturated solution (negative control). (Bar = 50 μm.)

Figure 8. Visualization of skin penetration of NR-MPEG-dihexPLA and fluorescein after application for 24 h of (a) fluorescein loaded in NR-labeled fluorescent micelles and (b) fluorescein water saturated solution (negative control). (Bar = 50 μm.)

the skin surface, fluorescein was released from the carrier and was able to diffuse to deeper layers in the skin as seen by the presence of more distinct red and green bands and the relative absence of the yellow coloration. Visualization of Micelle Penetration into Hair Follicles. The ability of CLSM to provide optical images from different focal planes in the sample allows the three-dimensional reconstruction of the observed fluorescent signal. Thus, the combination of XY-plane images from different skin depths (Figure 9a) enabled the three-dimensional reconstruction of the skin surface showing a hair follicle (Figure 9b and c). Optical sections (XZ-plane) of the hair follicle can also be seen in Figure 9d. The images clearly indicate a preferential 2996



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Figure 9. Three-dimensional reconstruction of a hair follicle. (a) XY-plane images of the hair follicle at progressively increasing skin depth, (b) threedimensional reconstructions of the hair follicle, side view, and (c) three-dimensional reconstructions of the hair follicle, view from the top, and (d) series of optical sections (XZ-plane) into the hair follicle. The images clearly show the accumulation of the NR-MPEG-dihexPLA micelles around the base of the hair shaft and in the follicle. (Bar = 50 μm.)

micelle formation. Others have argued that the enhancement might be due to micelle disassembly when in contact with skin and action of individual polymer chains as chemical penetration enhancers.45 The superiority of MPEG-dihexPLA micelles over the marketed liposomal formulation (Pevaryl) was attributed to the small diameters of the carriers and their increased contact area with the skin and the possibility of depot formation in the appendages.36 Furthermore, the thermodynamic activity in the micelle may also be superior and thus affects release from the carrier and partitioning into the stratum corneum. Porcine skin is considered as one of the best in vitro models for human skin,46 and yet the results indicate that similar drug deposition in porcine and human skins was found only for the commercial formulation: the micelle formulation yielded better delivery in porcine skin. Human skin samples used in the experiment originated from abdominoplasty patients; therefore, skin presented only small vellus hair follicles, whereas hair in porcine skin resembles the morphology of larger terminal hairs.47 Moreover, hair follicles in pig ears can extend up to 1.2 mm into the dermis, whereas human vellus hair follicles penetrate far less into this layer.46 It is probable that micelles penetrate the larger, deeper porcine hair follicles far more easily than the hair follicles of abdominal human skin. Similar differences between human and porcine skins were observed

without any transdermal permeation, thus limiting the risk of undesirable systemic exposure in vivo. Indeed, the optimized 0.1% micelle formulation showed a 9fold increase in delivery to porcine skin and a smaller but still impressive 4-fold improvement with human skin. The delivery efficiency of the micelle formulation (percentage of the applied dose delivered) was 4.0% and 1.5% in porcine and human skins, respectively, whereas the delivery efficiency of Protopic (0.1% w/w) remained low (0.47 and 0.36% for porcine and human skins, respectively). The poorer delivery observed with the ointment might be due to the nature of its excipients. Given that Protopic contains only highly lipophilic excipients, different sorts of paraffin, propylene carbonate, and beeswax,37 it is likely that this reduces TAC thermodynamic activity and, as a consequence, drug partitioning from the formulation into the skin. Few studies have been conducted using polymeric micelles as drug delivery systems for dermal applications; therefore, there is no real consensus concerning the mechanism by which they enhance skin delivery. Several hypotheses have been proposed to explain the possible interactions between micelles and skin. Some authors have suggested that intact micelles can permeate through intact skin,44 but this does not seem to be plausible given the high molecular weight of the polymers used for 2997



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whereas in normal tissue, skin-homing T-cells are present only in the dermis.48 It has already been mentioned that the lack of efficacy of Protopic ointment in the treatment of psoriatic lesions was attributed to the inability of the formulation to deliver TAC through the thickened stratum corneum. Similarly, it can be supposed that an inflamed epidermis may modify the skin penetration profile of TAC. However, Meingassner et al.49 demonstrated that skin inflammation increased only the transdermal permeation of TAC without affecting its skin deposition. The TAC containing MPEG-dihexPLA micelles studied here resulted in a significantly greater cutaneous bioavailability than Protopic (0.1% w/w), targeting the stratum corneum, viable epidermis, and upper dermis with equivalent TAC levels in the deeper dermis. It is envisaged that when applied to diseased skin, these innovative carriers will deliver TAC across a thickened stratum corneum to the epidermis and the dermis where it can act on infiltrated and skin-homing Tcells, respectively. Given that no transdermal permeation was observed, they have a significant potential to improve topical psoriasis treatment. Several delivery systems to increase the bioavailability of TAC have already been investigated: nanoparticulate systems include microemulsions,22 liposomes, ethosomes,23,24 liquid crystalline nanoparticles,25 and nanolipid carriers;26,27 so how do the micelles developed in this study compare? Table 4 shows the permeation and skin deposition of TAC from different nanoparticulate systems (to facilitate comparison, only studies employing porcine or human skins have been presented here22,26,27). On the basis of the experiments conducted using porcine skin, modified nanolipid carriers developed by Pople et al.27 yielded better delivery than the polymeric micelles studied here. This performance was probably due to the longer formulation application time. After 48 h of contact with the formulation, the skin is completely hydrated, and the barrier function of the SC may be somewhat compromised. Indeed, further support for this hypothesis comes from the observation that under these conditions, transdermal permeation of TAC from Protopic exceeded 1 μg/cm2. Although the microemulsions developed by Goebel et al.22 were applied to human skin for a slightly longer time than the micelles in the present study, the overall results are comparable. Skin delivery yielded by the best microemulsion was similar to that of the MPEG-dihexPLA micelles (1.42 ± 0.50 and 1.50 ± 0.59 μg/cm2, respectively). The delivery enhancement of microemulsions was not only due to their nanoparticulate nature but also to their ability to solubilize the drug and the presence of several formulation

when MPEG-dihexPLA micelles were used to deliver econazole nitrate.36 It has already been reported that for nanoparticulate systems, the follicular pathway is one of the main routes of skin penetration.43 Therefore, the different deliveries yielded by MPEG-dihexPLA micelles in porcine and human skin may indicate that the follicular pathway is a preferential penetration route for these nanocarriers. Given the known thicknesses of the different human skin layers (10−20 μm for stratum corneum, ∼70−100 μm for the epidermis, and approximately 1 mm for the dermis)46 and knowing the number of cryotomed slices removed (20), the investigation into the biodistribution profile of TAC in human skin allowed the quantification of TAC as a function of position in the individual skin layers (Figure 10).

Figure 10. TAC deposition in the upper layers of human skin (stratum corneum, viable epidermis, and superficial dermis, to a total depth of 400 μm) following a 12 h application of a 0.1% micelle formulation (■) and Protopic (0.1% w/w) (□). TAC deposition from the micelle formulation was significantly superior to that from Protopic with respect to total delivery (p = 0.0053) and deposition in the stratum corneum (p = 0.0107) and in the viable epidermis (p = 0.0103). (Data are expressed as the mean ± SD; n = 5.)

TAC deposition in the first 400 μm of skin was doubled by the application of the 0.1% micelle formulation as compared to Protopic (0.1% w/w), which is consistent with the results obtained in total skin delivery experiments. Upon closer examination, micelles yielded significantly higher delivery into the stratum corneum and the viable epidermis, increasing TAC deposition 4- and 2- fold, respectively. TAC delivery to the dermis (∼160−200 μm and below) was not significantly different from that with Protopic (0.1% w/w). Psoriatic skin is recognized to be very different from healthy tissue.48 It is characterized by a thickened stratum corneum and inflamed epidermis. Moreover, the pathohistological changes in psoriatic skin lead to the presence of T-cells in the epidermis,

Table 4. Comparison of TAC Delivery from Different Nanoparticulate Systems Across Porcine and Human Skin study

formulation

particle size (nm)

application time (h)

skin

TAC permeation after application time (μg/cm2)

TAC skin delivery (μg/ cm2)

Pople et al.27

modified nanolipid carrier

60

48

porcine

MNLC Protopic

7.5 ± 0.9 1.3 ± 0.7

16.88 ± 3.74 0.04 ± 1.78

Goebel et al.22

micro emulsions

4−11

16

human

ME

from 0.42 ± 0.27 to 0.74 ± 0.52

Protopic

N.D.

from 0.87 ± 0.35 to 1.42 ± 0.50 0.96 ± 0.17

micelles Protopic micelles Protopic

N.D. N.D. N.D. N.D.

4.03 0.47 1.50 0.36

present work

polymeric micelles

10−50

12

porcine human

2998

± ± ± ±

0.88 0.20 0.59 0.05



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after application for 12 h, micelles increased TAC delivery into the stratum corneum and viable epidermis. Given that immune cells, responsible for the inflammatory response in psoriatic lesions, are located in this latter layer and the dermis, the results suggested that the micelle formulation might be able to increase clinical efficacy without increased risk of systemic exposure. Comparison of the results using porcine and human skin suggested that the follicular pathway plays an important role in micelle-mediated delivery. This was visualized and confirmed by CLSM, which also demonstrated that the application of the MPEG-dihexPLA copolymer was safe not only because of its biocompatibility but also because of its inability to penetrate intact skin. In conclusion, this study suggests that MPEGdihexPLA micelles are innovative, safe biomaterial based nanosized carriers that enable targeted cutaneous delivery of TAC. Further studies will be performed to optimize the ease of application of the micelle formulation and to test efficacy in vivo, first in a psoriatic skin model and then in patients. In preparation for the in vivo studies, the polymeric material is currently undergoing validation (including toxicity testing) prior to its registration as an excipient in a number of therapeutic formulations. In addition, the micelles’ potential to target the follicular pathway will also be explored.

components including excipients with penetration enhancing properties.50 The increased thermodynamic activity of the drug and the presence of penetration enhancers such as 1,2alkanediols in the microemulsions may have improved skin deposition of TAC but also increased permeation. In contrast, the MPEG-dihexPLA micelles did not result in detectable TAC permeation. Therefore, MPEG-dihexPLA micelles, a “single excipient” formulation, can be considered as both efficient and safe as they provide superior TAC levels in the skin while reducing the risk of undesirable systemic exposure. Visualization of Micelle Penetration Pathways. CLSM showed that the NR-MPEG-dihexPLA copolymer remained mainly on the skin surface. As Nile Red fluoresces only when in contact with lipophilic media, two hypotheses were formulated: (i) micelles were deposited on skin in their intact form so that Nile Red fluoresced inside the micelle core as it was linked to the lipophilic moiety of the copolymer; and (ii) the micelles disassembled upon skin contact, and NR-MPEG-dihexPLA copolymer fluoresced when in contact with the stratum corneum lipids. Although the exact micelle penetration enhancement mechanism is not clear, it is unlikely that the copolymer, given its high MW (6080 g/mol), can cross an intact stratum corneum even after application for 24 h. Therefore, and in addition to its already demonstrated biocompatibility,33 this biomaterial can also be considered as safe for topical use on the grounds of its lack of penetration into the skin. The CLSM study showed that the two dyes used to visualize delivery had different behaviors. DiO, like the copolymer, remained at the skin surface, whereas fluorescein penetrated deeper into the skin (Figures 7 and 8). This difference can be explained by their different physicochemical properties. DiO possesses two C18 chains rendering it exceptionally lipophilic (log P = 21.2; estimated using SciFinder). Its absence from the viable epidermis below the stratum corneum can be attributed to its strong interaction either with the copolymer lipophilic chain or with the stratum corneum lipids. In contrast, fluorescein, owing to its more moderate lipophilicity (log P = 2.68; again estimated using SciFinder) and better water solubility (4.3 μg/mL), was able to partition into the stratum corneum and diffuse into the epidermis. The distribution of both dyes in the skin appeared to be homogeneous, evidencing no preferential penetration pathway. Visualization of hair follicles (Figure 9) showed that the NRMPEG-dihexPLA copolymer tended to accumulate inside these structures, releasing the dye (fluorescein) into the surrounding tissue. It has already been reported in the literature that nanoparticles preferentially penetrate into hair follicles.51,52 Indeed, the polymeric particles were used to increase the intrafollicular delivery of hinokitiol or minoxidil.53,54 In light of these findings, it is suggested that MPEG-dihexPLA micelles can not only increase topical delivery to skin but also preferentially target hair follicles; this is currently being tested in follow-up studies.

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ASSOCIATED CONTENT

S Supporting Information *

Additional data concerning the validation of HPLC-UV, UHPLC MS/MS, and skin extraction procedures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +41 22 379 3355. Fax: +41 22 379 3360. E-mail: Yogi. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the University of Geneva for a teaching assistantship for M.L. and for providing financial support for the purchase of the Waters Xevo TQ-MS detector. We also thank the Fondation Ernst and Lucie Schmidheiny and the Société Académique de Genève for providing equipment grants. We acknowledge Dr. Christoph Bauer and Jerome Bosset at the Bioimaging Center of the University of Geneva for their help with confocal and electron microscopies. We also express our thanks to Professor Brigitte Pittet-Cuénod and her colleagues from the Department of Plastic, Aesthetic and Reconstructive Surgery, Geneva University Hospital (Geneva, Switzerland) for providing human skin samples.

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ABBREVIATIONS BSA, bovine serum albumin; CAM, chorioallantoic membrane; CLSM, confocal laser scanning microscopy; CMC, critical micelle concentration; DLS, dynamic light scattering; EtOAc, ethyl acetate; LOD, limit of detection; LOQ, limit of quantification; MPEG-dihexPLA, methoxy-poly(ethylene glycol)-dihexyl substituted polylactide; NR-MPEG-dihexPLA, Nile-Red labeled methoxy-poly(ethylene glycol)-dihexyl substituted polylactide; PBS, phosphate buffered saline; PLA, poly(lactic acid); TEM, transmission electron microscopy;

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CONCLUSIONS A stable 0.1% TAC formulation using MPEG-dihexPLA micelles was successfully developed and characterized. Micelle diameters were on the nanometer scale (∼20 nm) and displayed a homogeneous size distribution. Experiments demonstrated that the 0.1% micelle formulation significantly increased the cutaneous bioavailability of TAC as compared to Protopic (0.1% w/w). The biodistribution study showed that 2999



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(19) Yamamoto, T.; Nishioka, K. Topical tacrolimus is effective for facial lesions of psoriasis. Acta Dermatol. Venereol. 2000, 80, 451. (20) Schon, M. P.; Boehncke, W. H. Psoriasis. N. Engl. J. Med. 2005, 352, 1899−912. (21) Nestle, F. O.; Kaplan, D. H.; Barker, J. Psoriasis. N. Engl. J. Med. 2009, 361, 496−509. (22) Goebel, A. S.; Neubert, R. H.; Wohlrab, J. Dermal targeting of tacrolimus using colloidal carrier systems. Int. J. Pharm. 2011, 404, 159−68. (23) Li, G.; Fan, C.; Li, X.; Fan, Y.; Wang, X.; Li, M.; Liu, Y. Preparation and in vitro evaluation of tacrolimus-loaded ethosomes. TheScientificWorld 2012, 2012, 874053. (24) Li, G.; Fan, Y.; Fan, C.; Li, X.; Wang, X.; Li, M.; Liu, Y. Tacrolimus-loaded ethosomes: physicochemical characterization and in vivo evaluation. Eur. J. Pharm. Biopharm. 2012, 82, 49−57. (25) Thapa, R. K.; Yoo, B. K. Evaluation of the effect of tacrolimusloaded liquid crystalline nanoparticles on psoriasis-like skin inflammation. J. Dermatol. Treat. 2014, 25, 22−25. (26) Pople, P. V.; Singh, K. K. Targeting tacrolimus to deeper layers of skin with improved safety for treatment of atopic dermatitis. Int. J. Pharm. 2010, 398, 165−178. (27) Pople, P. V.; Singh, K. K. Development and evaluation of colloidal modified nanolipid carrier: application to topical delivery of tacrolimus. Eur. J. Pharm. Biopharm. 2011, 79, 82−94. (28) Trimaille, T.; Mondon, K.; Gurny, R.; Möller, M. Novel polymeric micelles for hydrophobic drug delivery based on biodegradable poly(hexyl-substituted lactides). Int. J. Pharm. 2006, 319, 147−154. (29) Möller, M.; Trimaille, T.; Gurny, R. Polylactides Compositions and Uses Thereof. US 8466133 B2, 2013 (filed 2006). (30) Mondon, K.; Gurny, R.; Möller, M. Colloidal drug delivery systems - recent advances with polymeric micelles. Chimia 2008, 62, 832−840. (31) Mondon, K.; Zeisser-Labouebe, M.; Gurny, R.; Möller, M. MPEG-hexPLA micelles as novel carriers for hypericin, a fluorescent marker for use in cancer diagnostics. Photochem. Photobiol. 2011, 87, 399−407. (32) Trimaille, T.; Gurny, R.; Möller, M. Poly(hexyl-substituted lactides): Novel injectable hydrophobic drug delivery systems. J. Biomed. Mater. Res. A 2007, 80A, 55−65. (33) Mondon, K.; Zeisser-Labouebe, M.; Gurny, R.; Möller, M. Novel cyclosporin A formulations using MPEG-hexyl-substituted polylactide micelles: a suitability study. Eur. J. Pharm. Biopharm. 2011, 77, 56−65. (34) Mondon, K.; Zeisser-Labouebe, M.; Gurny, R.; Möller, M. MPEG-hexPLA micelles as novel carriers for hypericin, a fluorescent marker for use in cancer diagnostics. Photochem. Photobiol. 2011, 87, 399−407. (35) Di Tommaso, C.; Behar-Cohen, F.; Gurny, R.; Möller, M. Colloidal systems for the delivery of cyclosporin A to the anterior segment of the eye. Ann. Pharm. Fr. 2011, 69, 116−123. (36) Bachhav, Y. G.; Mondon, K.; Kalia, Y. N.; Gurny, R.; Möller, M. Novel micelle formulations to increase cutaneous bioavailability of azole antifungals. J. Controlled Release 2011, 153, 126−132. (37) EMA, The European Medicines Agency Protopic Product Information. http://www.ema.europa.eu/docs/en_GB/document_ library/EPAR_-_Product_Information/human/000374/ WC500046824.pdf (accessed May 27, 2013). (38) Trimaille, T.; Möller, M.; Gurny, R. Synthesis and ring-opening polymerization of new monoalkyl-substituted lactides. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 4379−4391. (39) Trubitsyn, G.; Di Tommaso, C.; Gurny, R.; Möller, M. Novel PEG-hexPLA Micellar Drug Carriers with a Covalently Labeled Nile Red Fluorescence Marker. In PolyColl; Division of Polymers and Colloids of the Swiss Chemical Society and University of Geneva: Geneva, Switzerland, 2011. (40) FDA.gov Neoral Medication Guide. http://www.accessdata.fda. gov/drugsatfda_docs/label/2009/050715s027,050716s028lbl.pdf (accessed Jul 8, 2013).

UHPLC-MS/MS, ultrahigh performance liquid chromatography with tandem mass spectrometry detection

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Polymeric Micelle Nanocarriers for the Cutaneous Delivery of

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