European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 75–84

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Research paper

Polymeric nanoparticles-embedded organogel for roxithromycin delivery to hair follicles Eliza Główka a,⇑, Hanna Wosicka-Fra˛ckowiak a, Kinga Hyla a, Justyna Stefanowska b, Katarzyna Jastrze˛bska c, Łukasz Klapiszewski d, Teofil Jesionowski d, Krzysztof Cal b a

´ , Poland Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Poznan ´ sk, Poland Department of Pharmaceutical Technology, Medical University of Gdansk, Gdan ´ , Poland NanoBioMedical Centre, Adam Mickiewicz University, Poznan d ´ , Poland Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan b c

a r t i c l e

i n f o

Article history: Received 5 April 2014 Accepted in revised form 30 June 2014 Available online 9 July 2014 Keywords: Roxithromycin Nanoparticles Poly(epsilon-caprolactone) Pluronic lecithin organogel Hair follicle targeting, Apparatus 4 dialysis adapter

a b s t r a c t Drug delivery into hair follicles with the use of nanoparticles (NPs) is gaining more importance as drug-loaded NPs may accumulate in hair follicle openings. The aim was to develop and evaluate a pluronic lecithin organogel (PLO) with roxithromycin (ROX)-loaded NPs for follicular targeting. Polymeric NPs were evaluated in terms of particle shape, size, zeta potential, suspension stability, encapsulation efficiency and in vitro drug release. Lyophilized NPs were incorporated into the PLO and rheological measurements of the nanoparticles-embedded organogels were done. The fate of the NPs in the skin was traced by incorporation of a fluorescent dye into the NPs. As a result, ROX was efficiently incorporated into polymeric NPs characterized by the appropriate size (approximately 300 nm) allowing drug delivery to hair follicles. In ex vivo human skin penetration studies, horizontal skin sections revealed fluorescence deep in the hair follicles. Although the organogel has higher affinity to the lipidic follicular area than an aqueous suspension of NPs, it did not seem to improve penetration of the NPs along the hair shaft. The results proved that it was possible to achieve preferential targeting to the pilosebaceous unit using polymeric NPs formulated either into the aqueous suspension or semisolid topical formulation. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction On the one hand, stratum corneum is the main barrier for drug absorption, but on the other hand, the epidermis is still regarded as the main pathway for drug penetration and permeation in the skin. However, many earlier studies confirmed that in addition to the transepidermal route, hair follicles may contribute significantly to topical or even transdermal drug delivery as transappendageal pathway. Initially, follicular drug penetration was underestimated due to the fact that hair follicles occupy less than 0.1% of the total skin area. Recently, it has been depicted that this is true for the inner side of the forearm which is typically used as a model skin area for drug penetration experiments. Currently, it is known that Abbreviations: NPs, nanoparticles; PLO, pluronic lecithin organogel; ROX, roxithromycin; PCL, poly(epsilon-caprolactone); LOs, lecithin organogels; SEM, scanning electron microscopy; PDI, polydisperisty index. ⇑ Corresponding author. Department of Pharmaceutical Technology, Poznan University of Medical Sciences, ul. Grunwaldzka 6, 60-780 Poznan´, Poland. Tel.: +48 61 854 66 59; fax: +48 61 854 66 66. E-mail address: [email protected] (E. Główka). http://dx.doi.org/10.1016/j.ejpb.2014.06.019 0939-6411/Ó 2014 Elsevier B.V. All rights reserved.

there are significant variations of hair follicle density, diameter of hair follicle orifices, and volume of the follicular infundibula on different body sites. In case of scalp and face, the combined areas of follicular openings may constitute as much as 10% of the total skin area in this body region. Transfollicular drug delivery or targeting to the hair follicles and sebaceous glands is not a new concept. For the last two decades many investigations have been done and many attempts are still being made to exploit the full potential of follicular pathway [1–6]. Drugs applied on the skin surface in conventional topical preparations may be transported within the skin through both the stratum corneum and the hair follicles indistinctively. Preferential drug deposition in hair follicles is dependent on a type of vehicle used in the preparation and physicochemical properties of the drug itself [2]. In order to enhance topical or transdermal delivery of active pharmaceutical ingredients, many drug delivery systems, such as liposomes, polymeric or lipid nanoparticles (NPs) and microparticles, have been extensively studied [7–9]. However, it is generally recognized that the potential for skin penetration of NPs or microparticles with the aim of targeting into the deeper skin layers is

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negligible unless the skin barrier is disrupted or mechanical stressors are introduced. There is evidence of skin penetration into viable tissues for very small NPs such as quantum dots [10,11]. Although a gray zone may exist concerning the passive skin penetration possibility of extremely small NPs with a size of small molecules across the intact skin, there is no evidence for skin penetration into viable tissue for particles of about a few dozen nanometers in size or larger. All available data suggest that the far larger NPs used e.g. in sunscreens as physical UV-filters do not penetrate the barrier of intact skin, nor reach the viable epidermis or produce systemic exposure, even though they can penetrate into the hair follicle upper regions or the superficial layers of the stratum corneum [8,12]. Generally, entrapment of drugs into particulate carriers reduces the transepidermal transport and increases the drug concentration in the hair follicles as particles have tendency to penetrate and accumulate preferentially in hair follicle orifices. In this way, hair follicles represent a reservoir where particles create a high local drug concentration. The particle depot in the follicular duct also ensures a prolonged drug release potentially enabling the reduction in the applied dose and the frequency of applications. The particles are depleted from the follicles only by slow processes such as hair shedding and sebum flow [9,13]. It is known that NPs penetrate down the follicular duct to different depths in a size- and timedependent manner [14,15]. Therefore, the use of the adequate particle size would allow the selective targeting of specific structures and cell populations in the follicles [9,15–17]. It should be mentioned that under physiological conditions not all hair follicles are open for the penetration process. In the work of Otberg et al. [18] penetration experiments proved the presence of open and closed hair follicles where only 74% of the hair follicles on the upper arm were open for the penetration of the curcumin emulsion. The phenomenon of closed follicles is explained by the presence of a plug consisting of desquamated corneocytes and dried sebum, which is characteristic for hair follicles being in the telogen phase. However, most of hair follicles exhibit hair growth and/or sebum production, implying a mass flow out of the openings, which enables topically applied substances to enter the appendages [9,18]. Follicular targeting into the pilosebaceous unit may be a promising tool in topical therapy of pathologies associated with sebaceous gland dysfunctions such as seborrhoeic eczema, acne or androgenetic alopecia [16]. Roxithromycin (ROX), a semi-synthetic macrolide antibiotic, is one of the actives follicular delivery of which would be beneficial. This derivative of erythromycin belongs to the IV class of Biopharmaceutical Classification System meaning poor water solubility and low permeability [19]. Roxithromycin has an in vitro spectrum of antimicrobial activity similar to erythromycin but greater potency and longer action. Clinically, ROX is used to treat infections of the respiratory tract, genitourinary tract, skin, soft tissues and orodental infections. The mechanism of action involves inhibition of RNA-dependent bacterial protein synthesis [20]. The antibiotic exhibits bacteriostatic action toward Propionibacterium sp. and is effective in oral therapy of acne [21]. Propionibacterium acnes, although indigenous to the pilosebaceous unit, plays an important role in etiology of acne by inducing formation of reactive oxygen species and inflammation process. However, ROX inhibits the production of extracellular inflammation-inducing agents showing anti-inflammatory potential in addition to bacteriostatic action [21,22]. Furthermore, ROX has been proved to possess anti-apoptotic and anti-oxidative action against UVB-irradiated keratinocytes [23]. Additionally, Ito et al. [24] showed that ROX prevented apoptosis of hair follicle keratinocytes in mice and humans and suggested its potential role as a hair restoration agent. They observed hair growth restoration in about half of individuals with androgenetic alopecia treated with topical 5% ROX solution. Lecithin organogels (LOs) are the most investigated organogels for topical delivery of active agents. They are thermodynamically

stable, clear, viscoelastic gels composed of lecithin, an appropriate organic solvent (e.g. cyclooctane, isopropyl palmitate or myristate), and a polar solvent (usually water). They consist of a threedimensional network of entangled reverse cylindrical phospholipid micelles which immobilize the external organic phase, thus, turning a liquid into a gel. The hydrogen bonding network is built up by molecules of polar phase and phosphate groups. Such organogels are very promising in drug delivery owing to their biocompatibility, amphiphilic and solubilizing properties, possibility of dissolution of both hydrophilic and lipophilic drugs, as well as their permeation enhancing properties. The lecithin and organic solvent can efficiently partition within the skin and provide an enhanced permeation as it has been shown for several model drugs. Additionally, synthetic polymers (pluronics) are often incorporated into LOs as cosurfactants or stabilizers making the organogelling feasible with lecithin of relatively lesser purity [25,26]. Thus, LOs containing pluronics are classified as pluronic lecithin organogels (PLOs) or poloxamer organogels. Due to amphiphilic nature, PLO does not leave greasy after feel on the skin while it is still able to mix well with sebum filling the pilosebaceous units. Organogels have been also proved to deliver actives transdermally [27]. In the present work, the objective was to develop and evaluate a semisolid formulation containing polymeric NPs suitable for follicular targeting of ROX. Poly(epsilon-caprolactone) (PCL) was used to prepare NPs. Apart from its biodegradability and biocompatibility, PCL seems to be a good candidate to make NPs due to hydrophobic properties and compatibility with a wide range of drugs enabling uniform drug distribution in the formulation matrix [28]. To date, there have been no data reporting on PCL NPs as drug delivery system for ROX targeting to hair follicles. In our study, the follicular targeting effect of the polymeric NPs was investigated in ex vivo human skin after application in the form of either a nanoparticle suspension or an organogel containing NPs. For this purpose, fluorescence microscopy was used to visualize the follicle uptake of PCL NPs loaded with a lipophilic fluorescence dye. 2. Materials and methods 2.1. Materials Roxithromycin (ROX) was purchased from Pol-Nil S.A. (Warsaw, Poland). Poly(epsilon caprolactone) (PCL) (average Mw  10,000, average Mn  14,000), poly(vinyl alcohol) (PVA) (average Mw 30,000–70,000, 87–90% hydrolyzed) and Nile red, suitable for fluorescence, were purchased from Sigma–Aldrich Sp. z o.o. (Poznan´, Poland). Methylene chloride (pure for analysis) and the components of pH 5.8 phosphate buffer (monobasic potassium phosphate and sodium hydroxide, both pure for analysis) were obtained from POCH (Avantor Performance Materials, Gliwice, Poland). Methanol and acetonitrile were HPLC isocratic grade by J.T. Baker. PluronicLecithin Organogel kit (Fagron Sp. z o.o., Cracow, Poland) was used as supplied and consisted of 2 phases: an aqueous phase (water, Poloxamer 407, propylene glycol, methyl parahydroxybenzoate, potassium chloride, propyl parahydroxybenzoate) and an organic phase (soybean lecithin, isopropyl palmitate, propylene glycol, tocopherol and methyl parahydroxybenzoate). Spectra/Por Biotech regenerated cellulose ester dialysis membranes (10 kDa molecular weight cut off) were purchased from Spectrum Laboratories, Inc. (USA). Ultrapure water was used in all studies (Simplicity UV Water Purification System, Millipore, Germany). 2.2. Preparation of nanoparticles Polymeric NPs were prepared with PCL using a simple emulsion solvent evaporation technique. The polymer and ROX were mixed

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in various ratios (formulation types: F10, F25, F50 and F100) and 500 mg of the mixture was dissolved in 10 ml of methylene chloride. The organic solution was then added to 50 ml of 0.2% (w/v) aqueous PVA solution and sonicated for 3 min with an amplitude of 50% and 20 kHz (VCX 130 Sonics and Materials, Inc., USA). The resulting o/w emulsion was immediately transferred to a rotary vacuum evaporator for the removal of the organic solvent and solidification of particles (Rotavapor R-215 with Vacuum Pump V-710 and Vacuum Controller V-850, Buchi, Switzerland). After evaporation of methylene chloride, the pressure was additionally reduced in order to evaporate a large portion of water. Finally, the volume of the concentrated suspension of NPs was adjusted with ultrapure water to 15 ml. The suspension was frozen at 20 °C and lyophilized (freeze dryer Beta 1-16, Christ, Germany). In order to prepare blank NPs, the same procedure was used with no addition of the drug. Nile red was used as a fluorescent dye for labeling of particles. The NPs loaded with both ROX and Nile red were prepared by the same procedure except that a very small amount of the dye was added and dissolved directly in the organic solution containing the polymer and ROX. After lyophilization, all the formulations were stored in a desiccator at 4 °C. In case of NPs loaded with ROX and Nile red, the powder was protected from light. 2.3. Preparation of nanoparticles-embedded organogel Pluronic-lecithin organogel was prepared according to the manufacturer’s recommendations by thoroughly mixing by hand the organic phase with the aqueous phase in ratio 1:5 (v/v). When preparing the PLO with ROX-loaded NPs, an appropriate amount of the lyophilized NPs was mixed with the aqueous phase to wet the powder and after 30 min the organic phase was added and mixed well to ensure uniform distribution of NPs in the gel. A placebo PLO gel and four PLO formulations embedded with NPs were prepared according to Table 1. The final ROX concentration in all the PLO formulations was 0.1%. The gels were stored at 4 °C in sealed containers. 2.4. Scanning electron microscopic studies Scanning electron microscopy (SEM) was used to analyze the particle morphology of the following samples: (i) fresh suspension of NPs obtained after evaporation; (ii) dry powder of NPs after lyophilization; (iii) suspension of lyophilized NPs in ultrapure water. In case of samples in suspension, 20 ll was washed twice in 1 ml of ultrapure water, centrifuged and resuspended with 50 ll of ultrapure water. Then, the homogenous suspension was air-dried on a glass slide and sputtered for 120 s with gold/palladium under an argon atmosphere (Q150T ES Sputter Coater, Quorum Technologies Ltd., United Kingdom), whereas the lyophilized powder was fixed on a microscope stage with carbon tape and sputtered as described above. Samples were then observed with a JSM-7001F field emission scanning electron microscope (JEOL Ltd., Japan) at 15 kV accelerating voltage. For each formulation type (fresh NPs before lyophilization), average particle size was calculated by determining the diameter of 60–90 individual particles per image. In addition, light microscopy was employed to detect the presence of particle aggregates or drug crystals.

2.5. Particle size, polydisperisty index and zeta potential of nanoparticles The Zetasizer Nano ZS analyzer (Malvern Instruments Ltd., United Kingdom) was used to determine particle size and zeta potential using either dynamic light scattering technique or laser Doppler micro-electrophoresis, respectively. Mean particle size (z-average), size distribution and polydisperisty index (PDI) were measured before lyophilization by appropriate dilution of the concentrated nanoparticle suspension with ultrapure water. The zeta potential was determined either in ultrapure water or in the original medium i.e. 0.67% PVA solution (as the concentration of PVA changed to 0.67% after evaporation and volume adjustment of the suspension). All the measurements were taken in duplicate. 2.6. Short term stability study To examine the physical stability of nanoparticle suspensions, short term stability studies were carried out for F50 formulation. The batch (fresh suspension of NPs obtained after evaporation, volume adjusted to 15 ml) was divided into three vials and stored at three different temperatures (4 °C, 25 °C or 40 °C) for 4 weeks. Particle size analysis was performed on the day of production, and on the 7th, 14th and 28th day of storage. Zeta potential was measured on the day of production and on 28th day of storage. The study was done in triplicate. 2.7. Determination of actual drug loading and encapsulation efficiency The actual drug loading and encapsulation efficiency of ROX in NPs was determined upon particle separation from the remaining PVA solution by centrifugation. The amount of the encapsulated ROX was determined from concentrations of free ROX in supernatants. HPLC method was developed using Hyperchrome Prontosil 120-5-C18 H column (150  4.6 mm i.d., 5 lm) and precolumn (10  4.0 mm i.d., 5 lm) with a UV detection (k = 205 nm) and isocratic elution. The injection volume was 20 ll, and the column temperature was maintained at 50 °C. ROX was eluted with acetonitrile – pH 5.8 phosphate buffer (50:50 v/v) at a flow rate of 1 ml/ min. The retention time was about 6 min. A stock ROX solution was prepared at a concentration of 200 lg/ml in ultrapure water with the addition of methanol. Standard ROX solutions in a concentration range 10–100 lg/ml were prepared by the dilution of the stock solution with ultrapure water. Samples (supernatants) after appropriate dilution in ultrapure water were directly injected into the column. The actual drug loading was calculated as follows: actual drug loading (%) = [weight of encapsulated drug/(weight of encapsulated drug + weigh of polymer)]  100%. Encapsulation efficiency was calculated using the following equation: encapsulation efficiency (%) = (weight of encapsulated drug/total weight of drug used during preparation)  100%. 2.8. In vitro drug release studies To determine the in vitro ROX release from NPs a method was developed with the use of USP Apparatus 4 dialysis adapters (Sotax AG, Switzerland). Originally, such dialysis adapters are to be used

Table 1 Formulations of pluronic-lecithin organogel (PLO) containing roxithromycin (ROX)-loaded polymeric nanoparticles (NPs) with different drug loadings. Final ROX concentration in the gel: 0.1%. PLO formulation

Placebo

PLO F10

PLO F25

PLO F50

PLO F100

Type of embedded NPs Amount of lyophilized NPs used to prepare the gel (mg)

– –

F10 300

F25 120

F50 60

F100 30

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with the Sotax flow through dissolution system in conjunction with the standard 22.6 mm flow cells. The adapter was designed by Sotax and the University of Connecticut to fulfill a growing application requirement for drug release testing from drug delivery systems such as liposomes and NPs and allows to accurately discriminate different formulations under standardized conditions [29]. A dialysis membrane is placed over the main body of the adapter and sealed with O-rings at the top and bottom. In our study, an appropriate amount of the lyophilized NPs equivalent to 5 mg of ROX was extemporaneously suspended in 1 ml of the release medium, placed inside the Sotax dialysis adapters equipped with cellulose ester dialysis membrane, and then, the adapters were sealed with O-rings. Before the experiment, the dialysis membranes were washed and soaked in ultrapure water for 24 h in order to remove glycerol from the dialysis tubing. The dialysis adapters loaded with the nanoparticle suspension were placed in 100-ml glass bottles containing 49 ml of the release medium maintained at 37 °C in the incubator under horizontal stirring at 250 rpm (KS 130 control orbital shaker, IKA-Werke GmbH & Co. KG, Germany). One milliliter aliquots were withdrawn at each time point and replaced with the fresh medium. Sink conditions were maintained throughout the experiment. Ultrapure water was used as the release medium and was filtered and deaerated by drawing a vacuum. A stock solution of ROX was freshly prepared in the release medium at a concentration of 200 lg/ml and contained 5% of methanol. Then, a series of five ROX standard solutions was prepared by the dilution in the release medium in the concentration range of 10–100 lg/ml. The absorbance of the standard solutions and samples was measured at 205 nm (Evolution 300 UV–VIS spectrophotometer, Thermo Fisher Scientific, Inc., USA). Results were expressed as cumulative release of ROX ± SD (standard deviation) of three replicates. 2.9. Rheological behavior of organogel Rheological measurements were carried out at 32 °C on a R/S Plus Rheometer (Brookfield Engineering Laboratories, Inc., USA) coupled with Lauda E 200 thermostat (Lauda, Germany). Cone and plate geometry was used (cone radius 25 mm, cone angle 0.9936°, gap 0.048 mm). Rheological properties of the gels were studied by controlled shear rate investigations to evaluate the shear stress and viscosity as a function of shear rate. The shear rate was first increased from 0 to 200 [1/s] for 120 s, then decreased from 200 to 0 [1/s] again for 120 s. Flow behavior and hysteresis loop were assessed. All measurements were taken in duplicate on the day of sample preparation. For each measurement, 0.7 ml of the sample was placed on the plate using a syringe and was allowed to equilibrate for 10 min. Placebo PLO and four formulations containing ROX-loaded NPs (PLO F10, PLO F25, PLO F50 and PLO F100) were taken under investigation. 2.10. Ex vivo human skin penetration studies Ex vivo skin penetration study was done in order to visualize the distribution of Nile red/ROX-loaded NPs in the skin. The written

consent from the local ethical committee was obtained. Human scalp skin was provided by the Department of Pathomorphology (Medical University of Gdansk, Poland) and stored at 20 °C for three months. Pre-treatment of the skin included: thawing, rinsing with cold water, drying, abscising hairs and removing fat. Then, the skin was cut into small specimens (2  2 cm) using a scalpel. The aqueous suspension of Nile red/ROX-loaded NPs, PLO containing Nile red/ROX-loaded NPs or Nile red solution in grape seed oil (reference) was applied (0.2 ml of each formulation) on the skin surface and massaged for 5 min using a massage appliance (Atom Massager, HoMedics Group, United Kingdom). After a penetration time of either 5 min or 1 h, during which the skin specimens were stored on glass plates at controlled temperature (37 °C), the surplus of the formulation was removed. Vertical or horizontal sections of the frozen skin tissue were obtained using a Cryotome E (Thermo Fisher Scientific, Inc., USA) and distribution of NPs in the skin was observed using a Nikon e50i fluorescence microscope (Nikon Corp., Japan).

3. Results and discussion 3.1. Particle size and shape The preparation of ROX-loaded NPs was based on the wellknown simple emulsion solvent evaporation method which is intended particularly for encapsulation of lipophilic active agents. NPs were prepared with 4 different theoretical drug loadings from 2% to 20% (Table 2). All the formulations were composed of biodegradable PCL polymer which is a good candidate for controlled and long-term drug delivery and appears to improve drug residence in the skin. Most studies used lipid-based carriers for topical delivery but biodegradable polymers have also been shown to have the efficacy and advantages as delivery systems in dermatology [30–32]. There are only few studies investigating the influence of particle size, surface charge or particle composition on the follicular targeting [15]. Currently, it is not well known to what extent such parameters are relevant to uptake into the hair follicles. In addition to size, physicochemical properties of the topically applied active component and its vehicle also seem to play a role [33]. During preparation process several factors impact the formation of NPs with acceptable size, polydispersity, and good entrapment efficiency. In preliminary studies, some critical factors such as volume ratio of organic solvent phase to external aqueous phase and sonication time were selected for the optimization of mean particle size and entrapment efficiency (data not shown). As a result, a monomodal distribution with a PDI below 0.2 was obtained and the size of all the particles was sub-micron. It was seen that the mean particle size decreased with increasing the drug loading (Table 2). It may be explained by different polymer concentrations in the dispersed phase depending on the formulation type. Blank NPs were ca. 380 nm in size as the polymer concentration was the highest resulting in the increased viscosity of the dispersed phase and formation of larger particles. In comparison with the blank formulation, there was a considerable decrease in the

Table 2 Characteristics of different nanoparticle formulations (mean ± SD). Formulation

Drug– polymer (mg)

Theoretical drug loading (%)

Actual drug loading (%) (n = 6)

Encapsulation efficiency (%) (n = 6)

Particle diameter (nm) (n = 3)

Polydispersity index (n = 3)

Zeta potential (mV) (n = 3)

Blank F10 F25 F50 F100

0–500 10–490 25–475 50–450 100–400

– 2 5 10 20

– 0.3 ± 0.1 1.4 ± 0.1 4.0 ± 0.5 13.4 ± 2.1

– 15.8 ± 3.5 26.6 ± 1.4 37.5 ± 5.2 61.7 ± 10.9

384 ± 7 338 ± 5 279 ± 15 277 ± 6 283 ± 1

0.155 ± 0.020 0.194 ± 0.018 0.176 ± 0.015 0.170 ± 0.018 0.171 ± 0.005

5.3 ± 3.1 3.3 ± 0.5 3.6 ± 1.1 8.5 ± 4.5 4.0 ± 0.6

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particle size with increasing the amount of ROX in the NPs as the lower the polymer concentration, the lower the viscosity and particle diameter. It should be mentioned that the particle size of around 300 nm is suitable for penetrating into the hair follicles, mainly into the region of sebaceous glands [15]. Recently, the studies concerning selective follicular targeting were performed for PLGA and silica particles of different sizes from 122 to 1000 nm. The particles penetrated down to different depths of the follicles depending on their size. Since both particle types showed the same tendency (similar penetration depths into the hair follicle for similar particle size), it can be presumed that this observation may be explained by a mechanical effect rather than by an effect specific to composition of particles. The results revealed that the particle sized circa 650 nm penetrated deepest into the porcine hair follicles [15]. Fig. 1 shows the electron micrographs of unloaded PCL NPs (left row) or NPs loaded with ROX (right row). Most of the particles were spherical and some were irregular in shape. All particles had rather smooth surface with no visible pores. The external structure looked like an orange peel at a high magnification (micrographs not shown). After reconstitution of lyophilized NPs in water, it was seen that the particles showed similar morphology but some small aggregates appeared in case of ROX-loaded NPs (Fig. 1F). Moreover, average particle size was calculated by

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determining the diameter of 60–90 individual particles per image in order to compare the results with those obtained by dynamic light scattering. However, the estimated average size from SEM micrographs was higher for each formulation type (approx. 400 nm). In addition, light microscopy was employed to get a fast indication of the presence of microparticles, aggregation of particles or drug crystals. However, large particles or crystals were not detected in fresh suspensions of NPs. 3.2. Zeta potential and short term stability The zeta potential is an important parameter as it may impact the skin penetration ability and effective targeting to hair follicles. Zeta potential varies according to formulation parameters such as the stabilizer, drug and polymer. In this work, all formulations exhibited slightly negative zeta potential values (Table 2). The negative charge of the NPs can be explained by the properties of PCL. PCL shows an anionic character and PCL NPs prepared without any drug and surfactant would have a high negative zeta potential [28,32]. However, in the emulsion solvent evaporation method addition of a stabilizing agent is necessary as it is well known that it decreases mean particle diameters and PDI values, contributing for increased stability of NPs. Nevertheless, a stabilizer influences zeta potential of the final particles. It was demonstrated that

Fig. 1. Scanning electron micrographs of blank nanoparticles (NPs) (left row) or roxithromycin-loaded NPs (F10 formulation) (right row): (A) and (B) nanoparticle suspension before lyophilization and after washing; (C) and (D) lyophilized NPs; (E) and (F) suspension after reconstitution of lyophilized NPs in water and washing. The bar represents 1 lm.

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PVA, a nonionic surfactant, shields the surface charge of PCL NPs because of its physical entrapment within the surface layer of the polymer and it would be adsorbed on the surface of PCL NPs like a coating layer. Since PCL is a hydrophobic polymer, the main chain of PVA would interact with the external surface of NPs with hydroxyl groups facing water phase rendering NPs more hydrophilic [34]. Therefore, the surface potential would be determined by the presence of adsorbed PVA which reduces mean zeta potential of the NPs to less negative values. In the study of Rosado et al. [32] it was reported that the use of other nonionic surfactants (Poloxamer 188 and Polysorbate 80) also significantly reduced the zeta potential of PCL NPs. According to the data shown in Table 2, the zeta potential values are not high enough to prevent aggregation of NPs by electrostatic repulsions as a colloidal stability is obtained when the zeta potential reaches greater absolute values. Additionally, there is no significant difference between mean zeta potential of blank and ROX-loaded NPs. As mentioned, electrostatic charge may modulate the interactions between the NPs and the skin. Since the skin itself and the hair are negatively charged on their surface, a cationic charge seems to be helpful for penetration. For instance, cationic and amphoteric liposomes are described as capable of penetrating deeply in the follicles reaching approximately 70% of the full hair follicle length [35]. On the other hand, another study conducted by Kohli and Alpar [36] showed that latex particles that were negatively charged were able to permeate the skin while neutral and positively charged particles were not. The results observed in their study were proposed to be due to permeation via the hair follicles and sweat glands [36]. Since the data are contradictory, it is still not clear how and to what extent the particle charge promotes penetration through the skin and hair follicles. The particle charge is one of the factors that determines the physical stability of emulsions and suspensions. Due to the low values of the zeta potential obtained for all the formulations, short term stability study was conducted at different temperatures by measuring the zeta potential, particle size and PDI values. As shown in Table 3, when an ultrapure water was used as dispersion medium for the measurement of the zeta potential, the zeta potential slightly increased from approximately 8 mV to 3 mV after 28 days of storage at each storage temperature. However, when a PVA solution was used as dispersion medium, the obtained values were very similar and close to zero on the day of production and after 28 days independent of storage temperature. It can be concluded that almost one month after preparation no significant change in the zeta potential values was observed indicating stability as changes should only occur in case of chemical alterations (e.g. decomposition of a stabilizer, formation of charged molecules) [37]. The stability could be explained by a steric stabilization due to the presence of adsorbed PVA layer. The stability during the study was also confirmed by negligible increase in the mean particle diameter (Fig. 2). At each temperature all the batches remained stable during the storage time with PDI values below 0.2 indicating

Table 3 Zeta potential of roxithromycin-loaded polymeric nanoparticles (F50 formulation) measured either in ultrapure water or in the original medium on the day of production and after 28 days of storage at various temperatures (mean ± SD, n = 3). Time and storage temperature

Zeta potential (mV) Ultrapure water

Original medium (PVA solution)

Day of production

8.5 ± 5.3

0.8 ± 0.2

28th day of storage at 4 °C 25 °C 40 °C

4.1 ± 1.7 2.6 ± 0.4 2.9 ± 0.5

0.3 ± 0.2 0.4 ± 0.3 0.3 ± 0.3

Fig. 2. Mean particle size of roxithromycin-loaded nanoparticles (F50 formulation) in suspension after storage at various temperatures.

no aggregation. However, the temperature of 4 °C seems to be the best for long term storage. 3.3. Actual drug loading and encapsulation efficiency Table 2 presents the actual drug loading and encapsulation efficiency values obtained for different formulations. It should be highlighted that with an increase in the drug/polymer ratio (from 0.02 to 0.25 mg of ROX per mg of the polymer), the polymer content was decreasing in the formulation. Emulsion solvent evaporation is one of the mostly used methods to prepare PCL-based NPs where a drug entrapment efficiency is found to be primarily influenced by PCL concentration [28]. In our study it was clearly seen that with increase in the drug/polymer ratio, both the drug loading and encapsulation efficiency were also increased. The encapsulation efficiency was acceptable for the F100 formulation (around 62%). However, it seemed that the polymer saturation by the drug was not achieved. In case of F100 formulation, the quantity of the polymer was still sufficient to cover most of the drug and encapsulation efficiency was the greatest. PCL has a great capacity to enhance encapsulation of lipophilic drugs through hydrophobic interactions and entrapment efficiency may reach more than 90% [32,38]. The ratio of drug-to-polymer is a key factor influencing the drug loading and encapsulation rate. However, high drug/polymer ratio generally results in lower drug entrapment efficiency [38,39]. In the present study, for the lowest drug polymer/ratio (F10 formulation), the encapsulation efficiency was only 16%. This could be explained by ROX solubility. ROX is a very slightly soluble drug and according to Biradar et al. [19] saturation solubility of ROX in water at 25 °C is 0.28 mg/ml. Thereby, during the preparation of F10 formulation, the ROX concentration calculated per volume of the external aqueous phase was under its solubility. It was possible that ROX saturation solubility in 0.2% PVA solution was even higher than in water as PVA may have a solubilizing effect for poorly water-soluble drugs [40]. This could be the reason of drug leakage from the organic internal phase to continuous aqueous phase during the preparation process resulting in poor entrapment efficiency. For other formulations (F25, F50, F100), the ROX concentrations were greater than solubility. The higher the concentration of a poorly soluble drug in the organic internal phase, the greater the drug amount insoluble in water and the better its entrapment in the polymeric matrix [41]. It is also possible that ROX could diffuse from the polymer matrix, before evaporation of methylene chloride as the drug has also an affinity for this organic solvent. However, no drug crystals were observed under both light and electron microscopy indicating that this hypothesis is less probable. Finally, no correlation was found between the drug/polymer ratio and mean particle diameter or zeta potential.

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3.4. In vitro drug release studies The release of ROX from the NPs followed a biphasic process typical of PCL with an initial fast drug release (approx. 35–55% within 1 h depending on the formulation) and the remaining ROX being released in a sustained manner over a further period up to 24 h (Fig. 3). The initial fast ROX release is probably attributed to the leakage of surface-bound and/or poorly entrapped drug which rapidly diffused from external pores into the release medium. After the burst effect a remarkable decrease in drug release rate has been observed. The slow and sustained release phase corresponds to the ROX fraction (i.e. about 40%) that was deeply embedded into the polymeric matrix, and had to follow a longer diffusion path before release. From the drug release profiles the differences in drug release rates are clearly seen for various formulations. The correlation was observed i.e. the lower the drug loading/encapsulation efficiency, the faster the drug release rate. Due to various drug loadings and encapsulation efficiencies between formulations, different amounts of lyophilized NPs were taken for the release study in order to have the same drug dose. For example, in case of F10 formulation about 10 times larger amount of the NPs was used as compared with F100 formulation. This could explain the difference in release between F10 and F100 formulations (Fig. 3) as the same dose was dispersed in different amounts of NPs resulting in different surface areas available for release. PCL is known to prolong the release of lipophilic drugs due to hydrophobic character and long degradation. Thereby, rather lower values of ROX amount released from NPs to water were expected. For instance, in the work of Rosado et al. [32] PCL NPs loaded with hydrocortisone acetate, a very poorly soluble drug, released only 7% of the drug after 24 h in pH 7.4 phosphate buffer indicating a great ability of PCL to retain highly hydrophobic drugs. A very slow drug release could be accelerated by PCL degradation but it may take several months. The in vitro drug release studies conducted in an aqueous environment cannot predict the behavior of NPs in contact with the skin. Generally, the release of lipophilic drugs from PCL particles is a slow process in water which occurs mainly upon drug diffusion and further polymer hydrolysis and particle erosion. On the contrary, a rapid release of lipophilic drugs from polyester NPs after contact with a lipophilic solvent/sebum is expected due to the partition of the drug between the lipophilic cores of the particles and the lipophilic medium as well as to the dissolution of the polymer in the lipophilic phase. In some reports, the in vitro release studies carried out in a two-phase (hydrophilic/lipophilic) system have

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been described. In such two-phase systems typically a phosphate buffer as hydrophilic phase and a lipophilic solvent are used such as hexane because of its high lipophilicity or isopropyl myristate due to its similarity with sebum and to simulate the lipophilic nature of the stratum corneum [13,42]. Although PCL NPs are biodegradable in an aqueous environment, they are rather stable in the follicular ducts, which contain mainly lipophilic material. Therefore, the drug has to be released from the polymeric NPs mainly by diffusion. Besides, as the NPs, after entering the hair follicles, are eliminated by sebum excretion, drug release from the NPs has to be much faster than 8 days, which is the sebum excretion time in humans [1]. 3.5. Rheological studies For any vehicle to be used for topical delivery applications, it is essential to study its rheological behavior. The latter is important not only for practical use but also for its efficacy in delivering the molecules onto or across the skin. Water dispersions of PCL NPs possess very low viscosity so they are rather inappropriate for topical administration. Therefore, in our study, the dispersions were

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Cumulative drug release (%)

100 90 80 70 60 50 40 F10 F25 F50 F100

30 20 10 0 0

1

2

3

4

5

6

24

Time (hours) Fig. 3. Cumulative percentage roxithromycin (ROX) release from different formulations of ROX-loaded nanoparticles in water at 37 °C (mean ± SD, n = 3).

Fig. 4. Shear stress (A) or viscosity (B) measured on the day of production as a function of shear rate for placebo pluronic lecithin organogel (PLO) or PLOs containing different formulations of nanoparticles (F10, F25, F50 or F100). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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incorporated into a convenient topical formulation having the desired semisolid consistency. In order to determine the influence of PCL NPs on rheological properties of PLO organogel, placebo gel and the organogels having different amounts of lyophilized ROXloaded NPs were evaluated. The influence of temperature was eliminated as the temperature was maintained at 32 °C during all the experiments. From the shape of viscosity profiles (Fig. 4B) it can be stated that all PLO gels showed non-Newtonian, shear-thinning (pseudoplastic) behavior. Thixotropy was also observed as the up and down curves on the rheograms (Fig. 4A) did not overlap (the up curve was always over the down curve). However, the hysteresis loop area was significantly greater for the placebo gel than for the gels with embedded NPs, indicating that the addition of lyophilized particles makes the PLO gel less prone to deformation. A correlation can be seen between the amount of added lyophilized powder and the hysteresis loop area of the gel i.e. the greater the content of the powder, the smaller the area. As seen in Table 1, the amounts of lyophilized particles used to prepare PLO gels differ between the formulations, but the amount of ROX is constant. Finally, it can be concluded that ROX itself does not change rheological properties of the organogels, but the influence of the whole lyophilized formulation and its amount was clearly seen. In search of a vehicle to deliver a drug into deeper skin layers or through the skin, varied kinds of formulation systems and strategies have been evolved over the years. LOs have been shown to provide a very promising topical drug delivery vehicle because of

the ease of preparation and scale-up, stability, biocompatibility and safety upon applications for prolonged period. LOs provide opportunities for incorporation of a wide range of substances with diverse physicochemical characters. There are several drugs such as hormones, non-steroidal anti-inflammatory drugs and antipsychotic drugs that have been incorporated within PLO with confirmed systemic absorption [27]. Permeation enhancing properties of LOs have been well recognized as being well balanced in hydrophilic and lipophilic character, and LOs can efficiently partition with the skin and, therefore, enhance the skin penetration and transport of the molecules. However, the use of PLOs for drug delivery to hair follicles has not been widely tested. For instance, LO or PLO formulation containing extract of saw palmetto along with acyl carnitine and coenzyme Q10 has been reported as an effective formulation for selective delivery into the pilosebaceous units for the topical treatment of androgenic alopecia [26]. However, incorporation of drug-loaded NPs into LOs may combine the advantageous properties of both NPs and the organogel. It was demonstrated that incorporation of coenzyme Q10-loaded NPs into a hydrogel was more efficient in comparison with the gel prepared with free coenzyme Q10 [43]. 3.6. Skin penetration study In ex vivo skin penetration study, human scalp skin sections were evaluated for distribution of Nile red/ROX-loaded NPs applied either in the form of water suspension or in the PLO gel. Nile red

Fig. 5. Fluorescence images of Nile red disposition within the hair follicles after 5-min penetration time (left row; horizontal scalp skin section, depth: approx. 300 lm, exposure time: 600 ms) or 1-h penetration time (right row; vertical scalp skin sections, exposure time: 1.5 s): (A and B) aqueous suspension of Nile red/roxithromycin (ROX)loaded nanoparticles (NPs); (C and D) pluronic lecithin organogel containing Nile red/ROX-loaded NPs; (E and F) Nile red oily solution. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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solution in grape seed oil served as a reference. Nile red was chosen because of its emission in the red region of the light spectrum, where skin has a low auto-fluorescence [13]. Horizontal sections prepared after 5-min penetration time (Fig. 5A, C and E) revealed fluorescence specifically in the follicular areas at the depth of approx. 300 lm. The fluorescence intensity appeared definitely greater for Nile red incorporated into particles both in the form of suspension and PLO gel as compared with the oily solution of Nile red at the same depth. After 1-h penetration time, vertical skin sections were done to evaluate the path of NPs penetration in the skin. Again the images revealed fluorescence specifically in the areas of the follicles, however, NPs applied in the form of suspension seemed to have made the longest distance along the hair shaft. This proved the ability of NPs to reach the hair bulb of the terminal hair follicles (Fig. 5B). For the PLO formulation strong fluorescence can be noticed at the skin surface around the hair shafts. It can be supposed that less deep penetration of NPs from the PLO may result from significantly higher viscosity of the gel in which particles are trapped. Although the organogel should have higher affinity to the lipidic follicular area, its consistency seems to impede NPs penetration along the hair shaft. The water suspension of NPs is very fluid and in our study this was advantageous for follicular targeting. Moreover, its aqueous character did not prevent follicular penetration as could be supposed. On the contrary, in the study of Suwannateep et al. [33] the lipophilicity of the vehicle promoted follicular targeting. The curcumin-loaded NPs made of cellulose derivatives were formulated into either a water suspension, or oil in water (o/w) lotion or water in oil (w/o) lotion. From those formulations, the w/o lotion enhanced most the follicular penetration of the particles into porcine skin [33]. However, Korkmaz et al. [43] evaluated the dermal delivery of coenzyme Q10 using either a solution, gel or Q10-loaded lipid nanoparticles in the form of suspension or gel. They concluded that the lipid carriers both in the suspension or gel increased significantly Q10 skin targeting [43]. Finally, it is also important to emphasize the role of the massage in skin penetration studies. It has been reported that the massage is used to improve follicular penetration as some authors claim that it can mimic in vitro the hair movement that occurs under in vivo conditions [4,44–45]. Thus, the massage was also included in our application protocol. Most probably, the same result could not be obtained without the massage. Based on the results received in previous studies, it is now known that the massage has a vital penetration enhancing effect. Lademann et al. [44] studied the follicular penetration of a dye in particle (diameter 320 nm) and non-particle form, with and without the massage. Penetration of particle-containing formulations was enhanced by mechanical massage, reaching significantly deeper penetration depths than without the massage. Toll et al. [45] also proved the positive effect of the massage while studying microspheres penetration in human terminal hair follicles. Overall, it is extremely difficult to compare the results of skin/ follicular penetration experiments conducted by different research groups, as instead of human skin, animal skin (porcine, rat, murine) is often used and the application way differs significantly between the studies. The follicular penetration has been extensively studied for over two decades, however, there is still much to be done to draw some general conclusions.

4. Conclusions Drug delivering into the hair follicles and sebaceous glands is gaining more importance, especially in the treatment of acne or hair loss. In the treatment of androgenetic alopecia, topical drug administration is the first-line therapy. Minoxidil is the only drug

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for local application accepted by the FDA as effectively counteracting androgenic hair loss in men and women. Due to the lack of topical formulations of ROX the opportunity to apply the drug externally would be highly desired. In the present study, sitespecific delivery of ROX into the human terminal hair follicles has been achieved by employing two approaches: (i) encapsulation of ROX into polymeric NPs with adequate size allowing preferential follicular targeting, and (ii) the use of the PLO as the vehicle for ROX-loaded NPs in order to increase their concentration at the pilosebaceous units as high lipophilicity of the vehicle promotes follicular deposition. In conclusion, the encapsulation of ROX into biodegradable, biocompatible and inexpensive polymeric NPs was successfully performed. The results obtained in ex vivo human scalp skin penetration studies proved that it is possible to achieve preferential targeting to the pilosebaceous unit by using polymeric NPs. The particles containing ROX and Nile red showed significantly improved follicular penetration behavior in water suspensions as well as in the organogel in comparison to the oily solution. However, as not expected, the PLO formulation did not prove to promote follicular penetration more efficiently than the suspension of NPs. The particles in the water dispersion form appeared to reach the hair bulb with the dermal papilla so it may suggest that the formulation may be effective in the treatment of androgenic hair loss. This disease is caused mainly by apoptosis of the sensitive papilla cells, thus, ROX with its anti-apoptotic properties seems to be the proper candidate to inhibit the process.

Declaration of interest The authors report no declaration of interest. Acknowledgements The authors thank Prof. Janina Lulek (Poznan University of Medical Sciences, Poland) for useful comments. The authors wish to thank Dr Bartłomiej Milanowski (Poznan University of Medical Sciences, Poland) and the SOTAX AG (Switzerland) for providing A4D dialysis adapter kit for drug release testing. This work was supported by the grants for young scientists at the Faculty of Pharmacy, Poznan University of Medical Sciences, Poland (Grant Numbers: 502-14-03314429-09664 and 502-14-03314429-09229). References [1] A. Rolland, N. Wagner, A. Chatelus, B. Shroot, H. Schaefer, Site-specific drug delivery to pilosebaceous structures using polymeric microspheres, Pharm. Res. 10 (1993) 1738–1744. [2] A.C. Lauer, L.M. Lieb, C. Ramachandran, G.L. Flynn, N.D. Weiner, Transfollicular drug delivery, Pharm. Res. 12 (1995) 179–186. [3] V.M. Meidan, M.C. Bonner, B.B. Michniak, Transfollicular drug delivery – is it a reality?, Int J. Pharm. 306 (2005) 1–14. [4] F. Knorr, J. Lademann, A. Patzelt, W. Sterry, U. Blume-Peytavi, A. Vogt, Follicular transport route – research progress and future perspectives, Eur. J. Pharm. Biopharm. 71 (2009) 173–180. [5] H. Wosicka, K. Cal, Targeting to the hair follicles: current status and potential, J. Dermatol. Sci. 57 (2010) 83–89. [6] H. Wosicka, J. Stefanowska, K. Cal, The potential of drug targeting to hair follicles, Treat Strategies Dermatol. 1 (2011) 62–66. [7] T.W. Prow, J.E. Grice, L.L. Lin, R. Faye, M. Butler, W. Becker, E.M.T. Wurm, C. Yoong, T.A. Robertson, H.P. Soyer, M.S. Roberts, Nanoparticles and microparticles for skin drug delivery, Adv. Drug Deliv. Rev. 63 (2011) 470–491. [8] D. Papakostas, F. Rancan, W. Sterry, U. Blume-Peytavi, A. Vogt, Nanoparticles in dermatology, Arch. Dermatol. Res. 303 (2011) 533–550. [9] J. Lademann, H. Richter, S. Schanzer, F. Knorr, M. Meinke, W. Sterry, A. Patzelt, Penetration and storage of particles in human skin: perspectives and safety aspects, Eur. J. Pharm. Biopharm. 77 (2011) 465–468. [10] J.P. Ryman-Rasmussen, J.E. Riviere, N.A. Monteiro-Riviere, Penetration of intact skin by quantum dots with diverse physicochemical properties, Toxicol. Sci. 91 (2006) 159–165.

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