Tacrolimus-loaded lecithin-based nanostructured lipid carrier and nanoemulsion with propylene glycol monocaprylate as a liquid lipid: Formulation characterization and assessment of dermal delivery compared to referent ointment

International Journal of Pharmaceutics 569 (2019) 118624

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Tacrolimus-loaded lecithin-based nanostructured lipid carrier and nanoemulsion with propylene glycol monocaprylate as a liquid lipid: Formulation characterization and assessment of dermal delivery compared to referent ointment

T

Vedrana Savića, Tanja Ilića, Ines Nikolića, Bojan Markovićb, Bojan Čalijaa, Nebojša Cekićc,d, ⁎ Snežana Savića, a

Department of Pharmaceutical Technology and Cosmetology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Faculty of Technology, University of Niš, Leskovac, Serbia d DCP Hemigal, Leskovac, Serbia b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Lecithin-based nanostructured lipid carriers Nanoemulsion Tacrolimus Precirol® ATO 5 Capryol™ 90 Dermal drug delivery

Nanostructured lipid carriers (NLC) and nanoemulsions (NE) are colloid carriers which could improve dermal delivery of tacrolimus. The aims of this study were to evaluate effects of different formulation and process parameters on physicochemical characteristics and stability of lecithin-based NLC with glyceryl palmitostearate as solid and propylene glycol monocaprylate as liquid lipid and to compare the influence of different inner structure of tacrolimus-loaded NLC and corresponding NE on physicochemical characteristics, stability, entrapment efficiency, in vitro drug release and overall skin performance. Solid/liquid lipid ratio, total amount of lipids, homogenization pressure and cooling after the preparation were identified as critical variables in NLC development. Moreover, tacrolimus-loaded NLC emerged as more stabile carrier than NE. Differential stripping performed on porcine ear skin revealed significantly higher tacrolimus amount in stratum corneum from nanocarriers compared to referent ointment (Protopic®). Similarly the highest amount of tacrolimus in hair follicles was obtained using NLC (268.54 ± 92.38 ng/cm2), followed by NE (128.17 ± 48.87 ng/cm2) and Protopic® (77.61 ± 43.25 ng/cm2). Contrary, the highest permeation rate through full-thickness porcine ear skin was observed for Protopic®, implying that the selection of experimental setup is critical for reliable skin performance assessment. Overall, developed NLC could be suggested as promising carrier in a form of lotion for tacrolimus dermal delivery.

1. Introduction Nanostructured lipid carriers (NLC) are the second generation of solid lipid nanoparticles (SLN), developed in order to overcome the problems related to SLN. Namely, the lipid phase of SLN consists only from solid lipid(s), while the lipid phase of NLC contains a mixture of solid and liquid lipid(s) in different ratios, which is solid at body and room temperature. The disadvantages of SLN, such as low drug loading capacity and the expulsion of active substance during storage due to the transformation from higher energy modifications (α and β') into the lower energy β modification with reduced number of imperfections, are resolved by incorporation of liquid lipid into the solid matrix, which

leads to the increased number of imperfections, allowing the incorporation of higher amount of active substance and reducing its expulsion (Beloqui et al., 2016; Montenegro et al., 2016; Müller et al., 2002). Moreover, when formulated with non-irritant, non-toxic and biodegradable excipients, NLC were suitable for use even on inflamed or damaged skin and they were superior in dermal delivery of drugs compared to conventional dosage forms (e.g. (Mitri et al., 2011; Shinde et al., 2019; Zhao et al., 2016). After the topical application, due to the small particle size, NLC adhere to the lipid film of stratum corneum, allowing increased or modulated skin penetration of drugs (Montenegro et al., 2016). Further, Patzelt et al. (2017) highlighted that nanoparticles can be very useful in dermal drug delivery if they are able to

⁎ Corresponding author at: Department of Pharmaceutical Technology and Cosmetology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia.\ E-mail address: [email protected] (S. Savić).

https://doi.org/10.1016/j.ijpharm.2019.118624 Received 10 May 2019; Received in revised form 8 August 2019; Accepted 12 August 2019 Available online 13 August 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.

International Journal of Pharmaceutics 569 (2019) 118624

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evaluate the effects of different inner structure on the physicochemical properties, stability, tacrolimus entrapment efficiency, in vitro release and dermal drug delivery. In this regard, the amount of tacrolimus penetrated in the stratum corneum and hair follicles of porcine ear skin, was assessed by differential stripping. In addition, in vitro permeation studies through full-thickness porcine ear skin were performed and the amount of tacrolimus deposited into the skin at the end of experiment was determined. The developed nanocarriers (NLC and NE) were compared to the referent ointment (Protopic®) in order to assess their potential therapeutic benefits.

release drug at specific times and locations within the hair follicle where the released drug can then penetrate in the follicle-surrounding tissue. Tacrolimus is a potent immunosuppressive macrolide with high molecular weight (804.02 g/mol) and poor water solubility (4–12 µg/ mL, Patel et al., 2012). Due to these properties, it is a great challenge to make a suitable formulation for topical application. Although there are different topical ointments with tacrolimus which are registered today, they are all generic versions of Protopic® ointment (0.03% and 0.1%), the first approved product for topical application of tacrolimus. In Protopic®, tacrolimus is dissolved in the droplets of propylene carbonate, which are uniformly dispersed in paraffin- and beeswax-based vehicle. Although the application of ointments in general increases skin hydration and provide greater drug penetration, the patient compliance can be reduced due to the greasy texture, difficulty of applying and spreading onto the skin (especially in hairy areas) and difficulty to wash them off (Tan et al., 2012). There are still no registered creams or liquid preparations for cutaneous application of tacrolimus. As a result, different approaches have been employed aiming the better skin penetration and localization of tacrolimus effects, such as formulation of polymeric nanocarriers (Gabriel et al., 2016), natural oil based nanoemulsions (Sahu et al., 2018), lecithin based microemulsions (Savic et al., 2017), etc. To the best of our knowledge, lecithin-based NLC for dermal delivery of tacrolimus have not yet been formulated or examined. Aiming the development of stabile NLC which could manifest satisfying dermal delivery of tacrolimus, one has to balance between practical solubility of tacrolimus in a lipid mixture, stability of formulations in general and their interaction with skin. Incorporation of high amount of liquid lipid with high tacrolimus solubilization capacity would be beneficial to minimize the risk of drug expulsion. However, it could lead to the reduction of skin occlusion followed by altered topical bioavailability of drug (Teeranachaideekul et al., 2008). Although there are a lot of literature data about SLN and NLC containing glyceryl palmitostearate (Precirol® ATO 5) as a solid lipid, just few of them evaluated the stability of NLC containing combination of Precirol® ATO 5 with propylene glycol monocaprylate (Capryol™ 90), as a liquid lipid (Date et al., 2011). Capryol™ 90 is an especially interesting excipient due to the ability to act as liquid lipid and as nonionic water-insoluble cosurfactant. No literature data can be found for lecithin-stabilized Precirol® ATO 5-Capryol™ 90 NLC. The aim of our study was firstly to elaborate which of the evaluated factors (solid/liquid lipid ratio, amount of lipid phase, amount of surfactants, number of homogenization cycles, pressure during homogenization, cooling after the preparation) are the critical ones for the development of lecithin-based NLC with biocompatible lipids (Precirol® ATO 5 and Capryol™ 90). Secondly, promising NLC formulations were characterized in term of particle size, polydispersity index, zeta potential, pH, conductivity and viscosity, and their stability was assessed during six months of storage at room temperature. The best NLC candidate was compared to the corresponding nanoemulsion (NE), consisting of liquid lipid dispersed in an aqueous phase, in order to

2. Materials and methods 2.1. Materials Tacrolimus (98% pure) was purchased from Ontario Chemicals, Inc (Guelph, Canada). Soybean lecithin (Lipoid S75, fat free soybean phospholipids with 70% phosphatidylcholine) was obtained from Lipoid GmbH (Ludwigshaften, Germany). Propylene glycol monocaprylate, type II (Capryol™ 90) and glyceryl palmitostearate (Precirol® ATO 5 ATO 5) were kindly donated from Gattefosse (Lyon, France). Butylated hydroxytoluene (BHT) and polysorbate 80 were purchased from Sigma–Aldrich Lab. GmbH (Germany). Ultra-purified water (water) was obtained with a GenPure apparatus (TKA Wasseranfbereitungs-systeme GmbH, Neiderelbert, Germany). Protopic® ointment, 0,1% (Astellas Ireland Co. Ltd) was purchased from a local pharmacy. 2.2. Differential scanning calorimetry (DSC) – preformulation evaluation In order to assess the optimal solid/liquid lipid ratio for the formulation of NLC, the change in the melting points of different solid/ liquid lipid mixtures was assessed using DSC. Samples of mixtures with different ratios of Precirol® ATO 5 and Capryol™ 90 (from 9:1 to 2:8) were heated up from 20 °C to 180 °C at a heating rate of 10 °C/min, using Mettler Toledo DSC 1, STARe System (Mettler Toledo AG, Analytical, Switzerland). Measurements were conducted under constant nitrogen flow of 50 mL/min. 2.3. Formulation development and preparation In order to develop an optimal NLC formulation as a potential carrier for tacrolimus, different placebo formulations were prepared, varying formulation composition (Table 1, solid/liquid lipid ratio (4:6, 5:5, 6:4), amount of lipid phase (5 vs. 10%, w/w), amount of lecithinpolysorbate 80 mixture at the ratio 1:1 (1% vs. 2% vs. 4%), the presence/absence of polysorbate 80 as steric stabilizer) and process parameters (number of homogenization cycles (5 vs. 10), pressure during homogenization (500 bars vs. 800 bars), cooling after the preparation (rapid in an ice-bath vs. regular cooling at room temperature). Comparing physicochemical characteristics and stability of evaluated formulations, the factors which are critical for the development of lecithin-

Table 1 Composition of investigated formulations. Formulation

Lipoid S75 (%)

Precirol® ATO 5 (%)

Capryol™ 90 (%)

BHT* (%)

Polysorbate 80 (%)

Water (%)

PCLP 6:4 PCLP 5:5 PCLP 4:6 PCLP 2:3 PCLP 4:6 B PCLP 4:6 C PCLP 4:6 D CLP 10

1.00 1.00 1.00 1.00 2.00 0.50 1.00 1.00

6.00 5.00 4.00 2.00 4.00 4.00 4.00 –

4.00 5.00 6.00 3.00 6.00 6.00 6.00 10.00

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

1.00 1.00 1.00 1.00 2.00 0.50 – 1.00

87.95 87.95 87.95 92.95 85.95 88.95 88.95 87.95

* Butylated hydroxytoluene. 2

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2.4.4. Rheological analysis Rheological properties of the samples were determined on DV-III ULTRA Programmable Rheometer and Rheocalc software v.4.3 (Brookfield Engineering Laboratories, Middlesboro USA), coupled with cone and plate measuring device. All experiments were performed at 20 °C with the shear rate in the range of 375–1850 s−1 and vice verse for up and down curves.

stabilized NLC with Precirol® ATO 5 and Capryol™ 90 as lipid phase were identified. The hot high pressure homogenization method was used for the preparation of the formulations. Briefly, the lipid phase, containing mixture of solid and liquid lipid, lecithin and BHT, was heated to 80 °C on magnetic stirrer, while water phase (ultra-purified water with/ without polysorbate 80) was heated to 85 °C under slight stirring. When the solid lipid was completely melted and lecithin dissolved, water phase was added to the lipid phase and pre-homogenized for 1 min at 10,000 rpm with a rotor-stator homogenizer (Ultra-Turrax®T25 digital, IKA® Werke GmbH&Company KG, Germany) under continuous heating. The obtained emulsion was then homogenized at 800 bars during 5 cycles in pre-heated EmulsiFlex C3 (Avestin Inc, Ottava, Canada). All prepared formulations were immediately packed into glass vials, tightly closed with crimp seals, and left to cool down at the room temperature (except one cooled rapid in an ice-bath). To be precise, in order to compare the effect of diverse process factors, one formulation was additionally prepared using 500 bars during 5 cycles (PCLP 4:6 500 bars), while another was prepared using 800 bars during 10 cycles of homogenization (PCLP 4:6 10 cycles). Tacrolimus (0.1% w/w) was incorporated in the best NLC candidate, by dissolving it in lipid phase prior to mixing with water phase and pre-homogenization (PCLP 4:6 TAC). Additionally, corresponding placebo and drug-loaded formulations without solid lipid (nanoemulsions, CLP 10 and CLP 10 TAC, containing 10% of Capryol™ 90 as lipid phase) were prepared using above described method in order to compare the effect of solid lipid incorporation on physicochemical characteristics, drug incorporation, in vitro drug release and dermal drug delivery.

2.4.5. Entrapment efficiency In order to destabilize formulations and separate the lipid phase, sodium chloride was added to the selected NLC formulation and corresponding NE formulation with incorporated tacrolimus (PCLP 4:6 TAC and CLP 10 TAC), mixed on vortex during 30 min at 100 rpm, left overnight, mixed again the following day and centrifuged 1 h at 14,500 rpm (MiniSpin® plus, Eppendorf, Hamburg, Germany). Test tubes were examined for the presence of undissolved tacrolimus and the amount of tacrolimus in water phase was measured using previously developed UPLC-MS/MS method (Savic et al., 2017). Entrapment efficiency (EE) was calculated according to the following equation (Date et al., 2011):

Minitial drug − Mfree drug ⎞ EE(%) = ⎜⎛ ⎟ × 100% Minitial drug ⎝ ⎠ where Minitial drug is the mass of the drug used for the fabrication of the formulations and Mfree drug is the mass of free tacrolimus, present as dissolved in water phase, or undissolved at the bottom of the test tubes. 2.4.6. Polarization microscopy In order to evaluate the presence of tacrolimus crystals in final formulations, drug-loaded formulations and corresponding placebo ones were evaluated using polarization microscopy. Formulations were analyzed using Motic digital microscope DMB3-223ASC and Motic Images Plus v.2.0 software (Motic GmbH, Wetzlar, Germany) after one month of storage at room temperature.

2.4. Characterization 2.4.1. Particle size analysis Mean particle size and polydispersity index of formulations was measured at 20 °C by photon correlation spectroscopy (PCS), using Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK). All formulations were diluted with ultra-purified water 1:100 v/v prior to the measurement. Additionally, in order to examine the presence of larger particles/droplets, the laser diffraction analysis was performed using Malvern Mastersizer 2000 (Malvern Instruments Ltd.).

2.5. Stability studies Physical and chemical stability (particle size, polydispersity index, zeta potential, pH, conductivity) of formulations stored at room temperature (20 ± 2 °C) was assessed during six months. Additionally, all formulations were evaluated for the presence of eye-visible structures and/or separation of phases.

2.4.2. Zeta potential measurement Zeta potential (ZP) is a key indicator of the stability of colloid dispersion. The higher absolute value of ZP indicates the better stability of formulations due to the electrostatic repulsion between particles (Han et al., 2008). ZP was determined by measuring the electrophoretic mobility of particles/droplets in formulations using Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK). Ultra-purified water with constant conductivity was used for the dilution of formulations prior to the measurements (1:100 v/v).

2.6. In vitro release of tacrolimus In vitro drug release studies were carried out using modified Franz diffusion cells (Gauer Glas, D-Püttlingen, Germany). Final formulations with tacrolimus (PCLP 4:6 TAC, CLP 10 TAC) and referent ointment (Protopic® 0.1%) in quantity equivalent to 1 mg of tacrolimus were placed on previously activated dialysis membrane (pore size 2.4 nm, molecular weight cut-off 12,000) in the donor compartment and covered with Parafilm® to avoid evaporation. The receptor compartment contained a mixture of water and methanol (60:40 v/v, 12 mL) in order to maintain sink conditions for tacrolimus. During the entire experiment, temperature of diffusion cells was maintained at 32 °C, while stirring speed was 500 rpm. Samples (500 µL) of receptor medium were acquired and replaced with fresh pre-warmed medium after 30 min, 1 h, 2 h, 3 h, 6 h, 9 h and 24 h of formulation application. The amount of tacrolimus in all samples was determined by UPLC-MS/MS method (Savic et al., 2017). Although when employing a hydroalcoholic medium as a receptor phase there is possibility of back diffusion of alcohol through the synthetic membrane (Shah and Williams, 2014), no precipitation of tacrolimus or any change in investigated formulations was observed during their visual and microscopic examination at the end of the experiment, implying that addition of methanol had a

2.4.3. pH and conductivity measurements Bearing in mind the intended dermal application of formulations, as well as the fact that tacrolimus is prone to degradation in medium with pH > 7 (Skak and Hansen, 2016), it is important to consider the pH values of developed formulations. Additionally, significant changes in pH and conductivity values during storage could indicate instability of these carriers. The pH values of formulations were measured at a temperature of 20 ± 2 °C using HI 9321 pH meter (Hanna Instruments Inc., Ann Arbor, Michigan), by direct immersion of glass electrode into the samples. Measurement of the conductivity was performed at 20 ± 2 °C using CDM 23 conductometer (Radiometer, Copenhagen, Denmark) without addition of electrolytes. The presence of negatively charged phospholipids and different impurities in lecithin provided the charges necessary for the measurement of conductivity. 3

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Permeation rate of tacrolimus was calculated as a slope of linear portion of the plot which represents cumulative amount of tacrolimus (ng/cm2) against time (h). After 24 h the skin was dismantled from the cells, excess of formulation was carefully removed using cotton swabs, and the skin was cut into pieces. Tacrolimus was extracted by addition of 4 mL methanol and leaving it on orbital shaker KS 260 basic (IKA®-Werke GmbH & Co. KG, Staufen, Germany) at 250 rpm during 24 h followed by centrifugation at 4000 rpm for 30 min (Centrifuge MPW-56, MPW Med. Instruments, Warszawa, Poland). Previously developed UPLC-MS/MS method (Savic et al., 2017) was employed in order to measure the amount of tacrolimus in samples.

negligible effect on the sample integrity. In vitro release profiles were interpreted using different mathematical models, such as zero-order, first-order, Higuchi, Hixson–Crowell cube root law, and Korsmeyer–Peppas models, and the model with the highest correlation coefficient (R2) was selected. 2.7. In vitro skin penetration assessment by differential stripping In order to evaluate the penetration of tacrolimus in stratum corneum and the contribution of follicular uptake, differential stripping technique was employed on porcine ear. Briefly, on the day of experiment, porcine ears were defrosted, the skin was freed from visible hairs with scissors and ears were fixed on styrofoam plates. When TEWL reached ~15 gm−2h−1, marked areas without damage or pigmentation were treated with PCLP 4:6 TAC, CLP 10 TAC or Protopic® 0.1% in a quantity corresponding 50 µg/cm2 of tacrolimus. After two hours of treatment, excess formulation was removed using cotton pads and layers of stratum corneum were removed using 15 adhesive tapes (Dsquame®, CuDerm, Dallas, USA) for each treated site. In order to reduce the influence of skin wrinkles and furrows, all tapes were pressed onto the skin during 10 s by using a roller device (300 g) prepared according to Lademann et al. (2009). The removed strips were placed in test tubes, 4 mL of ethanol (70% v/v) was added to each tube and tacrolimus was extracted using ultrasonic bath (15 min), followed by centrifugation at 4000 rpm for 15 min (Centrifuge MPW-56, MPW Med. Instruments, Warszawa, Poland). After the removal of stratum corneum, two cyanoacrylate skin surface biopsies were performed on each site by applying a drop of cyanoacrylate superglue (UHU GmbH & Co. KG, Brühl, Germany), covering it with adhesive tape and leaving it to polymerize during 10 min. The amount of tacrolimus which could be removed by this procedure represents mainly the follicular content of tacrolimus, since our preliminary study indicated that the sufficient amount of tacrolimus localized in stratum corneum has been removed using 15 tape strips prior to the cyanoacrylate skin surface biopsy (the amount of tacrolimus which could be removed by more than 15 tape strips was negligibly low, i.e. under the limit of quantification (5 ng/mL). Both tapes from each site with dried cyanoacrylate superglue were placed in one test tube with 4 mL of acetonitrile. Tacrolimus was extracted using ultrasonic bath (15 min), followed by centrifugation during 30 min at 10,000 rpm (MiniSpin® plus, Eppendorf, Hamburg, Germany). The amount of tacrolimus in all samples was determined by UPLC-MS/MS method (Savic et al., 2017).

2.9. Data analysis All data are represented as mean values ± standard deviations (SD). Statistical analysis was performed using software package IBM SPSS Statistics 21. The normal distribution of the data was tested using Shapiro–Wilk test. Statistical significance between the groups (p < 0.05) was determined using one-way analysis of variance (ANOVA) with Tukey's post hoc analysis or Welch and Brown-Forsythe test followed by Games-Howell post hoc analysis for data with unequal variances. For data which deviated from normal distribution, MannWhitney U test and Kruskal-Wallis H Test were employed. 3. Results and discussion 3.1. DSC studies – preformulation evaluation During development of NLC formulations, DSC studies are often firstly employed in order to assess the crystalline behavior of lipid phase. Namely, the addition of a liquid lipid(s) increases the number of imperfections in crystalline structure of solid lipid(s), followed by depression of its melting point (Beloqui et al., 2016; Montenegro et al., 2016; Müller et al., 2002). Bearing in mind that the solubilization of tacrolimus is significantly greater in Capryol™ 90 (113.06 ± 6.4 µg/ mg) than in Precirol® ATO 5 (26.95 ± 3.36 µg/mg, Pople and Singh, 2011), and that the imperfect structure allows incorporation of higher amounts of drug and reduces drug expulsion, it is preferred to obtain NLC with higher amount of Capryol™ 90. On the other side, the prerequisite for the production of NLC is that the mixture of solid and liquid lipids has to be solid on body and room temperature. DSC thermograms (Fig. 1) of Precirol® ATO 5 and Capryol™ 90 mixtures in different blending ratios reveal, as expected, the significant depression of Precirol® ATO 5′s melting point upon the addition of liquid lipid. All mixtures, apart from 1:9, appeared solid, with relatively high melting onset and peak temperatures (Table 2). No oozing of liquid lipid could be visually observed. The mixture of Precirol® ATO 5 and Capryol™ 90 with ratio 1:9 exhibited semisolid characteristics at room temperature and therefore was not analyzed on DSC. Analyzing thermograms of lipid mixtures with the lowest Precirol® ATO 5/Capryol™ 90 (PC) ratios (PC 2:8, PC 3:7, PC 4:6, Fig. 1, right) two melting endotherms could be observed for PC 2:8 (Table 2). It is known that Precirol® ATO 5 has polymorphic forms, which can be observed by applying heating–cooling programs on DSC, i.e. when DSC thermograms were performed on the freshly solidified samples immediately after crystallization, especially after rapid cooling of melted lipid (Hamdani et al., 2003). However, it seems that the high amount of liquid lipid led to the formation of two polymorphs which could be observed days after crystallization. Two polymorphic forms of Precirol® ATO 5 cannot be observed for the mixture PC 4:6, while their presence in PC 3:7 could be speculated. Because of this reason, as well as the fact that low amount of liquid lipid could risk tacrolimus expulsion due to the relatively lower solubility of tacrolimus in Precirol® ATO 5 compared to Capryol™ 90 (Pople and Singh, 2011), the solid/liquid lipid ratio in formulations to be developed was restricted to 4:6, 5:5 and 6:4.

2.8. In vitro skin permeation In vitro skin permeation studies were performed in order to obtain the information about skin permeation rate of tacrolimus from examined formulations (PCLP 4:6 TAC, CLP 10 TAC, Protopic®). The experiments were conducted under the similar conditions as the in vitro release studies (Section 2.6), with the exception of membrane between donor and receptor compartment, which was in this case full-thickness porcine ear skin, and receptor medium more suitable for skin experiments (25% ethanol in phosphate-buffered saline, pH 7.4). The fullthickness porcine ear skin was selected as a skin model in order to evaluate the potential of systemic adverse effects due to the permeation of tacrolimus through all layers of the skin, and to assess the deposition of drug into the skin, where it should manifest its effect by binding to the immune cells localized in both epidermis and dermis (Pople and Singh, 2010, Raney et al., 2015). Similar to the in vitro release study, 1 g of formulations/referent ointment corresponding 1 mg of tacrolimus (infinite dose) was applied onto the skin (2,01 cm2) in the donor compartment of modified Franz diffusion cell, which was afterwards covered with Parafilm® to reduce the evaporation of water from the formulations. Samples of receptor medium were acquired after 3 h, 6 h, 20 h, 22 h and 24 h and replaced with fresh pre-warmed medium. 4

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Fig. 1. Left: DSC thermograms of Precirol® ATO 5, Capryol™ 90 and their mixtures in different Precirol® ATO 5/Capryol™ 90 (PC) ratios. Right: Detailed DSC thermograms of PC mixtures with high content of Capryol™ 90.

distribution (polydispersity index ≤0.26, Table 3), initially and during six months of storage. Likewise, the D(0,9) values obtained from laser diffraction measurements were below 226 nm, confirming the absence of particles in the micrometer range (data not shown). However, it should be emphasized that the evaluated variables (formulation composition and process parameters) had different effect on particle size, while polydispersity index was more uniform, regardless the formulation. Analyzing placebo NLC formulations which contain the same amount of lipid phase (10%), but different solid/liquid lipid ratio (PCLP 6:4, PCLP 5:5, PCLP 4:6) it can be observed that the higher amount of solid lipid resulted in higher particle size (Table 3). It is interesting to note that Date et al. (2011) examined the stability of lipid nanoparticles containing 5% mixture of Precirol® ATO 5 and Capryol™ 90 stabilized by Gelucire 50/13 (stearoyl polyoxyl-32 glycerides). In contrast to our results, no change in particle size due to the increased amount of Capryol™ 90 is observed in their experiments. However, this difference could be the consequence of the lower amount of lipid phase, as well as different surfactant used. Particle size and polydispersity index of PCLP 4:6 were not significantly altered during six months of storage, while the evaluation of PCLP 6:4 and PCLP 5:5 was discontinued during first month due to the gelling of these samples. Comparing two formulations with the same solid/liquid lipid ratio, but different total amount of lipids (10% in PCLP 4:6 vs. 5% in PCLP 2:3), as expected, significantly higher particle size is observed for

Table 2 Thermodynamic parameters of endothermic melting of solid and liquid lipid mixtures in different ratios. Precirol® ATO 5: Capryol™ 90 ratio

Onset (oC)

Peak (oC)

Enthalpy (J/g)

2:8 3:7 4:6 5:5 6:4 7:3 8:2 9:1 10:0

26.87/34.79 39.06 39.97 42.42 43.57 44.21 43.06 43.68 55.03

34.17/45.83 48.00 48.50 51.33 53.33 55.17 55.83 57.00 58.33

32.68 51.57 63.07 81.51 93.25 102.68 122.65 133.19 119.85

The effect of these ratios, as well as other examined factors on physicochemical characteristic of formulations will be discussed in following section. 3.2. Physicochemical characterization, stability testing and evaluation of critical formulation/process variables 3.2.1. Particle size analysis Analyzing data obtained using PCS, it can be noticed that all examined formulations had particle size below 200 nm with narrow size

Table 3 Particle size, polydispersity index and zeta potential of formulations (initial values and values after 3 (a) or 6 (b) months of storage, depending on stability). Mean ± SD, n = 3. Formulation

Mean particle size (nm) Initial

PCLP 6:4 PCLP 5:5 PCLP 4:6 PCLP 2:3 PCLP 4:6 B PCLP 4:6 C PCLP 4:6 D PCLP 4:6 TAC CLP 10 CLP 10 TAC PCLP 500 bars PCLP 10 cycles

182.23 154.53 143.00 112.17 137.87 151.27 151.97 143.23 127.03 135.13 147.80 144.67

Polydispersity index a,b

After the storage ± ± ± ± ± ± ± ± ± ± ± ±

1.54 2.32 1.42 0.35 2.92 2.40 2.02 1.08 2.52 1.15 1.15 0.51

140.33 108.47 153.63 149.50 154.03 140.33 124.20 143.43 137.57 147.93

± ± ± ± ± ± ± ± ± ±

2.12b 1.01b 0.67b 4.59b 2.72b 1.00b 1.66a 1.65a 0.57a 0.60b

Initial 0.26 0.25 0.23 0.25 0.23 0.20 0.19 0.21 0.20 0.19 0.21 0.23

± ± ± ± ± ± ± ± ± ± ± ±

Zeta potential (mV) After the storage

0.02 0.02 0.01 0.00 0.02 0.00 0.02 0.01 0.01 0.03 0.02 0.02

5

0.17 0.20 0.22 0.19 0.23 0.15 0.22 0.24 0.19 0.19

± ± ± ± ± ± ± ± ± ±

0.01b 0.01b 0.04b 0.03b 0.05b 0.01b 0.02a 0.00a 0.02a 0.02b

a,b

After the storagea,b

Initial −34.30 −34.49 −36.97 −36.53 −39.20 −31.60 −39.50 −35.60 −35.33 −34.10 −36.03 −35.83

± ± ± ± ± ± ± ± ± ± ± ±

0.26 0.13 0.45 1.10 1.22 0.26 2.48 1.50 0.55 0.36 1.80 0.78

−37.77 −35.57 −36.83 −36.80 −37.87 −37.30 −38.00 −37.00 −38.73 −37.90

± ± ± ± ± ± ± ± ± ±

0.90b 1.35b 0.40b 1.47b 0.58b 0.44b 1.55a 1.68a 0.35a 3.10b

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Table 4 pH, conductivity (initial values and values after 3 (a) or 6 (b) months of storage (depending on stability), mean ± SD, n = 3) and viscosity of formulations (mean ± SD, n = 2). pH

Conductivity (µS/cm)

Formulation

Initial

PCLP 6:4 PCLP 5:5 PCLP 4:6 PCLP 2:3 PCLP 4:6 B PCLP 4:6 C PCLP 4:6 D PCLP 4:6 TAC CLP 10 CLP 10 TAC PCLP 500 bars PCLP 10 cycles

4.52 4.58 4.61 4.77 4.62 4.58 4.52 4.42 4.22 4.38 4.55 4.54

± ± ± ± ± ± ± ± ± ± ± ±

After the storagea,b 0.01 0.01 0.01 0.02 0.03 0.02 0.01 0.02 0.02 0.02 0.02 0.02

4.06 4.06 4.11 4.10 3.95 3.89 4.07 4.10 4.30 3.79

± ± ± ± ± ± ± ± ± ±

After the storagea,b

Initial 52.53 68.13 69.60 63.13 95.70 65.83 65.43 70.73 76.87 74.93 66.37 67.30

b

0.02 0.02b 0.08b 0.02b 0.04b 0.02b 0.01a 0.02a 0.01a 0.08b

Viscosity (mPa*s)

± ± ± ± ± ± ± ± ± ± ± ±

0.67 0.40 0.98 0.85 0.17 0.51 0.14 0.15 0.70 0.12 3.00 0.14

90.33 ± 0.31b 75.57 ± 0.35b 107.87 ± 0.29b 89.93 ± 0.21b 65.37 ± 0.06b 75.80 ± 0.36b 84.73 ± 0.64a 85.93 ± 1.47a 74.57 ± 0.55a 81.80 ± 0.44b

10.40 ± 0.83 6.87 ± 0.09 3.01 ± 0.00 23.61 ± 0.28 5.62 ± 0.74 7.85 ± 0.55 6.80 ± 0.34 5.23 ± 0.00 4.38 ± 0.28 6.47 ± 0.09 7.26 ± 0.09

separation of oil droplets was observed for CLP 10 during the fourth month of storage. Since both formulations contain the same amount of surfactants and are produced using the same method, it seems that the incorporation of Precirol® ATO 5 and the presence of solid lipid matrix instead of oil droplets are responsible for better stability of PCLP 4:6.

higher amount of lipids (Table 3). Bearing in mind that both formulations were prepared using same homogenization pressure (800 bars) and 5 cycles of homogenization, by increasing the total amount of lipids less dispersion energy is available per unit of lipid, leading to the increase in particle size (Müller et al., 2002). Additionally, considering the fact that the same amount of lecithin and polysorbate 80 are present in these formulations, the relative amount of stabilizing agents is lower in the formulation containing higher amount of total lipids (PCLP 4:6). Although the formulation PCLP 2:3 was stabile during six months of storage, a slight change in color towards yellow was observed after three months of storage. The plausible explanation for color change is the oxidation of BHT, which gives yellow water soluble products (Nieva-Echevarría et al., 2014). Due to the same amount of BHT in all formulations, and thus higher concentration of BHT in lipid particles with lower content of lipid phase, this oxidation is more apparent in PCLP 2:3 than PCLP 4:6. Likewise, it could not be neglected that the oxidation products of lecithin in the examined formulations contributed to the color change. Different content of surfactants in formulations did not have such a significant effect on particle size, as the content of lipid phase. Namely, two times higher or lower amounts of surfactants resulted in particle size reduction or increase for only few nanometers (5–8 nm). Interestingly, formulation stabilized with only lecithin (without addition of polysorbate 80 as steric stabilizer, PCLP 4:6 D) showed satisfying particle size and polydispersity index without significant changes in the course of six months (Table 3). This is surprising due to the facts that phospholipids alone are not sufficient at forming or stabilizing nanoemulsions and that the addition of spacious surface-active agents as steric stabilizers is regarded as a successful strategy to optimize the stability of lecithin based nanoemulsions (Klang and Valenta, 2011; Salminen et al., 2014). One of the plausible reasons for this could be the amphiphilic properties of Capryol™ 90 due to which lecithin can incorporate better into the lipid phase. Long term stability studies should be conducted in order to confirm adequate stability of this formulation. Concerning evaluated process parameters, the change in pressure during homogenization and number of homogenization cycles did not significantly influence physicochemical characteristics of NLCs. However, formulation which was prepared using lower homogenization pressure (PCLP 4:6 500 bars) exhibited altered stability (formation of visible particles/aggregates) during the fourth month of storage, while aggregates were not detected for corresponding formulation produced using higher homogenization pressure (800 bars, PCLP 4:6). Comparing particle size and polydispersity index for placebo NLC and NE (CLP 10) containing 10% of lipid phase, it can be observed that the incorporation of solid lipid led to the increase in particle size, while droplets in NE were slightly smaller. Interestingly, while NLC formulation PCLP 4:6 was stabile during six months of storage, a visual

3.2.2. Zeta potential measurement Zeta potential, as a measure of particle charge and electrostatic repulsion, is an important parameter which could indicate the stability of the system. In order to obtain a stabile system, the electrostatic repulsion should dominate the attractive Van der Waals forces, according to the DLVO theory. Charged particles with absolute value of zeta potential higher than 30 mV are less likely to aggregate due to the electrostatic repulsion (Han et al., 2008). Zeta potential of developed formulations was below −30 mV (Table 3), indicating good stability. Comparing formulations with (PCLP 4:6) and without the addition of polysorbate 80 (PCLP 4:6 D), it can be noticed that the addition of steric stabilizer did not significantly influence the zeta potential. Additionally, comparing PCLP 4:6 and corresponding NE formulation without solid lipid (CLP 10), it seems that the incorporation of solid lipid also did not affect the zeta potential. Similarly, the variation in the amount of lipid phase or solid/liquid lipid ratio did not exert any significant influence on the particle surface charge. During the observation period, the absolute values of zeta potential remained stable or were slightly increased for most of the formulations, probably due to the formation of free fatty acids caused by the hydrolysis of phospholipids (Klang and Valenta, 2011). 3.2.3. pH and conductivity measurements All formulations had similar pH and conductivity values (Table 4), i.e., variation in formulation composition and process parameters did not remarkably affect the values of these parameters. The highest pH value (4.77) was observed for formulation with lowest amount of Capryol™ 90 (PCLP 2:3), while the lowest pH (4.22) vas observed for CLP 10 which has the highest amount of Capryol™ 90 (CLP 10). All these values are acceptable for intended dermal application, while mild acidity of formulations could improve tacrolimus stability considering its degradation in alkaline media (Skak and Hansen, 2016). The conductivity of formulations was highest for formulation PCLP 4:6 due to the highest amount of lecithin and lowest for PCLP 6:4 due to the high viscosity. During the storage, a decrease in pH values and an increase of conductivity was observed for all formulations. This is probably the consequence of hydrolysis of lecithin and mono- and diglycerides present in lipid phase, which results in formation of free fatty acids and should be solved through a proper selection of additional pharmaceutical excipients with the aim of reaching the optimal critical quality attributes for such a type of nanodispersed formulation, inevitably 6

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defined by final dosage form administration route.

It seems like the liquid oil core in NE was less robust to the incorporation of tacrolimus compared to the solid matrix in NLC. pH, conductivity and viscosity values of placebo and drug loaded formulations were similar for both NLC and NE (Table 3). This is not surprising, especially due to the absence of tacrolimus from the water phase.

3.2.4. Rheological analysis Viscosity values of the formulations (Table 4) were highly influenced by the solid/liquid lipid ratio, total amount of lipid and the amount of surfactants in formulations. Formulation PCLP 6:4, which has the highest ratio of solid/liquid lipid, had very high viscosity which could not be measured by described experimental settings. This formulation changed to semisolid, gel state during first month of storage and therefore, was excluded from further examination. Decreasing the solid/liquid lipid ratio led to the decrease of viscosity. However, formulation PCLP 5:5 also changed to semisolid state during first three months of storage and is excluded from further study. Comparing formulations with same solid/liquid lipid ratio (4:6), but different total amount of lipid phase (10% in PCLP 4:6 and 5% in PCLP 2:3) it can be observed that the reduction of total amount of lipid phase decreased viscosity values. Further, observing formulations with different amount of surfactants, two times higher amount of lecithin and polysorbate 80 in formulation PCLP 4:6 B compared to PCLP 4:6 increased viscosity value almost for four times. Similar findings are reported before (Zhou et al., 2010) and are not surprising due to the complex structure of lecithin and its gelling properties. On the other hand, two times lower amount of lecithin and polysorbate 80 (PCLP 4:6 C), or absence of polysorbate 80 (PCLP 4:6 D) did not drastically affected the viscosity of formulations. One of the possible reasons for this could be the relatively low number of surfactant molecules present in water phase of these formulations due to their predominant localization at the lipid-water interface. The change in process parameters (number of homogenization pressure and cycles) did not influence the viscosity of formulations. However, rapid cooling of formulation PCLP 4:6 in the ice bath after the homogenization resulted in formation of semisolid formulation and was therefore characterized as undesirable. The possible explanation for this could be the formation of different polymorphs of Precirol® ATO 5 due to the rapid cooling, which was previously described (Hamdani et al., 2003), leading to the instability of the system.

3.2.6. Entrapment efficiency Important advantage of NLC compared to SLN is the increased drug loading capacity due to the presence of liquid lipid, which increases number of imperfections in lipid matrix (Beloqui et al., 2016; Montenegro et al., 2016; Müller et al., 2002). One of the aims of this study was to develop stabile NLC with higher amount of liquid lipid which could minimize the expulsion of tacrolimus. During the determination of entrapment efficiency it was observed that the mixture of Precirol® ATO 5 and Capryol™ 90, as well as pure Capryol™ 90 have lower density than water. Thus, after the centrifugation of samples, the lipid phase was separated above the water phase. Since it was not observed sedimentation of tacrolimus crystals on the bottom of the examination tube, tacrolimus which was not incorporated in lipid phase could only be dissolved in water phase. However, concentration of tacrolimus in water phase was under the limit of quantification (5 ng/mL). Therefore we can suggest that the entrapment efficiency of tacrolimus in the selected NLC and corresponding NE was more than 99%, due to its good solubility in liquid lipid, and generally low amount which needs to be incorporated in formulations (0.1%). Owing to the presence of relatively high amount of liquid lipid, it seems that the incorporation of solid lipid did not lead to the expulsion of tacrolimus from the lipid matrix. Employing polarization microscopy, the absence of undissolved tacrolimus in drugloaded NLC and NE has been confirmed. 3.3. In vitro release of tacrolimus The release of active substance from a carrier is a prerequisite for its diffusion into the skin and consequently, pharmacological activity. Due to the different nature of NLC and NE, a different in vitro release of drugs could be expected. Namely, in NLC the complex solid lipid matrix, containing the mixture of solid and liquid lipids, is dispersed in water phase, while lipid phase of NE consists of plain oil droplets. Although in vitro release studies through a synthetic membrane cannot directly indicate the bioavailability of drug, in vitro release non-similarity of formulations could be a signal of altered in vivo performance (Shah et al., 2015). The in vitro release of tacrolimus from NE followed zero order kinetic, which implies that the release of drug from the carrier is not depended of its concentration. On the other hand, the release of tacrolimus from NLC could be better explained using Hixson-Crowell cube root law, which describes the release from systems where there is a change in surface area and diameter of particles. This type of release has been reported before for some nanostructured lipid carriers (Gadhave et al., 2019; Lee et al., 2014). The release of tacrolimus from the referent ointment followed Higuchi diffusion model which suggests that the diffusion through the ointment to the acceptor compartment is driven by the drug concentration gradient and is the rate-determining/ limitation step. The in vitro release profiles of tacrolimus are presented in Fig. 2. The highest release rate is obtained for formulation PCLP 4:6 TAC (1.44 ± 0.15 µg/cm2/h, significantly higher from other two formulations), followed by CLP 10 TAC (0.68 ± 0.27 µg/cm2/h) and Protopic® (0.09 ± 0.03 µg/cm2/h, significantly lower from NLC and NE). Additionally, significantly higher cumulative amount of tacrolimus was released from NLC compared to NE and the referent ointment for all sampling periods starting with 2 h, while NE exhibited higher cumulative release of tacrolimus compared to the referent ointment only after 6, 9 and 24 h after the application.

3.2.5. Physicochemical characterization and stability of selected tacrolimus-loaded NLC and NE Based on the previous physicochemical characterization and stability testing, it can be concluded that the most critical factors for the development of lecithin-based NLC with Precirol® ATO 5 and Capryol™ 90 are the solid/liquid lipid ratio, total amount of lipid phase and cooling after the preparation. Although the reduction of pressure during homogenization did not have a significant effect on physicochemical characteristics of formulations, it led to the formation of larger particles/aggregates during fourth month of storage and is therefore also regarded as an important process factor. The amount of lecithin and polysorbate 80, as well as the number of homogenization cycles did not significantly influence characteristics or stability of formulations and are regarded as factors of minor importance. In order to evaluate the best NLC candidate as a potential carrier for dermal delivery of tacrolimus, it was decided to limit the solid/liquid lipid ratio to 4:6, the total amount of lipid phase to 10% (ensuring tacrolimus dissolution), while the content of lecithin and polysorbate 80 was limited to the medium level (1% of each). Consequently, tacrolimus was incorporated in the selected NLC (PCLP 4:6 TAC) and corresponding NE (CLP 10 TAC). Bearing in mind challenging properties of tacrolimus (complex structure, high molecular weight and low solubility in water), altered physicochemical characteristics of nanocarriers could be expected after its incorporation. Analyzing particle size, polydispersity index and zeta potential of PCLP 4:6 and PCLP 4:6 TAC (Table 2), no notable difference between placebo and drug-loaded formulation was observed. On the other hand, while polydispersity index and zeta potential of drug-loaded NE did not differ, a slight increase in droplet size was noticed after the incorporation of tacrolimus. 7

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Fig. 2. In vitro release profiles of tacrolimus from nanostructured lipid carrier (PCLP 4:6 TAC), nanoemulsion (CLP 10 TAC) and the referent ointment (Protopic®); mean ± SD, n = 4.

Fig. 3. The penetration profiles of tacrolimus (TAC) from nanostructured lipid carrier (PCLP 4:6 TAC), nanoemulsion (CLP 10) and the referent ointment (Protopic®); mean ± SD, n = 3.

All obtained results suggest superiority of NLC in the release of tacrolimus compared to the NE and the referent ointment. The reason for this could be its inner structure. Müller et al. (2002) have suggested that NLC formulations which contain higher amount of liquid lipids can be classified in multiple type of NLC, where the lipid matrix contains small departments of oil. Bearing in mind that PCLP 4:6 TAC contains higher amount of liquid then solid lipid, multiply type NLC could be assumed. Because of its better solubility in Capryol™ 90 than Precirol® ATO 5, tacrolimus could be dissolved in these oil departments. Combining the release of tacrolimus from these compartments together with further erosion of lipid particles, overall high release of tacrolimus is observed. NE, which contains plane droplets of liquid lipid, has smaller area for diffusion compared to oil compartments in NLC and therefore exhibits slower release of tacrolimus.

and NLC compared to the referent ointment (Fig. 3). One explanation for this could be the pronounced adhesive properties of NE and NLC to skin surface, leading to the formation of dense film on the skin after the water evaporation (Zhou et al., 2010), which probably results in the formulation metamorphosis accompanied with the drug precipitation/ concentration at the application site. Although amounts of tacrolimus extracted from all tapes were relatively low, a trend of higher penetration of tacrolimus from NLC and NE compared to the referent ointment can be observed, especially in the upper layers of stratum corneum. On the other hand, comparing the total amount of tacrolimus extracted from stratum corneum after two hours of treatment with NLC, NE and Protopic® (Fig. 4, left), a significantly higher amount has penetrated after the application of PCLP 4:6 (495.84 ± 65.95 ng/cm2) and CLP 10 (564.12 ± 52.93 ng/cm2) compared to Protopic® (275.23 ± 53.54 ng/cm2). While Wolf et al. (2018a) observed higher penetration of curcumin from NLC compared to NE, there was no significant difference between the amount of penetrated tacrolimus in stratum corneum from NLC and NE in our study. Bearing in mind that it is difficult to apply ointment on hairy skin surface as well as that the developed nanocarriers could be especially suitable for these body regions, we have evaluated the amount of tacrolimus deposited in the hair follicles using cyanoacrylate skin surface biopsy after the removal of stratum corneum by tape stripping. The significantly higher amount of tacrolimus in hair follicles was observed for PCLP 4:6 (268.54 ± 92.38 ng/cm2, Fig. 4, right), while no significant difference was observed between CLP 10 (128.17 ± 48.87 ng/ cm2) and the referent ointment (77.61 ± 43.25 ng/cm2). Even though the amount of tacrolimus in hair follicles was more than two times higher after the application of NLC compared to NE, significant difference between these carriers was not obtained (p = 0.08), due to the relatively high variability of obtained data. In clinical setting, it could be expected even higher amount of tacrolimus in hair follicles after the application of developed formulations, due to the physiological movement of hairs, which may favor follicular uptake of drug (Ilic et al., 2018; Lademann et al., 2007; Radtke et al., 2017). Comparing all results obtained by in vitro differential stripping, it can be concluded that the intercellular route is the predominant diffusion route for tacrolimus, but the contribution of transfollicular route in overall skin penetration cannot be neglected, especially for NLC, since significant amount of tacrolimus retrieved by differential stripping was detected in hair follicles. In other words, the presence of solid

3.4. In vitro skin penetration assessment by differential stripping The penetration of tacrolimus into the stratum corneum and hair follicles of porcine ear skin was assessed by differential stripping. Taking into account that target site of tacrolimus action includes both a viable epidermis and dermis, the data obtained using tape stripping may provide a suitable surrogate for characterizing the rate and extent of drug absorption to the underlying tissues (EMA, 2018). Additionally, considering that nanoparticles can be very useful in dermal drug delivery if they are able to release drug at specific time intervals and at particular locations within the hair follicle, where the released drug can penetrate into follicle-surrounding tissue (Patzelt et al., 2017), it is of great importance to assess the amount of tacrolimus which can be found in hair follicles. Due to the fact that the trends observed for the penetration of drugs into porcine ear skin are highly representative for the in vivo situation on human skin (Klang et al., 2012) as well as an easier way of its supplying, the porcine ear skin was selected as a skin model in our study. The penetration profiles of tacrolimus 2 h upon corresponding samples of NLC, NE and Protopic® application are presented in Fig. 3. Having in mind the characteristics of ointments (greasiness, hard to wash off (Tan et al., 2012)), it could be expected that the first tape contains residuals of ointment which has not penetrated into the skin. However, we used standardized technique to remove residual formulations from the surface of the skin and it is observed that first tapes had relatively higher amounts of tacrolimus after the application of NE 8

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Fig. 4. Amount of tacrolimus (TAC) retrieved from stratum corneum (left) and hair follicles (right) after the application of nanostructured lipid carrier (PCLP 4:6 TAC), nanoemulsion (CLP 10 TAC) and the referent ointment (Protopic®); (mean ± SD, n = 3); * significantly higher (p < 0.05) compared to the referent ointment.

tacrolimus has permeated through the skin after 3 h and 6 h of application and no significant difference could be noticed between NLC, NE and the referent ointment. However, in the later sampling time points a superior permeation of tacrolimus was observed from the referent ointment compared to the developed formulations. Likewise, the highest permeation rate (177.46 ± 49.06 ng/cm2/h) was obtained for the referent ointment followed by NLC (39.87 ± 25.47 ng/cm2/h) and NE (8.02 ± 2.75 ng/cm2/h). These results are partially consistent with the results obtained by the in vitro release studies. Namely, comparing only nanocarriers, significantly higher permeation rate was observed for NLC, which also exhibited significantly higher release rate of tacrolimus compared to NE. However, comparing the referent ointment and the developed nanocarriers, permeation rate of tacrolimus was the highest using the referent ointment, although it manifested the slowest release of tacrolimus. The reason for this discrepancy could be ascribed to the fact that the synthetic membrane cannot reflex the complex interactions between skin and formulation (Ilic et al., 2018), especially for vastly different carriers as ointment and liquid nanodispersions. Furthermore, it is known that the skin occlusion enhances the percutaneous absorption, especially for very lipophilic substances (Hafeez and Maibach, 2013). Additionally, penetration of drugs increases with

matrix in NLC has promoted tacrolimus delivery into the hair follicles at the higher extent, without remarkable influence on its penetration into the stratum corneum when compared to the liquid oil core in nanoemulsion.

3.5. In vitro skin permeation of tacrolimus In order to evaluate the permeation of tacrolimus from the developed formulations, in vitro permeation studies were performed employing modified Franz diffusion cell, using full-thickness porcine ear skin as a barrier between donor and acceptor compartment. In general, in vitro permeation studies could provide information about the rate and the extent of drug delivery through the skin, as well as the potential for drug absorption into systemic circulation after topical application (OECD, 2004; SCCS, 2010; Sheshala et al., 2019). While transdermal formulations intend to deliver drug into system circulation, the targets for topical application of tacrolimus are T-lymphocytes in epidermis and dermis, while systemic absorption could lead to systemic adverse effects such as immunosuppression (Olson et al., 2014). The obtained permeation profiles of tacrolimus through porcine ear skin are shown in Fig. 5. It can be observed that a low amount of

Fig. 5. In vitro permeation of tacrolimus (TAC, left) through full-thickness porcine ear skin and skin deposition (right) after the application of nanostructured lipid carrier (PCLP 4:6 TAC), nanoemulsion (CLP 10 TAC) and the referent ointment (Protopic®); mean ± SD, n = 6, x significantly lower (p < 0.05) compared to the referent ointment. 9

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use conditions, should be conducted. Lastly, it is important to emphasize that obtained higher amount of permeated tacrolimus from the referent ointment could be regarded as a drawback, due to the higher risk of systemic absorption. Although minimal concentrations of tacrolimus were usually detected in the systemic circulation after topical application of ointment (Undre et al., 2009), there are few case studies which reported toxic levels of tacrolimus after application of ointment, especially during treatment under occlusion or treatment of large body surface (Olson et al., 2014). Having that in mind, an advantage could be given to the developed nanocarriers, which exhibited lower potential for systemic absorption, and higher amount of tacrolimus in stratum corneum and hair follicles, whence drug could slowly diffuse over time into the targeted skin layers (Lademann et al., 2007; Li and Ge, 2012; Vogt et al., 2016).

increasing duration of occlusion. Skin occlusion leads to increased skin hydration, while extended skin hydration (> 8h) causes a swelling of corneocytes, creating intercorneocyte ruptures, and causes microstructural changes in lipid self-assembly (Tan et al., 2010). Although all donor compartments were covered with Parafilm® in order to prevent an extensive evaporation of water from the formulations during 24 h, our study indicated that the referent ointment itself significantly contributed to the skin occlusion. Namely, after dismantling from the cells, all skin samples treated with the referent ointment appeared to be more swollen and hydrated compared to the samples treated with the developed formulations and untreated samples. This could be attributed to the presence of different types of paraffin and beeswax, which exhibit excellent occlusive properties and increase the hydration/swelling of stratum corneum making it more permeable for tacrolimus. It seems that this effect becomes more pronounced during the later part of the experiment since there were no significant differences in permeation of tacrolimus from all carriers after 3 h and 6 h of application. As mentioned before, comparing only developed nanocarriers, significantly higher permeation rate and higher amount of drug permeated after 20 h, 22 h and 24 h, is obtained using NLC. Since these formulations have the same amount of surfactants, similar particle sizes, pH and viscosity values, the possible reason for this could be the difference in their inner structure. Namely, Montenegro et al. (2017) have demonstrated the higher in vitro occlusive effect, as well as increased in vivo skin hydration after the application of NLC incorporated in gels compared to gels with NE. Having that in mind, the higher occlusive effect of PCLP 4:6 TAC compared to CLP 10 TAC, due to the presence of solid lipid matrix, could lead to increased skin hydration and enhanced permeation of drug. In addition, evaporation of water from NLC formulations could initiate the transition to higher ordered structure in lipid matrix, drug expulsion and increase in thermodynamic activity, influencing the increased penetration of drug into the skin (Müller et al., 2002). Our results were in line with findings of other researchers who observed the superiority of NLC compared to NE in dermal drug delivery (Li and Ge, 2012; Wolf et al., 2018b). Measuring the total amount of tacrolimus deposited in the skin after 24-hour in vitro skin permeation study, superiority of the referent ointment compared to NLC and NE (Fig. 5, right) is manifested. Although the permeation rate of tacrolimus was significantly higher from NLC compared to NE, the amount of deposited tacrolimus was slightly higher from NLC, however without statistical difference due to the high variation of obtained results. The results of in vitro penetration assessment by differential stripping are contradictory to the results obtained by in vitro permeation study. In our opinion, the main reason for this could be the difference in the exposure time (2 h vs. 24 h). In their research, Praça et al. (2018) stated that the physicochemical characteristics of the vehicle used in permeation studies can promote a reversible disruption of the skin layers and longtime used for in vitro skin permeation studies is able to promote higher degree of skin hydration and possible changes of this biological material reducing its barrier function. We hypothesize that the occlusion of skin in in vitro permeation study for a longer time compared to the differential stripping study led to the exaggerated skin occlusion by the ointment, altering its structure and allowing enhanced permeation of tacrolimus. In clinical setup, by using finite dose of ointment and shorter exposure time, this occlusion would be of minor importance. Furthermore, classical experimental set-up using Franztype diffusion cells may be influenced by interactions between the receptor medium containing ethanol and the model skin which may affect the barrier properties during the study (Sheshala et al., 2019). However, in our study ethanol had to be included in receptor medium in order to provide sink conditions for tacrolimus. Moreover, Klang et al. (2011) have concluded that the tape stripping experiments deliver more realistic data than permeation studies using Franz-type diffusion cell. In order to confirm the relevance of the obtained data, additional experiments utilizing finite dose setting, representative for common in-

4. Conclusion We have successfully developed novel lecithin-based NLC containing Precirol® ATO 5 and Capryol™ 90 as lipid phase with desirable physicochemical properties and stability. Factors such as solid/liquid lipid ratio and the total amount of lipid phase, homogenization pressure and cooling after the sample preparation have appeared as the critical formulation variables. Contrary, the amount of lecithin and polysorbate, as well as the number of homogenization cycles did not emerge as critical variables in our experimental setting. Comparable NE had slightly smaller droplet size, while other physicochemical characteristics were similar to the corresponding NLC. Nevertheless, the coalescence of oil droplets is noticed during the fourth month of storage, while NLC remained stabile until the end of the study period (six months). Tacrolimus was successfully incorporated in both NLC and NE in the targeted concentration, with high entrapment efficiency. Interestingly, the presence of tacrolimus did not significantly alter physicochemical properties and stability of developed carriers. Although different research groups have observed increased skin penetration or permeation of active ingredients after the application of NLC compared to NE, our studies failed to provide significant difference between these formulations for the delivery of tacrolimus in stratum corneum. However, increased accumulation in hair follicles and significantly higher permeation rate of tacrolimus from NLC compared to NE is observed. While it seems as the skin penetration of tacrolimus is increased by using NLC and NE compared to the referent ointment, in our experimental setting skin permeation data indicate superiority of ointment due to its pronounced occlusive effect, which could increase the risk of systemic absorption. Similar to what has been reported by Haque et al. (2017), we also conclude that potentially exaggerated values could be obtained in in vitro permeation studies due to the high occlusive effect of carrier itself, and that the difference in skin occlusion by carriers should be considered, especially in studies which utilize long exposure time and infinite dosage application. Because of the disagreement of the results obtained using different methodologies, further studies are necessary in order to clarify the clinical importance of developed carriers. However, having in mind better stability and skin performance of NLC than NE as well as a probably lower risk of systemic absorption it could be suggested that tacrolimus-loaded NLC has promising potential to be used as tacrolimus lotion, especially for the larger skin surfaces and hairy regions. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors would like to acknowledge the financial support from 10

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the Ministry of Education, Science and Technological Development, Republic of Serbia, through Project TR34031. The authors are grateful to Gattefosse for donating Capryol™ 90 and Precirol® ATO 5 ATO 5. The authors would like to thank Dr. Dominique Lunter and Mr Klaus Weyhing, Department of Pharmaceutical Technology at University of Tuebingen for their experimental assistance in laser diffraction measurements.

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