Formulation development of lipid nanoparticles: Improved lipid screening and development of tacrolimus loaded nanostructured lipid carriers (NLC)

Journal Pre-proofs Formulation development of lipid nanoparticles: improved lipid screening and development of tacrolimus loaded nanostructured lipid carriers (NLC) Anđelka B. Kovačević, Rainer H. Müller, Cornelia M. Keck PII: DOI: Reference:

S0378-5173(19)30963-9 https://doi.org/10.1016/j.ijpharm.2019.118918 IJP 118918

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

28 May 2019 24 November 2019 28 November 2019

Please cite this article as: A.B. Kovačević, R.H. Müller, C.M. Keck, Formulation development of lipid nanoparticles: improved lipid screening and development of tacrolimus loaded nanostructured lipid carriers (NLC), International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.118918

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Formulation development of lipid nanoparticles: improved lipid screening and development of tacrolimus loaded nanostructured lipid carriers (NLC)

Anđelka B. Kovačević1,2*, Rainer H. Müller1, Cornelia M. Keck3 1

Department of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Institute of Pharmacy,

Free University of Berlin, Kelchstraße 31, 12169 Berlin, Germany 2

Department of Pharmaceutical Technology, Institute of Pharmacy, Friedrich-Schiller

University Jena, Helmholtzeg 4, 07743 Jena, Germany 3

Institute of Pharmaceutical Technology and Biopharmacy, Faculty of Pharmacy, The Phillips

University of Marburg, Robert-Koch-Straβe 4, 35037 Marburg, Germany

Corresponding author: Anđelka B. Kovačević Department of Pharmaceutical Technology Institute of Pharmacy Friedrich-Schiller University Jena Helmholtzeg 4 07743 Jena Germany Phone: 0049 (0) 364 194 99 29 E-mail address: [email protected]; [email protected] (A. Kovačević) Abstract Lipid nanoparticles are well-known nanocarriers for improved drug delivery. Their formulation development typically involves three formulations steps. In the first part a 1

suitable lipid mixture which enables a high loading capacity and high encapsulation efficacy of the active needs to be identified (lipid screening). In the second step suitable stabilizers that enable the production of small-sized lipid nanoparticles with narrow size distribution and sufficient physical stability need to be identified (stabilizer screening, optimization of production parameters) and in the third step the biopharmaceutical efficacy needs to be evaluated. Based on the results obtained the formulations will require further optimization. The classical formulation development of lipid nanoparticles and especially the classical lipid screening is tedious. Therefore, in this study, a novel approach for the lipid screening that was based on the determination of the Hansen solubility parameters was evaluated and the results obtained were compared to the results from the classical model. Tacrolimus was used as a model drug. Results showed that both lipid screenings led to similar results, indicating that the new approach can be used for future developments. The optimized formulation was composed of a lipid matrix system that contained waxes, triglycerides and monoacylglycerols with various carbon chain lengths (C8, C10, C16, C18) and enabled an encapsulation efficiency of ~99%. The stabilizer screening showed that surfactants with high HLB values, lower molecular weight, and shorter alkyl chain length tended to form smaller particles with narrower size distribution and better physical stability. The most suitable surfactant was found to be a caprylyl/capryl glucoside (Plantacare® 810), a PEG-free stabilizer, that is extremely mild for atopic skin. It led to particle sizes of about 200 nm and a zeta potential well above │30│ mV. The optimized formulation contained 0.1% tacrolimus and possessed good physical stability. In conclusion, an optimized method for the selection of lipids that results in a limited number of experiments could be established and tacrolimus loaded lipid nanoparticles with similar drug load as a marketed formulation was successfully developed in this study.

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Keywords: tailor-made lipid nanoparticle, polyhydroxy surfactant, tacrolimus, Hansen solubility parameter, particle size

1. Introduction Tacrolimus monohydrate (USAN/INN name: tacrolimus) (TAC) (Fig. S1, Supplementary material) is a macrolide lactone that inhibits calcineurin phosphatase activity and the early expression of genes following T-cell stimulation. Marketed pharmaceutical preparation for the topical application of TAC (Protopic® 0.03%, 0.1% ointment) is recommended for moderate to severe atopic dermatitis in children (>2 years old) and adults. However, current usage of Protopic® ointment cannot be considered as safe due to variable and low dermal drug bioavailability and local adverse reactions (e.g., skin burning and itching with subsequent pain and redness) especially in the case of chronic therapy or large skin areas (Chong and Fonacier, 2015). Additionally, the occurrence of systemic adverse reactions was addressed in patients with markedly impaired skin barrier function (Nakagawa, 2002). In general, development of topical formulations with TAC is challenging due to unfavorable physicochemical properties like size (Mr = 804.02 g/mol), solubility in water (4-12 μg/ml) and permeability coefficient (-4.92) (https://www.drugbank.ca/drugs/DB00864; Potts and Guy, 1992) (Table 1). Although lipophilic (logP = 3.3), TAC is slightly soluble in solid and liquid lipids and prone to degradation in media with pH > 7 (Hane et al.., 1992; Skak and Hansen, 2016), which additionally complicates the development of topical formulations (Khan et al., 2016). Lipid nanoparticles were intensively investigated during the last twenty five years for improved and safer topical delivery of various actives, including TAC (Beloqui et al., 2016; Ding et al., 2015; Müller et al., 1997; Nam et al., 2011; Pople et al., 2011). A large number of in vitro and in vivo studies demonstrated prospective advantages of the lipid nanoparticles for 3

dermal application, including enhanced dermal drug penetration without skin barrier disruption, drug targeting to the skin with no systemic absorption, capacity for prolonged drug release, allowing less frequent administration (Chen-yu et al., 2012; Gomes et al., 2014; Gupta and Vyas, 2012; Vitorino et al., 2013; Vitorino et al., 2014). To use these benefits sufficient encapsulation efficiency must be ensured (Keck et al., 2014a). This could be realized using the complex lipid matrix with high solubility potential for the selected active. The particle matrices consisting of the mixture of solid and liquid lipid (NLC ®) or multiple solid and liquid lipids with various carbon chain lengths (smartLipids®) were developed in 1999 and 2014, respectively (Müller et al., 1999; Müller et al., 2015). In contrast to the solid lipid only, the lipid mixtures give special properties of the lipid nanoparticles such as increased drug loading capacity/encapsulation efficiency and improved physical stability. In order to get a suitable lipid nanoparticle matrix the first step is lipid screening, meaning the measurement of drug solubility in different lipids. However, the solubility screening of actives in various lipids is a time and resource consuming process, since there are numerous potential lipids that need to be investigated. Thereby, instead of that, it may be helpful to apply a theoretical approach based on mathematical calculations that provide information about drug-lipid miscibility at the molecular level. A concept of the solubility parameters was initially defined by Hildebrand and Scott (1950) and extended later on by Hansen (1967). It gained widespread attention in the pharmaceutical development (Benazzouz et al., 2014; Bordes et al., 2010; Forster et al., 2001; Hancock et al., 1997; Mohammad et al., 2011) but despite wide use, to the best of our knowledge, the concept of Hansen solubility parameters (HSP) has not yet been integrated into the lipid nanoparticles technology, i.e. lipid screening. In the second step of the formulation development of the lipid nanoparticles suitable stabilizers that enable the production of small sized particles with narrow size distribution and 4

sufficient physical stability need to be identified. We have recently reported that polyhydroxy surfactants tend to be widely recognized as an alternative for conventional polyethoxylated surfactants in the lipid nanoparticle formulations (Kovacevic et al., 2011; Kovačević et al., 2014; Keck et al., 2014b). The emerging use of polyhydroxy surfactants has been accentuated by their ability to: (i) enable formation and stabilization of the particles at lower concentration than polyethoxylated surfactants; (ii) facilitate the production of “tailor-made” lipid nanoparticles with optimized properties with respect to size, charge, and crystallinity; (iii) provide complimentary dermatological properties, especially in the case of dermal formulations for skin inflammation, such as atopic dermatitis; (iv) possess highly favorable environmental profile (rapid biodegradation and low toxicity) and (v) promote percutaneous drug penetration to the some extent without causing skin irritation (Ayala-Bravo et al., 2003; Balázs et al., 2015; Cázares-Delgadillo et al., 2005; Csizmazia et al., 2012; ElMeshad and Tadros, 2011; Kovacevic et al., 2011; Kovačević et al., 2014; Keck et al., 2014b; Tadros, 2005). From the therapeutic point of view, for pharmaceutical dermal products, it is important that the active pharmaceutical ingredient penetrate the skin, without being systemically absorbed. Among others, improved dermal uptake might be achieved by an increased contact surface of the carriers with the corneocytes and increased skin hydration (Schäfer-Korting et al., 2007). The small size ensures close contact with the stratum corneum and thus increases drug penetration into the skin. At the same time, with a decrease in the particle size densely packed lipid film onto the skin will be formed. This lipid film will reinforce a too thin natural lipid film of the stratum corneum, and repair damages, e.g. in the case of atopic dermatitis (Müller et al., 2014). Higher occlusion and skin hydration of the lipid nanoparticles can be achieved by using highly crystalline and very small particles (Wissing et al., 2001). However, the crystallinity of the particles should be finely tuned to avoid drug expulsion from the particles 5

over time. Therefore, the development of “optimal” lipid nanoparticle formulation concerning size and crystallinity relies on achieving a fine balance among mutually contradictory requests. Based on above considerations, the present study aimed to (i) create a suitable lipid matrix with high encapsulation efficiency for TAC and (ii) identify the most suitable surfactant to enable the production of physically stable TAC-loaded NLC with various sizes. For that, twenty lipids differing in the chemical structure and alkyl chain length (Table 2) were compared. Since there are currently no standard methods for the determination of the solubility of a drug molecule in the lipid excipients, in this study we used theoretical mathematical models complementary with the classical experimental approach. Secondly, hypothesizing that the differences in the size can confer distinct characteristics of the lipid nanoparticles such as different ability to penetrate the skin, we investigated the influence of five structurally different polyhydroxy surfactants (Table S1, Supplementary material) on the formation and characteristics of TAC-loaded NLC. [Insert Table 1 about here] [Insert Table 2 about here]

2. Materials and methods 2.1. Materials TAC monohydrate was purchased from Sandoz, Austria. Tables 2 and 3 list the lipids and surfactants used in the study. The purified water was obtained by reverse osmosis from a MilliQPlus, Millipore system (Schwalbach, Germany).

2.2. Methods 2.2.1. Lipid screening study 6

The next step in the development of lipid nanoparticle formulations includes the lipid screening study, i.e. determination of the drug solubility in different lipids. This is because the solubility of a compound in lipid media invariably influences the drug loading capacity, encapsulation efficiency, and subsequent usefulness of solid lipid drug carriers that may be produced therefrom (Müller et al., 1995; Müller et al., 2000; Jaspart et al. 2005). As expected, a higher loading capacity and encapsulation efficiency will be obtained if the drug is dissolved or solubilized in the melted lipid phase (Souto et al., 2007).

2.2.1.1. Theoretical lipid screening HSP were developed by Charles M. Hansen in 1967 as a way of predicting if one material will dissolve in another and form a solution (Hansen, 1967). They are based on the idea that like dissolves like where one molecule is defined as being 'like' another if it bonds to itself in a similar way. Specifically, each molecule is given three Hansen parameters, each generally measured in MPa0.5: partial HSP for dispersion (van der Waals) forces between molecules (δd), partial HSP for polarity (related to dipolar intermolecular force between molecules) (δp) and partial HSP for hydrogen bonding (δh). These three parameters can be treated as co-ordinates for a point in three dimensions also known as the Hansen space. The nearer two molecules are in this three-dimensional space, the more likely they are to dissolve into each other (Hansen, 2007). HSP are not available in the literature for TAC and all excipients used in the study. For the calculation of partial HSP δd, δp and δh, following equations were used: (1)

(2)

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(3) where Fdi, Fpi and Ehi refer to the specific functional group contributions: van der Waals dispersion forces, dipole interactions and hydrogen bonding of structural groups reported in the literature at 25°C and V is the molar volume of each structural group in the compound (van Krevelen and te Nijenhuis, 2009). The molecules are divided into smaller chemical groups and their partial solubility parameters were calculated using Fdi, Fpi, Ehi and V values. An example of the calculation of the solubility parameter is given for beeswax (For detailed information see Supplementary material). In the calculation of the solubility parameters for the other lipids, the same considerations was applied (Table S2, Supplementary material). Euclidean distance (D) (i.e., square root of the sums of the squares of the differences between the coordinates of the points in each dimension) between solute and solvent follows the classical “like dissolves like” rule, i.e., the smaller D is, the greater is the affinity between the compounds. It was calculated using Eq. 4 and the obtained values were used to predict interactions between TAC and lipid.

(4) where δd(solute), δp(solute) and δh(solute) are partial HSP of TAC and δd(solvent), δp(solvent) and δh(solvent) are partial HSP of lipids.

2.2.1.2. Classical lipid screening

8

The solubility of TAC in the lipids was determined experimentally. For that, 0.05 g of TAC was placed in a 40 ml glass vial, and 4.95 g of the lipid was added. In the case of liquid lipids (oil), the active was added into the oil and the mixture was stirred at 125 rpm at 80°C using an Innova 4230 Refrigerated Benchtop Incubator Shaker (New Brunswick Scientific, New Jersey, USA) during 1h. Oil solutions were cooled down to the room temperature and the solubility was checked afterward as well as after one day of storage (in the case of delayed crystallization). The classification of the solubility behavior was done visually and by light microscope (Orthoplan Leitz, Wetzlar, Germany). The lipid was considered as a good solvent if a clear solution was formed; while a lipid is denoted as a non-solvent if a two-phase system was obtained. In the case of solid lipids, the lipids were melted in glass vials, TAC was added and the mixture was stirred using the procedure described for liquid lipid. The drug-lipid mixture was cooled down and checked for the solubility. During the solidification glass vials were rotated by hand, to enable that the mixture solidifies in a thin layer on the vial wall and the lipid film was inspected for solubility. Additionally, the drop of the melted drug-lipid mixture was placed on a microscope slide, covered with a cover glass and pressed to make a thin film. After solidification, the mixture was inspected for the presence of drug crystals by light microscopy.

2.2.2. Chemical stability of TAC Since TAC has a large molecular structure and seems to be sensitive for high temperature during NLC preparation, chemical stability of TAC was initially assessed. For that, 4.95 g Imwitor® 308 was melted in glass vials, 0.05 g TAC was added and the mixture was stirred at 125 rpm and temperature of 75°C using an 206 Innova 4230 Refrigerated Benchtop Incubator Shaker (New Brunswick Scientific, New 207 Jersey, USA) over 1h, which is five time longer than the heat exposure during preparation of the lipid nanoparticles. The mixture was then 9

subjected to hot high pressure homogenization at temperature of 75°C, over ten homogenization cycles at the pressure of 500 bar. The concentration of TAC in drug lipid mixture before and after homogenization was determined by developed HPLC method. For HPLC analysis of drug-lipid mixture, solution of TAC in Imwitor® 308 was mixed with methanol HPLC grade at the final drug concentration of 10 µg/ml. TAC solution in methanol HPLC grade (10 µg/ml) was used for a comparison. For evaluation of the chemical stability of TAC HPLC system (Kroma System 2000, Kontron Instruments, Berlin, Germany) composed of an autosampler model 560, a pump system model 525 and a diode array detector model 540 (Kontron Instruments, Groß-Zimmern, Germany) was employed. This system was linked to Kroma System 2000 v. 1.70 data acquisition and process system, which also controlled the HPLC modules. 20 μl of the sample was injected onto a Nukleosil 100 C18 (5 μm) endcapped 250 mm × 4 mm column (Macherey-Nagel, Düren, Germany) which was kept at 50°C. The temperature during the analysis was controlled using a water bath Haake W90 (Haake, Karlsruhe, Germany). As a solvent for sample preparation methanol HPLC grade was used. The mobile phase, which was run with a flow rate of 1 ml/min, was a mixture of acetonitrile HPLC grade, MilliQwater and isopropanol HPLC grade in the ratio 70:28:2. The UV-spectrum was recorded at a wavelength of 210 nm. The calibration curve of TAC was linear (R2 = 0.99) within the concentration range 3–27 μg/ml. All samples were prepared in triplicate and mean values and standard deviation (SD) are given.

2.2.3. Thermal analysis of tacrolimus and bulk lipids The melting behavior of TAC, bulk lipids and lipid mixtures was investigated by Mettler DSC 821e apparatus (Mettler Toledo, Gieβen, Switzerland). TAC-free and TAC-loaded lipid mixtures were prepared by mixing of lipids and TAC at various ratios (Table 3). The mixtures 10

were stirred at 125 rpm using an Innova 4230 Refrigerated Benchtop Incubator Shaker during 1h at 80°C. Accurately weighted amounts of the lipids, TAC and lipid mixtures (1–2 mg) were placed in aluminum pans, cooled and scanned through a temperature range of 20–90°C (for bulk lipids and TAC-free lipid mixtures), i.e. from 20–150°C (for TAC and TAC-loaded lipid mixtures) at a heating rate of 10K/min, under constant flushing with nitrogen (80 ml/min). An empty aluminum pan was used as a reference. The instrument was calibrated for temperature and energy using indium standard.

[Insert Table 3 about here]

2.2.4. Preparation of tacrolimus-loaded NLC For the preparation of TAC-loaded NLC, lipid mixtures containing 1% TAC (TLM1–TLM3, Table 3) was melted at approximately 5–10°C above the melting point of the lipid (60–75°C, depending on the lipid mixture). The obtained melted phase was dispersed in an aqueous surfactant solution heated to the same temperature. Five polyhydroxy surfactants were employed to stabilize NLC as listed in Table 4. The dispersing was performed with highspeed stirring using an Ultra-Turrax (Janke & Kunkel, Staufen, Germany) for 30 seconds at 8000 rpm. The obtained pre-emulsion was subjected to the hot high pressure homogenization using a Micron LAB 40 (APV Deutschland GmbH, Unna, Germany). Five homogenization cycles at a pressure of 500 bar were applied. In order to prevent recrystallization of the lipid phase during homogenization, production temperature was kept 5–10°C above the lipid melting point. After homogenization, the obtained hot o/w nanoemulsions were filled in transparent glass vials, which were sealed immediately. The vials were put into a water bath adjusted to 20°C to control the cooling rate of the nanoemulsions and the velocity of

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crystallization. To find optimal process parameters, samples were withdrawn after the 1st, 3rd and 5th homogenization cycles and analyzed for particle size and size distribution.

[Insert Table 4 about here]

2.2.5. Characterization of tacrolimus loaded NLC 2.2.5.1. Particle size and zeta potential Particle size was determined by PCS, a dynamic particle size measurement technique, using a Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with a green laser. PCS yields the mean particle size and the polydispersity index (PI) as a measure of the width of the particle size distribution. The presence of particles in the micrometer range in the formulations was excluded by static light scattering (SLS), also known as laser diffractometry (LD) using a Malvern Mastersizer 2000 (Malvern Instruments, Malvern, UK). The volume weighted diameters d(v)0.10, d(v)0.50, d(v)0.90 and d(v)0.99 were used to characterize the dispersions. The zeta potential of lipid nanoparticles was determined via electrophoretic mobility measurements using a Zetasizer Nano ZS (Malvern Instruments, UK). Light microscopy (Orthoplan Leitz, Wetzlar, Germany) was used to detect the presence/absence of drug crystals and to confirm the results obtained by LD analysis (Keck and Müller, 2008; Keck, 2010). For detailed information, see Supplementary material.

2.2.5.2. Encapsulation efficiency The encapsulation efficiency (E.E.) of TAC in the NLC was determined by the ultrafiltration method. For that, 1,5 ml NLC dispersion was centrifuged at a rotation speed of 13000 rpm (Biofuge A, Heraeus, Hanau, Germany) for 12 h and aqueous supernatant was then filtered using Centrisart filters with a molecular weight cut-off of 300 000 Da (Sartorius, Göttingen, 12

Germany). The concentration of TAC in the NLC dispersion (total amount of TAC) and in the ultra-filtrate (the amount of free TAC) was analyzed using previously developed HPLC method (section 2.2.2). The E.E. was calculated using the following equation: EE(%)=

x 100

(5)

2.2.6. Physical stability In order to evaluate the physical stability, TAC-loaded NLC were stored at controlled room temperature (25±2°C) for one month, and the particle size was measured using PCS and LD.

3. Results and discussion 3.1. Lipid screening study 3.1.1. Theoretical lipid screening Twenty lipids, including waxes, partial glycerides, triglycerides, fatty acids and liquid lipids as presented in Table 2 were used for the theoretical determination of TAC solubility. The partial HSP (δd, δp, and δh) of TAC and lipids were calculated using the published values of the partial molar cohesive energies, designated as Fd, Fp or Eh resp., and molar volumes (V) of each structural group in the compound (van Krevelen and te Nijenhuis, 2009). The obtained values are visualized as coordinates in a 3D diagram, within which all three partial HSP could be represented at once (Fig. 1). In this way, any point within the cube represents the intersection of these three specific values. One can observe that the partial HSP are a function of the chemical structure of the examined components. Close HSP values reflect energetic similarity, i.e., a high degree of mutual miscibility between the components grouped into the small clusters. 13

[Insert Figure 1 about here]

TAC and lipids mainly differ in their polar (δp) and hydrogen bonding energies (δh), (Δδp from –2.28 to 2.55 MPa1/2; Δδh from –3.06 to 9.9 MPa1/2), whereas distinctions in dispersion forces (δd) are of smaller importance (Δδd from 0.84 to 2.27 MPa1/2) (Fig. 2). The smallest differences in the polar and hydrogen bonding energies (Δδp and Δδh) have been seen between TAC and monoglycerides (Imwitor® 308, Imwitor® 312, Imwitor® 900P). These lipids contain an alkyl chain (C8, C12 or C18) attached to the glycerol backbone by an ester bond. The two remaining carbons of the glycerol have active hydroxyl groups, giving polar characteristics to this portion of the molecule. The glycerol moiety may form hydrogen bonds and contribute to polar forces, including hydrogen bonding with OH groups of TAC. The solubility of TAC in monoglycerides was followed by liquid lipids (Miglyol® 812, Eutanol® G, Hemp seed oil), diglycerides (Compritol® 888 CG, Precirol® ATO), fatty acids (stearic acid, oleic acid) and triglycerides (Dynasan® 110, Dynasan® 114, Dynasan® 116, Dynasan® 118).

Among

triglycerides, the lowest Δδp and Δδh values were obtained for Dynasan® 110. Contrary, the highest Δδp, and Δδh values were obtained for waxes (Beeswax, Kahlwax® 6607L, SyncrowaxTM, Cutina® CP, Cetiol® MM) which implies again the lack of interaction of waxes and TAC. [Insert Figure 2 about here]

The euclidean distance, D in 3D space was used to reflect miscibility between drug and lipids (Fig. 3). According to the Hansen’s approach, the smaller the D value is, the better is the miscibility between two compounds (Vay et al., 2011). Again the smallest values were obtained between monoglycerides and TAC making them the most suitable excipients for TAC-loaded NLC. Monoglycerides were followed by diglycerides (Compritol® 888 CG and 14

Precirol® ATO with D values of 5.7 MPa and 6.2 MPa, resp.) and theoretically, they should also exhibit a good solubility potential for TAC. Among liquid lipids, Miglyol® 812, Hemp seed oil and Eutanol® G with D values of 5.4, 4.3 and 5.5 MPa, resp., were the closest to TAC which is an indication of their good miscibility.

[Insert Figure 3 about here]

3.1.2. Classical lipid screening The results of the lipid screening study are listed in Table 5. TAC is practically insoluble in waxes (Beeswax, Kahlwax® 6607L, SyncrowaxTM ERLC, Cutina® CP, Cetiol® MM), long chain triglycerides (Dynasan® 114, Dynasan® 116, Dynasan® 118), to a certain extent soluble in short chain triglycerides (Dynasan® 110, Miglyol® 812), fatty acids (stearic acid, oleic acid) and fatty alcohol (Eutanol® G), whereas partial glycerides (Imwitor® 308, Imwitor® 312, Imwitor® 900P, Precirol® ATO, Compritol® 888 CG) dissolved the highest amount of drug. Among partial glycerides, monoglycerides (Imwitor® 308, Imwitor® 312, Imwitor® 900P) possess superior solvent characteristics than diglycerides. For example, an experimentally determined solubility of TAC in Imwitor® 900P was ~30% (w/w) which is much higher than drug solubility in Precirol® ATO and Compritol® 888 CG (~2% (w/w)). The obtained data are in agreement with those reported by Pople and Singh (2011) and Thapa et al., (2013). Namely, TAC exhibits good solubility in glyceryl monooleate and can intercalate itself within the hydrophobic domain formed in glyceryl monooleate/oleic acid nanoparticles, resulting in high drug encapsulation efficiency. Among liquid lipids, almost identical solubility (~4% (w/w)) was obtained for oleic acid, Miglyol® 812 and Eutanol® G.

[Insert Table 5 about here] 15

In general, a good relationship between solubility data obtained experimentally and those calculated using HSP was obtained. This means that the data obtained using the theoretical model based are in a qualitative correlation with the data obtained in the experimental procedure. A certain discrepancy has been seen in the case of Hemp seed oil. The D value between oil and TAC was small (3.9 MPa) (Fig. 3) whereas the experimentally determined solubility pointed to the lack of drug-oil miscibility (Table 5). The observed inconsistency can be attributed to the variations in the composition of commercially available excipients and the presence of diverse compounds in Hemp seed oil (e.g. trace amounts of cannabidiol, oleic, stearic, palmitic acid, etc). Considering both the experimental and the theoretical approach, it was shown that mono- and diglycerides possessed the best solubility potential for TAC. Among them, Imwitor® 900P and Compritol® 888 CG were selected as solid lipids for further investigation because of their higher melting point (Table 2) (preferred for the preparation of the NLC). Since our efforts have been driven towards the development of the particle matrix consisting of lipids varying in the alkyl chain length, binary and ternary lipid mixtures were prepared and tested for TAC (Table 3). Cutina® CP (with C16 alkyl chain), Miglyol® 812 (having C8 and C10 fatty acids) as commonly used solid and liquid lipids in the preparation of the lipid nanoparticles, and Imwitor® 900P (with C18 fatty acids) with good solubility potential for TAC are considered as favorable excipients for the creation of ternary lipid matrix. Based on the results of preliminary experiments which showed that the excess of Imwitor® 900P might promote the lipid nanoparticles destabilization, Cutina® CP/Imwitor® 900P mass ratio of 9:1 was chosen to be initially tested. Additionally, miscibility between Cutina® CP and Imwitor® 900P in the selected mass ratio was confirmed and 10% Imwitor® 900P in the mixture with Cutina® CP was enough to enable sufficient TAC solubility (Kovačević, 2015; Kovacevic et al., 2015).

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3.2. Chemical stability of TAC For the production of TAC-loaded lipid nanoparticles the drug has been dissolved in the melted lipid mixture at 75ºC until an optically clear liquid was obtained. Since this high temperature might destroy sensitive compound, chemical stability of the drug need to be guaranted. In order to evaluate the chemical stability of TAC, HPLC analysis of pure drug in methanol and drug-lipid mixture before and after HPH has been performed. Since no loss in the drug content was detected (Table S3, Supplementary material), the result emphasizes that the drug is chemically stable under the applied production conditions.

3.3. Thermal analysis of tacrolimus and bulk lipids The crystalline structure of the lipids chosen in the previous part of the study, TAC and their physical mixture was analyzed, and the results are shown in Figs. 4 and 5. The thermoanalytical parameters as derived from the DSC measurements are given in Table 6.

[Insert Figure 4 about here]

The melting temperatures of bulk Imwitor® 900P, Compritol® 888 CG and Cutina® CP were found at expected values of 60.31°C, 71.80°C and 52.88°C, respectively (Doktorovova et al., 2011; Radtke, 2003; Saupe, 2004). The examination of the DSC thermograms of the binary mixture of Cutina® CP and Imwitor® 900P permits to observe the presence of two melting peaks belonging to Cutina® CP and Imwitor® 900P (Fig. 4). A small shift of the melting peaks of the binary mixture compared to the bulk lipid alongside with the decrease of the melting enthalpies indicates certain degree miscibility between Cutina® CP and Imwitor® 900P. The thermo-analytical parameters derived from the DSC curves of the lipid mixtures after oil addition were compared to those of the corresponding bulk lipids (Table 6). The broadening 17

of the melting peaks of both solid lipids (Imwitor® 900P and Compritol® 888 CG) as well as a binary mixture of Cutina® CP and Imwitor® 900P was observed, as postulated by an increase in the width of the melting event. This indicates that Miglyol® 812 is dissolved in the lipids and therefore the crystalline structure of the solid lipid is less pronounced (Saupe, 2004). It was interesting to note that after oil addition in a binary mixture of Cutina® CP and Imwitor® 900P melting peak corresponding to Imwitor® 900P disappeared, probably due to the dissolution of Imwitor® 900P in Miglyol® 812. Furthermore, a decrease in the melting points and melting enthalpies in both binary and ternary lipid mixtures compared to a single lipid only was detected, which can be attributed to the creation of lattice defects in the lipid matrices followed with a decrease in their crystallinity.

[Insert Table 6 about here]

Finally, as the main goal of this study was to develop TAC-loaded lipid mixtures, the ability of the previously selected lipid mixtures to dissolve TAC was studied. 1% (w/w) of TAC was dissolved in all mixtures. The results of thermal analysis of TAC-loaded lipid mixtures are summarized in Fig. 5. TAC revealed one endothermic peak with the extrapolated onset and melting temperatures at 124.91°C and 130.18°C, and melting enthalpy of 1031.12 J/g. The drug had high purity, and its range between melting and onset temperatures was 5.27°C. Since TAC has a crystalline character a melting event should have been detected in the lipid mixtures if the drug was not soluble. DSC study of TAC-loaded lipid mixtures revealed only the melting events of the lipid mixtures, whereas the melting peak of TAC is absent, which provides evidence that crystalline drug is dissolved and embedded in all lipid mixtures. This interpretation is supported by the visual observation and light microscopy, i.e. the absence of drug crystals in the sample. 18

[Insert Figure 5 about here]

3.4. Preparation of tacrolimus loaded NLC The next part of the study was devoted to the identification of the stabilizers which will enable the creation of the particles with tuneable size, satisfactory physical stability, and skin tolerability. As previously mentioned, local adverse reactions (e.g., skin burning, itching, pain, redness) are the drawbacks of the marketed TAC formulation. One way to increase skin compatibility of the lipid nanoparticles is to use "skin friendly" surfactants.

for interaction with the stratum corneum lipids. This ultimately reduces the probability of the skin barrier function damage. Non-ionic surfactants, represents for a long time the surfactant of choice, having the lower potential for skin irritation compared to anionic and cationic ones. The most often used nonionic surfactants for dermal lipid nanoparticles are polyethoxylated one (e.g. Polysorbates). However, the literature data document that Polysorbates are susceptible to oxidation by atmospheric oxygen during storage and handling at room temperature, whereby the oxidation products formed can act as skin irritants, especially in sensitized patients (Bergh et al., 1997; Karlberg et al., 2003). Today, nonionic PEG-free, polyhydroxy surfactants represent a competitive, trend-setting innovation and a new concept in dermal formulation for skin inflammation compared to PEGbased surfactants. In our previous studies a detailed analysis of the preparation, stabilization mechanism and structural characteristics of the lipid nanoparticles (SLN, NLC) stabilized with these surfactants was presented (Keck et al., 2013a,b; Kovacevic et al., 2009; Kovacevic et al., 2011). To extend these studies, in this study we have investigated the influence of five

19

structurally different polyhydroxy surfactants on size and physical stability of TAC-loaded NLC. Hot high pressure homogenization which is currently the simplest and the most developed technique for large scale production was used (Müller et al., 2008a). Since the homogenization process requires the optimization of production conditions, a various number of homogenization cycles at constant pressure was applied. The profiles of the particle size and PI decrease with an increasing number of the homogenization cycles were similar for all samples and corresponded to the typical profiles that can be found in the literature. Hence, exemplarily for the size decrease and narrowing of the size distribution relating to the number of applied homogenization cycle one figure is presented (Fig. 6a). After only one cycle, particles in the nanosize range (185 – 320 nm) were obtained in all samples. The smallest particles were obtained with alkyl polyglucoside surfactant (Plantacare® 810). Alkyl polyglucosides have better solubility in water (at the temperature of 75 °C) and lower molecular weight than other surfactants. It can be assumed that lower molecular weight surfactant having good aqueous solubility resulted in the faster diffusion to the new surfaces. Hence, after the first cycle, the surfactants with the highest HLB values and lower molecular weight exhibited the best functionality in the stabilization of the NLC. The particles in the lower nanosize range have better chances to adhere to the skin as a result of a large surface/volume ratio and their dispersions are more kinetically stable (Jores et al., 2004). Therefore, formulation development was continued, whereby a decrease in the particle size and PI was observed with an increasing number of the cycles at the constant pressure. However, the most pronounced decrease (sharp drop) in z-ave has occurred between the first and third cycle. Only very tiny changes in z-ave can be seen when moving from cycle three to five. The classic interpretation of the almost unchanged z-ave after cycle three can be explained with the reaching so-called “plateau” in the diameter of the bulk particles. Between 20

the first and third homogenization cycle, the energy in the system is used to decrease the particle size in the formulation. After cycle three, only reduction in the particle diameter of the bulk population, and partly decrease in the size of larger particles was observed. Although the average particle size practically did not change between the third and fifth cycles, the homogenization process was proceeded to see the changes in the homogeneity of the dispersion, i.e., decrease in PI. PCS data have confirmed no changes in PI between the third and fifth homogenization cycles. However, as it is well-known PCS can give misleading results because the large particle fractions are not “seen” by this technique (Keck, 2010; Souto, 2005). Therefore, to prove the presence/absence of larger particles and to distinguish nano- from microparticles, LD was used (Fig. 6b). Decrease in the volume diameters is very similar to the decrease in z-ave and PI, i.e., a reduction in the volume diameters by introducing the energy in the system was seen until they reached a “plateau.” In all investigated formulations, between the first and the third cycle, the volume diameters (d(v)0.10 – d(v)0.99) decreased in a linear fashion. With a further increase in the number of the homogenization cycles, the decrease of the volume diameters became exponential. Based on the results obtained it can be concluded that the maximum dispersibility of the bulk population (i.e. the minimum value of d(v)0.50) was reached after cycle three. Further input of energy into the system only slightly affected the decrease in the volume diameter of the bulk population. This result agreed with the result of the PCS study. In contrast to the volume diameter of the bulk population, which stayed practically unchanged between the third and fifth cycle, slight decreases in d(v)0.90 – d(v)0.99 were observed. This can be explained by a kind of two-step diminution process in the homogenizer. In the first step, most of the particles (bulk population) reached their maximum dispersibility relatively fast. In the second step, a little effect on the mean diameter of the bulk population could be seen, but the width of the distribution by eliminating remaining few large particles was further reduced. Therefore, even 21

the mean diameter of the bulk population had reached its minimum and stayed constant, additional cycles are recommended to improve the uniformity of the final formulations. Based on the results obtained, we can conclude that polyhydroxy surfactants at the concentration of 3 % (w/w)) exhibited good functionality in the preparation of NLC dispersions with small size and narrow size distribution. The optimal conditions for preparing dispersions were five homogenization cycles, at the pressure of 500 bar and the temperature of 75 °C.

[Insert Figure 6 about here]

3.5. Characterization of tacrolimus loaded NLC 3.5.1. Particle size and zeta potential As it is shown in Table 7, after fifth cycle z-ave in all dispersions was lower than 300 nm, but the size distribution changed from unimodal (Plantacare® 810, Plantacare® 2000, Plantacare® 1200, Plurol Stearique® WL 1009) to bimodal (Surfhope® C-1815). In the case of bimodal size distribution, z-ave was not considered as a reliable parameter because it does not have the physical meaning (Jillavenkatesa et al., 2001). Therefore, LD was applied as an additional characterization method. Volume diameters (d(v)0.10–d(v)0.99) were corroborated with PCS diameters and aggregation was observed in dispersions stabilized with Surfhope® C-1815 and Plurol Stearique® WL 1009 (Table 7). It is evident that after production Plantacare® 810 Plantacare® 1200 and Plantacare® 2000 showed the best potential in the stabilization, followed by Plurol Stearique® WL 1009, while the particle size in the presence of Surfhope® C-1815 was the highest. These results correlate well with previous observations that the nanoparticle size decreases as the alkyl chain length of nonionic surfactants decreases (Anarjan and Tan, 2013; Cheong et al., 2010; Tan and Nakajima, 2005; Yuan et al., 2008). In the study of Das et al., (2014) was also suggested that between the sugar esters with different 22

alkyl chain length, those with shorter alkyl chain led to the creation of the particles with smaller size and lower PI, presumably due to their more compact structure, resulting in better package on the nanoparticle surface. The smaller size of the hydrophilic moiety and the shorter alkyl chain in the molecule of Plantacare® 810 (caprylyl/capryl glucoside) are also responsible for its high critical micelle concentration (CMC = 20–25 mM) (Hill et al., 1997). Having in mind that a higher CMC of the surfactants decreases the formation of micelles and that the surfactant monomers adsorb to the particle surfaces rather than the micelles, we also assumed that the low number of monomers in the case of Plantacare® 810 might explains its best functionality in the stabilization of the NLC.

[Insert Table 7 about here]

The analysis of the functionality of the surfactants in our study also revealed that the changes in the particle size cannot be related to the HLB values of the surfactants. The order of HLB values of the investigated surfactants was Plantacare® 810 > Surfhope® C-1815 > Plantacare® 2000 > Plantacare® 1200 > Plurol Stearique® WL 1009, while the particle size increased in the following way Plantacare® 810 > Plantacare® 2000 > Plantacare® 1200 > Plurol Stearique® WL 1009 > Surfhope® C-1815. Namely, Plantacare® 810 showed superior performance in the particle stabilization than Surfhope® C-1815 despite the same HLB value (Table S1, Supplementary material and Table 7). The effect of the type of the lipid mixture on the particle size and size distribution is shown in Fig. 7. The large particle diameters (d(v)0.90–d(v)0.99) of the dispersions prepared with the TLM1 lipid mixture were well above 1 μm. The formulation containing TLM2 lipid mixture also did not meet the limit set for the lipid nanoparticles. Although the mean particle size was 208 nm, the size distribution was broad (PI>0.25) and more than 50% of particles had a 23

diameter higher than 1 μm. The obtained results could be explained by the insufficient ability of Imwitor® 900P and Compritol® 888 CG to create NLC in combination with the hydrophilic polyhydroxy surfactant. The stabilizing properties depend obviously on the specific interactions of the lipid mixture with the surfactant, e.g. anchoring of the stabilizer on the lipid surface of the particles.

[Insert Figure 7 about here]

The zeta potential of Plantacare® stabilized dispersions was surprisingly high for non-ionic surfactants, being in the range between −45 mV and -50 mV, indicating well-charged particle surface (Table 8). Nevertheless, Plantacare® is non-ionic surfactants and thus their main mechanism of stabilization is steric stabilization. In this case, the stability of the lipid nanoparticles is indicated by zeta potential values higher than │20│ mV and therefore the size of TAC-loaded NLC is expected to stay unchanged over time (Müller, 1996; Riddick, 1968). The observation is in agreement with the data obtained from particle size analysis. Surfhope® C-1815 and Plurol Stearique® WL 1009 stabilized particles possess the same composition as Plantacare® stabilized particles (TLM3), but lower zeta potential (Table 8). This lower zeta potential obviously results from a different type of stabilizer. Moreover, the surfactants can influence the crystalline structure of the lipid (Bunjes et al., 2003; Han et al., 2008; Siekmann and Westesen, 1994). As different crystalline structures may possess different charge densities, changes in the surface charges of the particles (Nernst potential) and thus in the measured zeta potential might occur with different surfactants. Despite the fact that the zeta potential of Surfhope® C-1815 and Plurol Stearique® WL 1009 stabilized particles was in the range of stable dispersions destabilizing effects were observed in these

24

formulations. Obviously, some changes in the composition of the particle matrix/surfactantlipid interaction and related surface composition and charge took place over time.

[Insert Table 8 about here]

3.5.2. Encapsulation efficiency The E.E. of TAC in the selected formulation (F3) calculated using Eq. (5) was around 99% at the day of production. Moreover, drug crystals could not be observed by light microscopy (Fig. 8.), indicating incorporation of TAC in the particles. This high encapsulation efficiency can be ascribed to Imwitor® 900P affording drug solubility. LogP of TAC is 3.96±0.84 signifying its high solubility in the lipids. The results obtained were in good agreement with previous findings where high encapsulation efficiency and drug loading were obtained for Imwitor® 900P based SLNs of highly lipophilic drugs cyclosporine A (Müller et al., 2008b). Additionally, mixing of the lipids with different chain length (C8, C10, C16, C18) presumably lead to a massive crystal order disturbance, providing enough space for drug accommodation and leading to a high E.E. So far, several studies were focused on the development of TAC loaded lipid nanoparticles (Dantas et al., 2018; Jain et al., 2019; Kang et al., 2019; Pople and Singh, 2010). These formulations were composed of solid lipid only or binary mixture of solid and liquid lipid. It is expected that the mixing of the lipids with different chain length (C8, C10, C16, C18) will lead to a massive crystal order disturbance, providing enough space for drug accommodation and leading to a high E.E. Due to the very different spatial forms of the various lipid molecules, polymorphic transitions during storage are minimized or can be completely avoided (Ruick, 2016). Moreover, the theoretical approach used for the selection of lipid

25

excipients opens the perspective for a prediction of the most suitable lipids for the particle matrix of NLC, reducing experimental work and accelerating development.

[Insert Figure 8 about here]

3.6. Physical stability of tacrolimus loaded NLC The physical stability of TAC-loaded NLC was assessed after storage of the formulations at 25°C±2°C for 30 days. While an increase of the particle size was seen in dispersions stabilized with Surfhope® C-1815 and Plurol Stearique® WL 1009, particle growth of the Plantacare® stabilized dispersions did not occur (Table 6). Several researchers pointed out (Bunjes and Koch, 2005; Bunjes et al., 2007; Helgason et al., 2009; Salminen et al., 2013) that the particle size is highly dependent on how the surfactants control the crystallization process of the particles during preparation and storage. Surfhope® C-1815 and Plurol Stearique® WL 1009 possess one i.e. two long rigid alkyl chain in the structure (“solid lipid” tail surfactants) and higher melting points than lipid particle matrix. Contrary, Plantacare® have one short mobile alkyl chain (“liquid lipid” tail surfactant) and notably lower melting point than the lipid matrix. As shown in our former study (Kovačević et al., 2014) the process of intercalation of the surfactant in the lipid matrix and the change in the crystallinity is generally suppressed in the case of “liquid lipid” tail surfactants due to the formation of fluid and “loose” layer around the particles. Contrary, the changes in the crystallinity of the particles are more likely for Plurol Stearique® WL 1009 and Surfhope® C-1815, due to the similarity of the alkyl chain length of the surfactants with the alkyl chain length of the lipid matrix and promoted penetration of the surfactants into the particle matrix. In line with previous observations, this can promote increased crystallinity of the particles and thus lead to physical instability (Freitas and Müller, 1999; Kovačević et al., 2014). 26

4. Conclusions TAC-loaded NLC for dermal delivery were developed. For drug-lipid miscibility/solubility determination, the theoretical model based on HSP provided a good fit with the experimental procedure. This allows extracting the maximum amount of information using mathematical calculations resulting in a limited number of experiments and opening the perspective for accelerated development. Besides drug-lipid miscibility/solubility, the composition of the lipid matrix and the properties of the surfactants had a significant impact on the particle size. The ternary lipid mixture comprising Imwitor® 900P/Cutina® CP/Miglyol® 812 revealed superior performance for TAC incorporation compared to the binary mixture Imwitor® 900P/Miglyol® 812 and Compritol® 888 CG/Miglyol® 812. Analysis of the functionality of nonionic polyhydroxy surfactants revealed that the stabilizer with the shortest alkyl chain length, the highest CMC and the lowest molecular weight (Plantacare® 810) provided the most optimal properties for particle stabilization. Mean particle size of around 120 nm with narrow and unimodal size distribution was obtained and the system was physically stable at 25°C for 30 days. Investigations in this study indicated that polyhydroxy surfactants obtained from natural sources would result in the new archive of excipients for stabilization of NLC. This may encourage further research on polyhydroxy surfactant stabilized NLC with tailormade particle matrix structure as prospective carriers for delivery of drugs to the skin not only for pharmaceutical purposes but also for consumer care products. Acknowledgements Anđelka Kovačević acknowledges the financial support obtained from Deutscher Akademischer Austausch Dienst (DAAD). The authors also would like to thank Ms. Corinna Schmidt and Ms. Inge Volz for technical assistance. Appendix A. Supplementary data

27

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Nakagawa, H., 2002. Treatment of atopic dermatitis with immunomodulatory drugs. JMAJ 45 (11), 477–482. Nam, S.H., Ji, X.Y., Park, J.S., 2011. Investigation of tacrolimus loaded nanostructured lipid carriers for topical drug delivery. Bull. Korean Chem. Soc. 32(3), 956–960. Pople, P.V., Singh, K.K., 2010. Targeting tacrolimus to deeper layers of skin with improved safety for treatment of atopic dermatitis. Int. J. Pharm. 398, 165–178. Pople, P.V., Singh, K.K., 2011. Development and evaluation of colloidal modified nanolipid carrier: Application to topical delivery of tacrolimus. Eur. J. Pharm. Biopharm. 79 (1), 83–94. Potts, R.O., Guy, R.H., 1992. Predicting skin permeability. Pharm. Res. 9 (5), 663–669. Radtke, M., 2003. Nanostructured lipid carriers (NLC): Untersuchungen zur Struktur, Wirkstoffinkorporation und Stabilität. PhD thesis, Freie Universität Berlin, Berlin, Germany. Riddick, T.M., 1968. Zeta-Meter Manual. Zeta-Meter Inc, New York. Ruick, R., 2016. smartLipids - die neue Generation der Lipidnanopartikel nach SLN und NLC, Ph.D. Thesis, Free University of Berlin, Germany Salminen, H., Helgason, T., Kristinsson, B., Kristbergsson, K., Weiss, J., 2013. Formation of solid shell nanoparticles with liquid ω-3 fatty acid core. Food Chem. 141 (3), 2934–2943. Saupe, A., 2004. Pharmazeutisch-kosmetische Anwendungen Nanostrukturierter Lipidcarrier (NLC): Lichtschutz und Pflege. PhD thesis, Freie Universität Berlin, Berlin, Germany. Schäfer-Korting, M., Mehnert, W., Korting, H.C., 2007. Lipid nanoparticles for improved topical application of drugs for skin diseases. Adv. Drug Deliv. Rev. 59, 427–443. Siekmann, B., Westesen, K., 1994. Thermoanalysis of the recrystallization process of melthomogenized glyceride nanoparticles. Colloids Surf B Biointerfaces. 3 (3), 159–175. Skak, N., Hansen, L., 2010. Stabilized tacrolimus composition. European patent 2575769 B1. Souto, E.B., 2005. SLN and NLC for topical delivery of antifungals. PhD thesis, Free University of Berlin, Germany. 35

Souto, E.B, Almeida, A.J., Müller, R.H., 2007. Lipid nanoparticles (SLN, NLC) for cutaneous drug delivery: structure, protection & skin effects. J Biomed Nanotechnol 3(4), 317–331 Tadros, T.F., 2005. Applied surfactants – principles and applications. Wiley VHC, Weinheim, Germany. Tan, C.P., Nakajima, M., 2005. Effect of polyglycerol esters of fatty acids on physicochemical properties and stability of β-carotene nanodispersions prepared by emulsification/evaporation method. J. Sci. Food Agr. 85 (1), 121–126. Thapa, R.K., Baskaran, R., Madheswaran, T., Rhyu, J.Y., Kim, J.O., Yong, C.S., Yoo, B.K., 2013. Effect of saturated fatty acids on tacrolimus-loaded liquid crystalline nanoparticles. J. Drug Del. Sci. Tech. 23 (2), 137–141. Van Krevelen, D.W., Te Nijenhuis, K., 2009. Cohesive Properties and Solubility. In: Van Krevelen, D.W., Te Nijenhuis, K.T. (Ed.), Properties of Polymers, 4th ed., Elsevier, Amsterdam, Netherland, pp. 189−227. Vay, K., Scheler, S., Frieβ, W., 2011. Application of Hansen solubility parameters for understanding and prediction of drug distribution in microspheres. Int. J. Pharm. 416 (1), 202–209. Vitorino, C., Almeida. J., Gonçalves, L.M., Almeida, A.J., Sousa, J.J., Pais, A.A., 2013. Coencapsulating nanostructured lipid carriers for transdermal application: from experimental design to the molecular detail. J. Control. Release 167 (3), 301–314. Vitorino, C., Almeida, A., Sousa, J., Lamarche, I., Gobin, P., Marchand, S., Couet, W., Olivier, J.C., Pais, A., 2014. Passive and active strategies for transdermal delivery using coencapsulating nanostructured lipid carriers: in vitro vs. in vivo studies. Eur. J. Pharm. Biopharm. 86 (2),133–144. Wissing, S.A., Lippacher, A., Müller, R., 2001. Investigations on the occlusive properties of solid lipid nanoparticles (SLN). J. Cosmet. Sci. 52, 313–324. 36

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37

Table captions Table 1. Molecular mass, solubility in water, logP and logKp of TAC monohydrate Table 2. Overview of physicochemical properties of lipid excipients Table 3. Composition of TAC-free and TAC-loaded lipid mixtures Table 4. Composition of TAC-loaded NLC formulations Table 5. The solubility of TAC (1%) in various lipids (L1−L20) [(+) dissolved, (−) not dissolved] Table 6. Thermoanalytical parameters derived from DSC curves for bulk lipid excipients and TAC-free lipid mixtures (onset temperature (To [°C]), melting temperature (Tm [°C]), melting enthalpy (ΔH [J/g]) and width of the melting event (WME [°C])) Table 7. Effect of the surfactant type on the particle size and size distribution of TAC-loaded NLC (PCD and LD data after production and after 1 month) Table 8. The values of zeta potential of TAC-loaded NLC (F3-F7)

38

Figure captions Fig. 1. Partial Hansen solubility parameters (δd, δp, δh) of TAC and lipids Fig. 2. Differences in partial Hansen solubility parameters (Δδd, Δδp, Δδh) between TAC and lipids Fig. 3. Euclidean distance (D) between TAC and lipids Fig. 4. DSC profiles of bulk lipids and binary lipid mixtures Fig. 5. DSC profiles of TAC and TAC-loaded lipid mixtures Fig. 6. Decrease in the particle size as a function of number of homogenization cycles for formulation TLN3 (pressure = 500 bar, temperature = 65°C) (PCS and LD data) Fig. 7. The effect of the lipid matrix type on the particle size and size distribution of TACloaded NLC (PCS-data (left) and LD-data (right) after production) Fig. 8. Photomicroraphs of TAC (pure drug) and optimized TAC loaded formulation (F3) after preparation and after one month (bar 10 μm): a) TAC (magnification, 160x); b) TAC (magnification 630x); c) F3 after preparation (magnification 630x); d) F3 after one month (magnification 630x).

39

Table 1

a

Molecular mass (g/mol)

Solubility in water (mg/ml)

Log (octanol-water partition coefficient) (logP)

Log of permeability coefficient (logKp)

804.02

0.00402

3.30

-4.92a

Trade name/ Supplier

Compound

as predicted according to Potts and Guy (1992)

Table 2 Lipid excipients Waxes

Code L1

Cera alba1 Triacontanyl palmitate (main component)2

Beeswax, white/ Roth, USA

40

Melting point [°C]

Properties at 25°C

62 ̶ 65

White or yellowish white solid

Monoglycerides

Triglycerides

Triglycerides

L2

Kahlwax® 6607L/ Kahl GmbH & Co. KG, Germany

Helianthus Annus (Sunflower Seed Wax)1 C42 ̶C60 esters2

74 ̶ 80

Pale hard, crystalline wax mass

L3

SyncrowaxTM ERLC/ Croda GmbH, Germany

C18–C36 Acid Glycol Ester1

70 ̶75

Crystalline wax

L4

Cutina® CP/ Cognis, Germany (now BASF, Germany)

Cetyl Palmitate1

46 ̶ 51

White coarse pellets or flakes

L5

Cetiol® MM/ Cognis, Germany (now BASF, Germany)

Myristil myristate

38 ̶ 42

White to light yellowish pelletized wax mass

L6

Imwitor® 308/ Sasol GmbH, Germany

Glyceryl Caprylate1

30 ̶ 34

White solid crystalline mass

L7

Imwitor® 312/ Sasol GmbH, Germany

Glyceryl Laurate1

~60

White solid mass

L8

Imwitor® 900 P/ Sasol GmbH, Germany

Glyceryl Stearate1

54 ̶ 64

L9

Dynasan® 110/ Sasol GmbH, Germany

Tricaprin1

31 ̶ 32

L10

Dynasan® 114/ Sasol GmbH, Germany

Trimyristin1

55 ̶ 58

White powder with slight odor

L11

Dynasan® 116/ Sasol GmbH, Germany

Tripalmitin1

61 ̶ 65

White fluffy powder

41

1

White to slightly yellow waxy flakes or powders Off-white, flaky, crystal mass or white powder

L12

Dynasan® 118/ Sasol GmbH, Germany

Tristearin1

70 ̶ 73

White powder

L13

Precirol® ATO/ Gattefossé, France

Glyceryl Palmitostearate1

50 ̶ 60

Off-white powder with slight odor

L14

Compritol® 888 CG/ Gattefossé, France

Glyceryl Behenate1

~70

Whitish powder

L15

Stearic acid/ Merck KGaA, Germany

Stearic acid1

68 ̶ 70

L16

Oleic acid/ Sigma Aldrich, Germany

Oleic Acid1

–

L17

Miglyol® 812/ Caelo GmbH, Germany

Caprylic/Capryc Triglyceride1 ̶

White or almost white waxy flaky crystals Colorless to very faint clear yellow viscous liquid Slightly yellowish, oily liquid

L18

Cetiol® V/ Cognis Germany (now BASF, Germany)

Decyl Oleate1

–

Light yellow liquid

L19

Eutanol® G/ Cognis, Germany (now BASF, Germany)

Octyldodecanol1 ̶

Clear light yellow oily liquid

L20

Hemp seed oil/ Gustav Hees GmbH, Germany

Diglycerides

Fatty acids

Liquid lipids

1

Hemp (Cannabis Sativa) Seed Oil1 Linoleic acid and α-linolenic acid2 (main components)

INCI name; 2Chemical description specified by the manufacturer

42

Colorless liquid, insoluble in water

Table 3 Compound (w/w, %) TAC

TAC-free lipid mixture LM1 LM2 LM3 -

Imwitor® 8.0 900P Cutina® CP Compritol® 888 CG Miglyol® 2.0 812 LM: lipid mixture

TAC-loaded lipid mixture TLM1 TLM2 TLM3

-

-

0.1

0.1

0.1

-

1.0

7.9

-

1.0

-

8.0

-

-

7.9

8.0

-

-

7.9

-

2.0

1.0

2.0

2.0

1.0

43

Table 4 Formulation

Compound (w/w, %)

F1

F2

F3

F4

F5

F6

F7

Tacrolimus

0.1

0.1

0.1

0.1

0.1

0.1

0.1

Imwitor® 900P

7.9

-

1.0

1.0

1.0

1.0

1.0

Cutina® CP

-

-

7.9

7.9

7.9

7.9

7.9

Compritol® 888 CG

-

7.9

-

-

-

-

-

Miglyol® 812

2.0

2.0

1.0

1.0

1.0

1.0

1.0

Plantacare® 810

3.0

3.0

3.0

-

-

-

-

Plantacare® 2000

-

-

-

3.0

-

-

-

Plantacare® 1200

-

-

-

-

3.0

-

-

Surfhope® C-1815

-

-

-

-

-

3.0

-

Plurol Stearique® WL 1009

-

-

-

-

-

-

3.0

100

100

100

100

100

100

100

MiliQwater

ad

44

Table 5 Lipids Time (min)

Waxes L1

L2

L3

Monoglycerides L4

L5

L6

L7

L8

Triglycerides L9

Diglycerides Fatty acids

Liquid lipids

L10

L11

L12

L13

L14

L15

L16

L17

L18

L19

L20

15

–

–

–

–

–

+

+

+

–

–

–

–

–

–

–

–

–

–

–

–

30

–

–

–

–

–

+

+

+

–

–

–

–

–

–

–

–

–

–

–

–

45

–

–

–

–

–

+

+

+

–

–

–

–

+

+

–

+

+

–

+

–

60

–

–

–

–

–

+

+

+

+

–

–

–

+

+

+

+

+

–

+

–

45

Table 6 Compound

To [°C]

Tm [°C]

ΔH [J/g]

WME [°C]

Imwitor® 900P

53.18

60.31

192.33

7.13

Compritol® 888 CG

69.40

71.80

120.58

2.40

Cutina® CP (main peak)

48.92

52.88

222.05

3.96

Cutina® CP + Imwitor® 900P

43.09

49.75

186.99

6.66

LM1 (Imwitor® 900P + Miglyol® 812)

51.37

58.82

157.75

7.45

LM2 (Compritol® 888 CG + Miglyol® 812)

62.92

68.00

91.89

5.08

LM3 (Cutina® CP + Imwitor® 900P + Miglyol® 812)

42.55

50.11

136.90

7.56

LM: lipid mixture

46

Table 7 Storage time

Formulation Surfactant code

Day 1 F3 Day 30 Day 1 F4 Day 30 Day 1 F5 Day 30 Day 1 F6 Day 30

PCS data z-ave (nm)

LD data d(v)0.10 d(v)0.50 d(v)0.90 d(v)0.95 d(v)0.99 (µm) (µm) (µm) (µm) (µm)

PI

Plantacare® 810 (caprylyl/capryl glucoside)

120±2

0.145±0.029 0.090

0.136

0.198

0.217

0.252

126±2

0.154±0.035 0.087

0.136

0.203

0.224

0.262

Plantacare® 2000 (decyl glucoside)

126±7

0.086±0.030 0.090

0.140

0.201

0.223

0.260

133±4

0.101±0.032 0.091

0.140

0.207

0.231

0.269

Plantacare® 1200 (lauryl glucoside)

142±3

0.079±0.036 0.091

0.151

0.213

0.239

0.317

151±4

0.117±0.046 0.093

0.150

0.218

0.242

0.321

Surfhope® C-1815 (sucrose stearate)

277±17

0.077±0.075 0.094

0.161

25.430

39.176

98.556

0.178

0.232

0.260

0.316

0.172

36.866

56.580

110.186

n.a.*

Plurol Stearique® 151±2 0.139±0.124 0.092 WL1009 F7 (polyglyceryl-6 Day 30 181±7 0.241±0.045 0.094 distearate) *the sample was like cream and the measurements were not performed Day 1

47

Table 8 Formulation code

Surfactant

Zeta potential (mV)

F3

Plantacare® 810 (caprylyl/capryl glucoside)

-49.6±0.7

F4

Plantacare® 2000 (decyl glucoside)

-48.4±0.8

F5

Plantacare® 1200 (lauryl glucoside)

-46.3±0.6

F6

Surfhope® C-1815 (sucrose stearate)

-42.4±1.2

F7

Plurol Stearique® WL1009 (polyglyceryl-6 distearate)

-32.3±0.8

Figure captions

48

TAC: Tacrolimus

16

Waxes: L1-L5

L6

Monoglycerides: L6-L8

14

Triglycerides: L9-L12

L7

Diglycerides: L13, L14

12

Fatty acids: L15, L16

1/2

h (MPa )

TAC

L20 L8

10 L19

8

L14 6

L16

4 2 16.0

L3 L17

L13 L15 L9 L18 L5 L10 L11 L12 L4 L1 L2

6 5

16.5

4

17.0 2

18.0

1

18.5

Pa

1/2

3

17.5  ( d M

Liquid lipids: L17-L20

1/2

)

Fig. 1.

49

Pa

(M

p

)

TAC: Tacrolimus Waxes: L1-L5 Monoglycerides: L6-L8 Triglycerides: L9-L12 Diglycerides: L13, L14 Fatty acids: L15, L16 Liquid lipids: L17-L20

Fig. 2.

50

TAC: Tacrolimus Waxes: L1-L5 Monoglycerides: L6-L8 Triglycerides: L9-L12 Diglycerides: L13, L14 Fatty acids: L15, L16 Liquid lipids: L17-L20

Fig. 3.

51

10

Normalized heat flow (W/g)

CompritolÒ 888 CG

5

CutinaÒ CP + ImwitorÒ900P

0

CutinaÒ CP

-5

ImwitorÒ900P

-10

20

30

40

50

60

Temperature (°C) Fig. 4.

52

70

80

90

15

Tacrolimus

Normalized heat flow (W/g)

10

Tacrolimus:ImwitorÒP:CutinaÒ CP:MiglyolÒ812 = 1:10:79:10 5

Tacrolimus:ImwitorÒP:MiglyolÒ812 = 1:79:20 Tacrolimus:CompritolÒCGMiglyolÒ

0

-5

-10

-15

20

40

60

80

100

120

Temperature (°C)

Fig. 5. a) z-ave (nm)

0.5

PI

400

0.4

300

0.3

200

0.2

100

0.1

0.0

0

1x500 bar

3x500 bar

5x500 bar

53

Polydispersity index (PI)

z-ave (nm)

500

140

b)

1.0

d(v)0.90 - d(v)0.99 d(v)0.50 - d(v)0.90 d(v)0.10 - d(v)0.50

Particle size (m)

0.8 0.6 0.4 0.2 0.0

1x500 bar

3x500 bar

5x500 bar

Number of cycles x pressure

Fig. 6.

a) 500

0,5 PI

400

0,4

300

0,3

200

0,2

100

0,1

0

0 TLM1

TLM2 Lipid mixture

54

TLM3

polydispersiti index (PI)

z-ave (nm)

z-ave (nm)

b)

Particle size (m)

150

d(v)0.90-d(v)0.99 d(v)0.50-d(v)0.90 d(v)0.10-d(v)0.50

100 50

0.2 0.1 0.0

TLM1

TLM2 Lipid matrix

Fig. 7.

a)

b)

c)

d)

55

TLM3

Fig. 8.

56

I hereby confirm that all authors equally contributed to the manuscript: Formulation development of lipid nanoparticles: improved lipid screening and development of tacrolimus loaded nanostructured lipid carriers (NLC)

Anđelka Kovačević, PhD

57

Declaration of interests

☒ 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.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

58

δd

p 

h 

 Fdi V

Equation (1)

F

2 pi

Equation (2)

V

E V

hi

Equation (3)

D  ( d ( solute)   d ( solvent) ) 2  ( p ( solute)   p ( solvent) ) 2  ( h ( solute)   h ( solvent) ) 2

EE (%) 

totalamountofTAC  freeamountofTAC x100 totalamountofTAC

59

Equation (4)

Equation (5)