Preparation and characterization of novel pseudo ceramide liposomes for the transdermal delivery of baicalein

Journal of Drug Delivery Science and Technology 52 (2019) 150–156

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Preparation and characterization of novel pseudo ceramide liposomes for the transdermal delivery of baicalein

T

A Rang Kim1, Nan Hee Lee1, Young Min Park, Soo Nam Park∗ Cosmetic R&D Center, Department of Fine Chemistry, Seoul National University of Science and Technology, 232, Gongneung-ro, Nowon-gu, Seoul, 01811, South Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Transdermal drug delivery system Pseudo ceramide Ceramide liposome Baicalein

To overcome the disadvantages of natural ceramides, pseudo ceramides (PO3C, 6C, and 9C) were synthesized and used to construct pseudo ceramide liposomes. The liposomes prepared using PO9C were the smallest in size, with a 130 nm particle size when the PO9C:PC content was 2:1. Also, the stability was higher than ordinary ceramide liposomes(CLs). The PC:ceramide ratio of 2:1, in which the particle size of the liposome as well as its stability was optimal, was finally selected to carry baicalein (BAI). Entrapment efficiency of the common CLs was 78.40%, whereas that of the pseudo CLs was over 80%. Notably, liposomes using ω9CL exhibited the highest entrapment efficiency. The morphology of BAI-loaded pseudo CLs were confirmed by TEM. The concentrations of BAI loaded in ordinary CLs present in the stratum corneum, epidermis and dermis, and transdermal permeation were 7.82, 38.59, and 49.46 μg/cm3, respectively. These were closely similar to concentrations of the pseudo CLs, BAI-ω9CL (8.37, 45.04, and 46.31 μg/cm3). In conclusion, it is suggested that the pseudo CLs carrying BAI (BAI-ω9CLs) are as effective as ordinary CLs in delivering the drug to the skin, but have the added advantage of being more stable.

1. Introduction The skin, consisting of the epidermis, dermis, subcutaneous fat layer, and epidermal keratinocytes, is differentiated into four layers—the stratum basale, stratus spinosum, granular layer, and stratum corneum. During differentiation, the nuclei and cell organelles disappear and the moisture content decreases, causing dead keratinocytes to accumulate as layers, forming the outermost layer of the skin. The stratum corneum not only plays an important role in the permeability barrier function of the skin, but has also recently been reported to be involved in cell signaling in response to external stimuli [1,2]. The stratum corneum is described as exhibiting a “Bricks and Mortar” structure, consisting of keratinocytes (Bricks) and the intercellular filling (Mortar) between them [3]. The keratinocytes are composed of keratin bundles, which give structural stability and elasticity to the skin and contribute to the production of natural moisturizing factors and signal transduction of anti-inflammatory cells. Unlike biological membranes, which are permeable to hydrophilic substances, interstitial lipids act as barriers to water permeation and hydrophilic materials because lipids such as ceramides, cholesterol, and fatty acids are arranged in a multilayered structure. It is known that ceramide, cholesterol, and fatty acids, which are major components of

intercellular lipids, exist at a 1:1:1 M ratio, and that the amount as well as the ratio of composition of each constituent plays an important role in the skin barrier [4]. Ceramide is a structure in which fatty acid is bonded to an amide of a sphingoid base. Ceramide of human skin is classified into 12 groups according to composition or esterificaion of 4 types of sphingoid base and 3 types of fatty acids. Ceramides account for about 50% of the intercellular lipid mass and are known to play an important role in damaged skin [5,6]. In skin irritation studies, a significant reduction of ceramides was observed in lesions as well as in normal skin, affected by skin diseases such as atopy, psoriasis, and LPS due to inflammation caused by chemicals. Transdermal water loss (TEWL) due to decreased exogenous ceramides has been reported [7]. In addition, reduction of ceramide was reported to be associated with abnormalities of skin barrier function, increased TEWL, decreased NMF content, and inflammatory cytokine induction [8]. The reduction of ceramide is reported to be due to a decrease in ceramide synthases, such as glucosylceramidase and sphingomyelinase, and an abnormal increase in ceramide metabolism due to ceramidase [9,10]. Ceramides have been used as cosmetic raw materials, and for the treatment of skin damage as well as for aging. However, ceramides have low solubility and cannot be economically extracted from natural

∗

Corresponding author. E-mail address: [email protected] (S.N. Park). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jddst.2019.04.009 Received 18 January 2019; Received in revised form 7 March 2019; Accepted 8 April 2019 Available online 13 April 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.

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formulation stability of liposomes, it is necessary to optimize liposome characteristics such as liposome composition, particle size, surface charge, and zeta potential size. It is known that the stability of the liposome bilayer is mainly determined by the properties of lipids such as degree of hydration, temperature, unsaturation of acyl groups, charge, and presence of cholesterol [25]. It is known that the addition of cholesterol to liposomes increases the fluidity and stability of phospholipid molecules [26]. Liposome surface charge inhibits bilayer fusion, antioxidants reduce oxidation, and cholesterol and sphingomyelin have been reported to reduce drug efflux and permeability [27,28]. Baicalein (BAI) is a polyphenolic flavonoid component found in the root of the medicinal plant, Scutellaria baicalensis Georgi, and has been reported to have antioxidant, antimicrobial, anti-inflammatory and anticancer effects [29,30]. It has been reported that it displays antioxidant effects and protects the skin from damage due to ultraviolet rays. It decreases the skin inflammation reaction in atopic dermatitis, and is involved in collagen synthesis in dermal cells [31]. However, a drug delivery system is needed to deepen the delivery of these active ingredients and to further improve their effectiveness. In this study, liposomes similar to skin constituents were prepared using the novel pseudo ceramides, PO3C, PO6C, PO9C, and their skin barrier function was tested. The physicochemical properties of the liposomes were investigated to determine the optimal composition ratio of the liposomes according to the number of double bonds of the novel pseudo ceramide and their 6-week stability. The liposomes were also loaded with baicalein, which exhibits antioxidant activity, and the drug absorption efficiency, in vitro drug release, and skin permeation were measured. Our results confirmed that the pseudo ceramide liposomes (CLs) carried baicalein well and effectively penetrated the skin. As a result, in summation, the liposome prepared using pseudo ceramide functions well and not only passed the skin barrier but also effectively transmitted the weakly soluble drug, baicalein, to the skin.

sources at a high purity, or made into preparations. Therefore, the possibility of producing synthetic ceramides and pseudo ceramides, structurally similar to ceramide is being explored [11–13]. Although synthetic ceramides such as ceramide-3 and ceramide have been successfully produced via bio-processes using enzymes, and used as raw material for cosmetics, synthetic ceramides carry a disadvantage due to cost competitiveness associated with high production costs. Ceramide has 2 asymmetric chains, and the polar head group has an amide structure and hydrogen bond structure around it. Pseudo ceramides that maintain structural similarity with ceramide have been developed by designing a group with different functions in the head group to maintain the effect as a skin barrier. The first Pseudo ceramide to be developed was Ceramide R4 (L'Oreal), followed by PC104 of (AmorePacific), PC-9S (Neo Parm) [14–16]. New pseudo ceramides, PO3C, PO6C, and PO9C, form a serine amide bond with double bonds 1, 2, and 3 of the C18 fatty acid, respectively. When applied to dry or damaged skin, it has a function almost similar to that of natural ceramide [17]. In addition, those with better barrier properties can be formulated by adding other ingredients such as cholesterol and fatty acids. In order for a drug or an efficacious substance to be absorbed into the skin, the stratum corneum, the outermost layer of the epidermis must be permeated. However, the stratum corneum is difficult to permeate because it acts as a barrier to absorption of external substances [18]. Thus, TDDS has developed various physical techniques and formulations to promote skin penetration for effective drug delivery [19,20]. The pathway through the stratum corneum has three channels; the transcellular pathway through the keratinocytes, the intercellular pathway through the interstitial lipid layer, and the follicular pathway through pores and glands [21]. The pathway through keratinocytes is considered as a passage for hydrophilic materials. Keratinocytes, are filled with hydrophilic proteins, and surrounded by a hydrophobic corneocyte lipid envelope (CLE), where hydrophilic material is unable to pass through the intercellular lipid filling between keratinocytes and cells. Therefore, lipid-based delivery systems, such as liposomes, elastic liposomes, etosomes, SLN, and NLC have been developed to pass through intercellular lipids [22–24]. Liposomes are spherical vesicles composed of phospholipids. They are composed of biocompatible components and can be decomposed in vivo and have low toxicity. They have been widely used to manufacture medicines, cosmetics, and food items among others. A phospholipid is an amphipathic molecule, which, when dispersed in water, forms an endoplasmic reticulum surrounded by a spherical closed double layer membrane capable of spontaneously collecting an aqueous solution inside. Due to the structure of the lipid bilayer membrane, it is able to carry a water-soluble substance in its core without bonding to the enclosed substance chemically. The lipid membrane is advantageous in that both hydrophilic substances and hydrophobic substances can be supported by dissolving or dispersing the lipophilic drug. Liposomes can be prepared by adding various substances such as cholesterol, surfactants, glycolipids, and cell membrane proteins, and manufactured in various sizes and multi-layer structures according to the composition of the material and the manufacturing method used [25]. Liposomes absorbed into the body are fused to the cell membrane, the phospholipids are released into the cell membrane and the drug is released into the cell. Despite these advantages, liposomes are beset with disadvantages such as physico-chemical instability, low skin absorption rate in normal skin, lipid oxidation, and hydrolysis. To overcome such problems and to enhance effective permeation, absorption, and

2. Materials and methods 2.1. Materials L-α-phosphatidylcholine (PC, from egg yolk, ≥99.0%), cholesterol, oleic acid, baicalein, Folin-ciocalteu's phenol reagent and 3-(4,5-dimethythiazol-2-yl)-2,5-di-phenytetrazolium bromide (MTT), sodium phosphate monobasic (NaH2PO4·2H2O) and sodium phosphate dibasic (Na2HPO4·2H2O) used in the phosphate buffer solution, were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Ceramide-3 was obtained from DS-CERAMIDE Y30 (Doosan, Korea) and pseudo ceramide PO3C, PO6C and PO9C were synthesized by Daebong LS Co.(Korea) [17]; (Fig. 1). Solvents such as ethanol, methanol, and chloroform were purchased from Daejung Chemical Co., Ltd. (Korea) and distilled water was purified with Milli-Q.

2.2. Preparation of pseudo ceramide liposomes The liposomes using ceramide were prepared by thin film hydration, and their constituents and compositions are shown (Table 1). The composition of BAI-loaded liposomes is shown (Table 2). First, the components were dissolved in 25 mL of chloroform-methanol (4:1). The solvent was then completely removed using a rotary evaporator (Buchi, Switzerland) to form a film. The film was then hydrated with 10 mL of phosphate buffer (PB; pH 7.4) and homogenized for 5 min using a probe sonicator. It was then passed through a 1.2 μm filter (Minisart CA Fig. 1. The molecular structures of pseudo ceramides (PO3C, PO6C, PO9C).

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calibration curve for the drug concentration was prepared using a standard solution. The wavelength of BAI was 270 nm. Concentration of the loaded drug was then calculated and the calculated value was substituted into eq. (1) to calculate entrapment efficiency.

Table 1 Composition of pseudo ceramide liposomes depending on ceramide concentration. Molar ratio

PC

Ceramide-3

PO3C

PO6C

PO9C

Chol

OA

CL1 CL2 CL3

2 2 2

0.5 1 2

– –

– –

– –

1 1 1

1 1 1

ω3CL1 ω3CL2 ω3CL3

2 2 2

– – –

0.5 1 2

– – –

– – –

1 1 1

1 1 1

ω6CL1 ω6CL2 ω6CL3 ω9CL1 ω9CL2 ω9CL3

2 2 2 2 2 2

– – – – – –

– – – – – –

0.5 1 2 – – –

– – – 0.5 1 2

1 1 1 1 1 1

1 1 1 1 1 1

Entrapment efficiency(%) =

2.6. Transmission electron microscopy (TEM) We used TEM (JEOL Ltd., Tokyo, Japan) to observe the morphology of pseudo CLs liposomes. Samples were adsorbed on a 200-mesh copper grid for 2 min, stained with 0.2% (w/v) phosphotungstic acid solution for 45 s, and dried. The analysis was performed at 80 kV. 2.7. Cell viability assay In order to apply prepared liposomes to the skin, it is necessary to confirm the absence of cytotoxicity. Therefore, cytotoxicity was evaluated using methylthiazoldiphenyl-tetrazolium bromide (MTT) assay. HaCaT cells were seeded at a density of 1 × 104 cells in 96-well plates, cultured in vitro at 5% CO 2 and 37 °C for 1 d, and the liposome solution was dispensed. Cytotoxicity was evaluated by comparing survival rates of HaCaT cells in the sample via the MTT assay. Absorbance at 570 nm was measured using an ELISA reader. Absorbance of the untreated group was determined as the negative control (100%). The cell survival rate relative to the treated group was determined using eq. (2).

Table 2 Composition of pseudo ceramide liposomes containing baicalein. PC

Ceramide-3

PO3C

PO6C

PO9C

Chol

OA

Baicalein

BAI-CL BAI-ω3CL BAI-ω6CL BAI-ω9CL BAI-ω9CL0

2 2 2 2 2

1 – – – –

– 1 – – –

– – 1 – –

– – – 1 1

1 1 1 1 1

1 1 1 1 0

0.5 0.5 0.5 0.5 0.5

(1)

Ci: Initial concentration of drug.Ce: Concentration of loaded drug.

PC: L-α-phosphatidylcholine, Chol: cholesterol, OA: oleic acid, CL: general ceramide liposome, ω3CL, ω6CL, ω9CL: pseudo ceramide liposomes based on Pro omega-3 ceramide, Pro omega-6 ceramide, and Pro omega-9 ceramide.

Molar ratio

Ce × 100 Ci

Cell viability (%) =

PC: L-α-phosphatidylcholine, Chol: cholesterol, OA: oleic acid, BAI-CL: BAIloaded general ceramide liposome, BAI-ω3CL, ω6CL, ω9CL: BAI-loaded pseudo ceramide liposomes based on Pro omega-3 ceramide, Pro omega-6 ceramide, and Pro omega-9 ceramide, BAI-ω9CL0: BAI-ω9CL not containing oleic acid.

Atreated group Auntreated gruop

×100

(2)

2.8. In vitro skin permeation study using Franz diffusion cells The in vitro skin permeation study of the ceramide liposomes with drug capture was performed using 9 mm Franz diffusion cells (Permegear, USA, receptor volume 5 mL). We purchased in vitro HuSKIN™ (HansBiomed Co., Ltd., Korea) from a 52 year old male with his consent. Subcutaneous fat and excess tissue of the skin was removed. The skin was fixed with the stratum corneum facing up between the donor and the receptor phase. The receptor chamber was filled with the receptor phase (HCO-60:ethanol:PBS = 2:20:78, weight ratio) and stirred at 150 rpm for 24 h. The temperature was maintained at 37 °C using a constant temperature water bath. Drug solutions dissolved at the same concentration in 1,3-BG/PB were used as a control. The receptor phase was sampled at each sampling port. After 24 h, the surface of the skin was washed with PBS solution, and the amounts of drug contained in the stratum corneum and skin was measured, by removing the stratum corneum by stripping thrice using 3 M scotch tape (3 M, Maplewood, MN, USA). Afterwards, the tape and drugs in the skin were dissolved by sonication with 100% ethanol. The concentration of the collected drugs was measured using a UV/Vis spectrophotometer.

26 mm; Sartorius, Göttingen, Germany) and stored. The final pseudo ceramide concentration of the prepared liposome formulation was 0.2% (w/v). 2.3. Particle size and zeta potential of pseudo ceramide liposomes Particle size of the liposome was measured via dynamic light scattering (Otsuka ELS-Z2; Otsuka Electronics, Osaka, Japan). The measurement temperature was 25 °C, the scattering angle was 165°, and an argon laser was used as the light source. Mean particle size was expressed using the cumulative method and its distribution was analyzed by the CONTIN method [32]. The particle size was measured as the average of 3 trials consisting of 70 measurements. The zeta potential was measured in 3 trials of 10 times using zetasizer (Otsuka ELS-Z2, Otsuka Electronics, Japan). 2.4. Stability of pseudo ceramide liposomes To confirm the stability of prepared liposomes, liposome suspensions were stored at 4 °C for 4 weeks [33]. Changes in particle size and polydispersity index (PI) values over this period were measured, and formation of precipitates was observed. Particle size, PI values, and zeta potential were measured in the same manner described above.

2.9. Statistical analysis The experiment was repeated thrice and all data were expressed as mean ± SD. Statistical significance was tested using the Graphpad Prism 7.0 program (San Diego, CA). One-way ANOVA was used to test for significance of results. Statistical significance was set at p < 0.05.

2.5. Entrapment efficiency 3. Results and discussion We removed unloaded BAI using a 0.45 μm filter, and sonicated 1 mL of the liposome suspension with 10 mL of ethanol to destroy the liposome membranes. The solvent was evaporated using a rotary evaporator and the drug was re-dissolved in 1 mL of ethanol. Drug concentration was measured using a UV–vis spectrometer, and a standard

3.1. Particle size and zeta potential of pseudo ceramide liposomes Liposomes were prepared using pseudo ceramide (PO3C, PO6C, PO9C), and ceramide-3. Ceramide-3 was used as a control (general 152

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3.2. Stability of pseudo ceramide liposomes In order to evaluate the stability of the prepared ceramide liposomes, the change in liposome particle size was measured following storage at 4 °C for 4 weeks (Fig. 2). In general, when the liposome is unstable, the particles agglomerate together and the average particle size increases [35]. The results showed that the stability of CLs decreased with higher ceramide content. On the contrary, when the PC:ceramide ratio was 2:0.5 (ω3CL1, ω6CL1, ω9CL1), the change in particle size of pseudo CLs after 3 weeks was remarkable. When the PC:ceramide ratio was 2:1 (ω3CL2, ω6CL2, ω9CL2) and 2:2 (ω3CL3, ω6CL3, ω9CL3), the change in particle size after 4 weeks was small and 153

0.24 ± 0.03 −35.07 ± 0.34 0.26 ± 0.00 −42.37 ± 0.40 0.23 ± 0.00 −39.76 ± 0.22 0.14 ± 0.02 −45.54 ± 0.11

Table 4 Particle size, polydispersity index, zeta potential of pseudo ceramide liposomes containing baicalein.

BAI-ω6CL

BAI-ω9CL

CLs). The particle size and polydispersity index (P·I.) of liposomes were measured (Table 3). PC, cholesterol, ceramide, pseudo ceramide, and oleic acid were added as constituents of liposome. To find the best composition for liposome formulation, the composition ratio of ceramide, cholesterol, and fatty acid was 0.5:1:1, 1:1:1, and 2:1:1, respectively. The ratio of phospholipid to cholesterol was fixed at 2:1 because the particle size decreased when the molar ratio of lecithin PC to cholesterol was 2:1. In general, it has been reported that a particle size of 100–150 nm is most suitable for the liposome to be easily absorbed into the skin [34]. As a result of comparing particle sizes of the liposomes, the particle size of the liposome using PO3C was remarkably increased. This is probably due to increased lipid intermolecular steric effect due to the twisted structure, as the number of double bonds of pseudo ceramides increased. The particle size also increased with increasing PO3C and PO6C contents. On the other hand, in the case of liposome using PO9C, when PC: PO9C content was 2:1, particle size was found to be 130 nm, which was confirmed as optimal size for skin absorbance. We therefore predict that in vitro skin permeation studies would further increase the skin permeability of liposomes using PO9C. The polydispersity index (P·I.), indicates the degree of dispersion of the particle size of the liposome. A P.I. of 0.3 or less indicates monodispersity, while polydispersity is indicated by a P.I. of 0.3 or more. All lipids showed a monodispersity of less than 0.3, but the content of ω3CL3 was greater than 0.3 when the ceramide content ratio was 2 and the particle size was the largest. Zeta potential represents the surface charge of the liposome and is a value that can predict the physical stability of the liposome by interfering with cohesion between liposomes via electrostatic repulsive force. Generally, a value of less than −25 mV has been reported to be stable [34]. The zeta potentials of the liposomes were all below −25 mV, indicating stability, and this value tended to increase slightly as the ceramide content increased. The particle size of BAI-loaded liposomes, P·I., and zeta potential after 1 d are shown (Table 4). Compared with the particle size of pre-BAI-loaded liposomes, there was no significant difference and the size tended to increase slightly. When oleic acid was not added (BAI-ω9CL0), the size of the liposome was remarkably decreased.

0.23 ± 0.00 −41.88 ± 0.43

0.50 0.02 0.50 0.68 0.50 0.32 0.61 0.26 0.28 0.55 0.51 0.37

137.75 ± 0.36

± ± ± ± ± ± ± ± ± ± ± ±

BAI-ω9CL0

0.01 0.00 0.00 0.02 0.02 0.01 0.02 0.02 0.00 0.01 0.01 0.01

210.45 ± 1.48

−46.40 −47.62 −44.99 −44.91 −43.76 −41.43 −43.51 −44.48 −40.44 −44.47 −44.08 −41.61

± ± ± ± ± ± ± ± ± ± ± ±

459.40 ± 7.14

0.27 0.25 0.25 0.19 0.20 0.34 0.22 0.16 0.14 0.23 0.19 0.19

146.35 ± 0.21

88.97 ± 0.85 146.73 ± 0.49 162.93 ± 3.30 292.50 ± 8.22 453.80 ± 3.04 697.13 ± 7.48 172.20 ± 2.90 193.20 ± 2.43 416.70 ± 2.52 140.40 ± 3.21 130.40 ± 1.85 410.33 ± 2.85

Particle size (nm) Polydispersity index (P·I.) Zeta potential (mV)

Zeta potential (mV)

BAI-ω3CL

Polydispersity index (P·I.)

BAI-CL

CL1 CL2 CL3 ω3CL1 ω3CL2 ω3CL3 ω6CL1 ω6CL2 ω6CL3 ω9CL1 ω9CL2 ω9CL3

Particle size (nm)

68.9 ± 1.24

Table 3 Mean particle size, polydispersity index, and zeta potential of pseudo ceramide liposomes.

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Table 5 Encapsulation efficiency of pseudo ceramide liposomes containing baicalein. Entrapment efficiency (%) BAI-CL BAI-ω3CL BAI-ω6CL BAI-ω9CL

78.40 80.04 81.56 81.71

± ± ± ±

0.10 0.11 0.10 0.14

the lipid bilayer, thus stabilizing the liposome. The measurement confirmed that stability was disrupted when oleic acid was not added, confirming that oleic acid contributes to the stability of the liposome. Fig. 2. Stability of pseudo ceramide liposomes.

3.3. Entrapment efficiency The encapsulation efficiency of BAI-CLs is shown (Table 5). Collection efficiency of the general CLs was 78.40%, whereas the collection efficiency of the pseudo CLs was higher than 80%, a marginal increased over that of CLs. Moreover, ω9CL showed the largest collection efficiency. This indicated that liposomes produced using PO9C, which has a single double bond, were least affected by the lipid-steric effect of double bonds. 3.4. Morphological analysis of BAI-loaded pseudo ceramide liposomes TEM was used for morphological analysis of BAI-CLs (Fig. 4). All liposomes were similar in size to those measured using dynamic light scattering. Lipid membranes of both liposomes were clearly observable. 3.5. Cell viability assay of BAI-loaded pseudo ceramide liposomes In order to be used as skin transporters, liposomes must not be cytotoxic. Therefore, the current study evaluated the cytotoxicity of BAI and BAI-loaded liposomes using HaCaT cells (Fig. 5). The concentration range of BAI and BAI-loaded liposomes was set at 15.6–250.0 μM. No cytotoxicity by the drug was observed. The cytotoxicity of BAI-loaded liposomes was not affected by BAI. Cell viability was 62.5 μM which was above 90%. When high concentrations of BAI were loaded, cell viability was reduced in pseudo CLs. This MTT assay provides meaningful data on the practical application of pseudo CLs using PO3C, PO6C, and PO9C.

Fig. 3. Stability of pseudo ceramide liposomes containing baicalein.

stability was established. It is believed that the double bond of pseudo ceramide contributed to the stability by inhibiting oxidation of the lipid membrane. PDI did not show a significant change for 3 weeks and showed values less than 0.3 except for ω3CL3. After 3 weeks, the zeta potential was measured, and the values for CL2, ω3CL2, ω6CL2, and ω9CL2 were −52.01, −50.79, −50.75, and −44.65, respectively. The absolute value increased slightly compared with the zeta potential value (−47.62, −43.76, −44.48, −44.08) after 1 day, and it was proposed that the surface charge may have increased due to a small amount of aggregation of liposome particles after 3 weeks. However, no significant changes were observed, confirming that the produced liposomes were stable. On the other hand, after 3 weeks, the absolute zeta potential value of CL was the highest, indicating low stability and high cohesion. As a result, when the ratio of pseudo ceramides was 2:1 or 2:2 with phospholipids, the stability was higher than that of common CLs and the stability of ω9CL was higher. Therefore, PC:ceramide ratio of 2:1, which is optimal for both particle size and stability of liposomes, was selected to support BAI. The change in particle size of BAI-loaded liposomes for 6 weeks is shown (Fig. 3). The sizes of common CLs (BAICLs) and pseudo ceramide liposomes, BAI-3CLs, BAI-6CLs, and BAI9CLs, were 222.30, 494.02, 245.52, and 155.35 nm, and 137.75 nm), respectively. This is attributed to the increase in volumetric capacity of the BAI due to the three-dimensional twist structure formed due to double bonds of the hydrophobic molecules. It was also confirmed that the size change of CLs was smaller than that of the general CLs for 6 weeks. Thus, it was confirmed that the stability of the pseudo CLs had further increased compared to that of the general CLs even after drug loading. The stability of the liposome (BAI-9CL0) without oleic acid was also measured. Oleic acid is a C18 fatty acid with one double bond, which increases the fluidity of the liposome and prevents oxidation of

3.6. In vitro skin permeation study using Franz diffusion cells The skin permeation study of BAI transported by pseudo CLs was carried out via Franz diffusion cell analysis. The amount of BAI permeation per hour is shown (Fig. 6). The amount of BAI that permeated

Fig. 4. TEM images of (A) BAI-CL, (B) BAI-ω3CL, (C) BAI-ω6CL, (D) BAI-ω9CL. 154

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and dermis) and transdermal permeation were 7.82, 38.59, and 49.46 μg/cm3, respectively. The concentrations of BAI loaded in ω9CLs present in the stratum corneum, skin (epidermis and dermis) and transdermal permeation were 8.37, 45.04, and 46.31 μg/cm3. However, the amount present in the skin was greater than that in BAI-ω9CLs. In conclusion, the findings indicate that BAI-ω9CLs may effectively deliver drugs to the skin as well as ordinary CLs. 4. Conclusion In this study, liposomes similar to skin constituents were prepared using novel pseudo ceramides (PO3C, PO6C, PO9C) and their role in skin barrier functions was investigated. Generally, an appropriate particle size between 100 and 150 nm is required for liposomes to be absorbed well by the skin. In a comparison of liposome particle sizes using 3 pseudo ceramides, the liposomes with PO9C showed the smallest size. Particularly, when the content of PO9C:PC was 2:1, the particle size was found to be 130 nm, which is the optimal size for highest skin absorption. The stability of pseudo CLs and general CLs related to the PC:ceramide ratio was measured. The results indicated that, when the ratio of the pseudo ceramide was 2:1 or 2:2, the stability was increased compared to that of general CLs. Therefore, a pseudo CL with a PC:ceramide ratio of 2:1 and optimal liposome particle size and stability was finally selected to carry BAI. It was confirmed that the stability of the pseudo CL was higher than that of the conventional CL even after drug loading. The collection efficiency of the common CL was 78.40%, whereas that of a pseudo CL was more than 80%, indicating a marginal increase over CL. Furthermore, liposomes using ω9CL exhibited the highest collection efficiency. In addition, morphological analysis of BAI-loaded pseudo CLs was performed using TEM, and the particle size of the CLs was similar to those measured using dynamic light scattering. The spherical morphology of liposomes was confirmed by TEM, and the lipid membrane was also visible. The result of the skin permeation study of BAI transported onto the CLs indicated that the amount of BAI that permeated through the skin was the highest for general CLs, and the amount of BAI permeation decreased in the order of ω9CL, ω6CL, and ω3CL. The concentrations of BAI transported on ω9CLs in the stratum corneum, skin (epidermis and dermis), and transdermal layer were 8.37, 45.04, 46.31 μg/cm3. This value was similar to that of ordinary CLs. In conclusion, BAI-ω9CLs are more stable than general CLs, and function as carriers that effectively transmit the poorly soluble drug baicalein to the skin.

Fig. 5. Cell viability of pseudo ceramide liposomes containing baicalein.

Fig. 6. In vitro skin permeation of BAI through 1,3-butylene glycol solution (1,3-BG), ceramide liposomes (BAI-CLs), and pseudo ceramide liposomes (BAIω3CLs, BAI-ω6CLs BAI-ω9CLs).

Declaration of interest The authors declare no conflict of interest. References [1] J.M. Waller, H.I. Maibach, Age and skin structure and function, a quantitative approach (II): protein, glycosaminoglycan, water, and lipid content and structure, Skin Res. Technol. 12 (3) (2006) 145–154. [2] P.M. Elias, Stratum corneum defensive functions: an integrated view, J. Investig. Dermatol. 125 (2) (2005) 183–200. [3] P.M. Elias, Epidermal lipids, barrier function, and desquamation, J. Investig. Dermatol. 80 (1983) 44–49. [4] S.J. van, M. Janssens, G.S. Gooris, J.A. Bouwstra, The important role of stratum corneum lipids for the cutaneous barrier function, Biochim. Biophys. Acta 1841 (3) (2014) 295–313. [5] M.H. Meckfessel, S. Brandt, The structure, function, and importance of ceramides in skin and their use as therapeutic agents in skin-care products, J. Am. Acad. Dermatol. 71 (1) (2014) 177–184. [6] M. Rabionet, K. Gorgas, R. Sandhoff, Ceramide synthesis in the epidermis, Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1841 (3) (2014) 422–434. [7] A. Sugiura, T. Nomura, A. Mizuno, G. Imokawa, Reevaluation of the non-lesional dry skin in atopic dermatitis by acute barrier disruption: an abnormal permeability barrier homeostasis with defective processing to generate ceramide, Arch. Dermatol. Res. 306 (5) (2014) 427–440. [8] J.W. Park, W.J. Park, A.H. Futerman, Ceramide synthases as potential targets for therapeutic intervention in human diseases, Biochim. Biophys. Acta 1841 (5) (2014) 671–681.

Fig. 7. Proportions of BAI permeation (%) in 1,3-butylene glycol solution (1,3BG), ceramide liposomes (BAI-CLs), and pseudo ceramide liposomes (BAIω3CLs, BAI-ω6CLs BAI-ω9CLs) after 24 h (Tape: stratum corneum, Skin: epidermis and dermis, Transdermal: permeated through skin).

the skin by time in the common CL was the highest, and ω9CL was similar to that of the ordinary CL but showed a slightly lower permeation amount. Then, the amount of skin permeation of BAI decreased in the order of ω6CL and ω3CL. This is due to the fact that the order of particle size increases in the order of ω9CL < ω6CL < ω3CL, where particle size affects the amount of skin permeation. The amount of BAI permeated per skin area is shown (Fig. 7). In the control group, the concentrations of BAI dissolved in 1,3-BG present in the stratum corneum, skin (epidermis and dermis) and transdermal permeation were 3.60, 16.47, and 21.88 μg/cm3, respectively. The concentrations of BAI loaded in ordinary CLs present in the stratum corneum, skin (epidermis 155

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