Nanoemulsion Vehicles as Carriers for Follicular Delivery of Luteolin

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Controlled Release and Delivery Systems

Nanoemulsion Vehicles as Carriers for Follicular Delivery of Luteolin Kyounghee Shin, Hayoung Choi, Sun Kwang Song, Ji Won Yu, Jin Yong Lee, Eun Ji Choi, Dong Hee Lee, Sun Hee Do, and Jin Woong Kim ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00220 • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Nanoemulsion Vehicles as Carriers for Follicular Delivery of Luteolin Kyounghee Shin,† Hayoung Choi, ‡ Sun Kwang Song, ‡ Ji Won Yu,‡ Jin Yong Lee,† Eun Ji Choi,§ Dong Hee Lee,‡ Sun Hee Do, *,§ and Jin Woong Kim *,†,∥ †

Department of Bionano Technology, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu,

Ansan 15588, Republic of Korea ‡

Biomaterial Research Center, Cellinbio, 88 Sinwon-ro, Yeongtong-gu, Suwon 16681, Republic

of Korea §

College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul

05029, Republic of Korea ∥Department

of Chemical and Molecular Engineering, Hanyang University, 55 Hanyangdaehak-

ro, Sangnok-gu, Ansan 15588, Republic of Korea

KEYWORDS: Luteolin, Nanoemulsion, Hair growth, Follicular delivery, Amphiphilic block copolymer

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected]

ABSTRACT Luteolin (3′,4′,5,7-tetrahydroxyflavone), a type of flavonoid found in medicinal herbs and vegetables, has been of great interest due to its anti-oxidative, anti-inflammatory, and anticarcinogenic effects. Despite these beneficial biological properties, the ease with which luteolin forms molecular crystals in conventional aqueous formulations has hampered much wider

ACS Paragon Plus Environment

1

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 19

applications. In this study, we introduce an oil-in-water (O/W) nanoemulsion vehicle system for enhanced follicular delivery of luteolin. The luteolin-loaded nanoemulsion, which had an average hydrodynamic size of approximately 290 nm, was produced by the assembly of poly(ethylene oxide)-block-poly(ε-caprolactone) and lecithin at the O/W interface. The luteolinloaded nanoemulsion showed outstanding stability against drop coalescence and aggregation. This was confirmed from the slight drop size increase after repeated freeze-thaw cycling and long-term storage. Moreover, in vivo hair growth evaluation demonstrated that the luteolinloaded nanoemulsions fabricated in this study possessed the hair growth-promotion activity, which is comparable with the case of using a luteolin solution in an organic solvent.

Introduction Hair follicles undergo repetitive regeneration, which is termed as the hair cycle. This cycle consists of the following phases: growth (anagen), regression (catagen), rest (telogen), and shedding (exogen).1 Recent studies have revealed that the activation pattern of alkaline phosphatase (ALP) is proportional to the activity of dermal papilla during the hair cycle; a maximal level appears in early anagen and a minimum level during catagen.2 Flavonoids, a type of polyphenolic compounds widely present in plants, are of scientific interest due to their excellent biological properties, including anti-oxidative, anti-carcinogenic, anti-inflammatory, anti-bacterial, anti-diabetic, and anti-proliferative actions.3 In addition to those biological benefits, some flavonoids, i.e., luteolin, baicalin, and alpinetin, are known to induce ALP activity in cultured rat osteoblasts.4 In particular, these specific flavonoids may even prevent the shedding of hair and promote hair growth. In fact, many biological benefits of flavonoid administration have been shown in vivo, while other beneficial biological functions of flavonoids are yet to be fully investigated, as flavonoids have low solubility and bioavailability in water.5 This intrinsic disadvantage stems from their intermolecular association, which is induced by statistical conformational disorder, π-π stacking interactions of aromatic rings, and strong hydrogen bonds between hydroxy and carbonyl groups.6 In order to overcome such limitations in formulation, while retaining biological efficacy, it is essential to block the molecular interactions between flavonoid molecules.5 When flavonoids are encapsulated in lipid-based nanocarriers, such as liposomes and solid lipid nanoparticles, their bioavailability can often be increased.7 However, assembled lipid structure is so fragile that it is


2

Page 3 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


eventually disrupted by growth of molecular crystals, thus producing phase separation.8 Solubility of flavonoids can be improved by using host-gest immobilization in cyclodextrin inclusions. In this case, the guest flavonoid molecules entrapped inside the cavity of the host cyclodextrin readily dissociate by small changes in medium solvency.9 Differing from conventional approaches in the scale of molecular length, recently, exploitation of amphiphilic polymers has been of great interest in the production of nanoscale dispersion of flavonoids for in vivo delivery.10 When nanoemulsions are formed by the assembly of amphiphilic polymers at the oil/water (O/W) interface, they provide convenient means for the encapsulation, protection, and delivery of poorly water soluble active ingredients.11 The small droplets stabilized with a thin resilient polymer membrane exhibit high thermodynamic stability against coalescence, aggregation, and Ostwald’s ripening. This nanoemulsion system is expected to lead to new opportunities for flavonoids to be formulated in aqueous phase, with better bioavailability and accurate dosing, rendering minimum side effects.11b. Here in this study, we introduce a nanoemulsion-based follicular delivery system, in which luteolin, a well-known flavonoid, is incorporated into O/W nanoemulsions, and its hair-growth promotion ability is evaluated in vivo. Stable luteolin-loaded nanoemulsions were prepared by the assembly of poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) and lecithin at the O/W interface.12 This emulsification method is based on an attractive nanoemulsion fluid system with a drop-percolated network, in which dipole-dipole interaction between phosphorylcholine groups of lecithin and methoxy end groups of poly(ethylene oxide)-blockpoly(ε-caprolactone) leads to a gel-like rheological behavior. We subsequently investigated the mechanism by which the encapsulation of luteolin in nanoemulsion drops renders assistance to the protection of molecular crystallization. Finally, we demonstrate through in vivo hair growth evaluation that the luteolin-loaded nanoemulsion enhances hair growth-promotion activity.

Materials and Methods Materials Luteolin was purchased from 4Chem Laboratory Co. (>98% determined by HPLC, Korea). PEOb-PCL was kindly supplied by ACT (Korea). The measured molar ratio of PCL to PEO was 1.07:1 and the average molecular weight was 10.4 kDa with a polydispersity index (PDI) of 1.37. Lecithin used in this study was the commercially available Lipoid P75-3 (PC content: 70%,


3


Page 4 of 19

Lipoid Kosmetik AG, Germany). Sweet almond oil was generously provided by Textron Tecnica, S.L. (Barcelona, Spain). Tetrahydrofuran (THF) (>98%, TCI, Japan) was used as a removable solvent. Minoxidil (≥99%, Sigma-Aldrich, USA) was used as a topical drug for the treatment of androgenetic alopecia. Ascorbic acid and e from Sigma-Aldrich Co. (≥99%, St Louis, MO, USA). For all experiments, deionized double distilled water was used.

Preparation of luteolin-loaded nanoemulsions Luteolin-loaded nanoemulsions were prepared by the phase inversion composition method and consecutive probe-type sonication. First, we carried out the solubility test for luteolin on various oils. 100 mg of luteolin was dissolved in 100 dL of oil at 80 °C. After cooling the mixture, we observed the precipitation of luteolin crystals. Finally, we selected sweet almond oil as the oil solvent for luteolin. In the next step, 5 wt% of PEO-b-PCL, 1 wt% lecithin, and 20 wt% of sweet almond oil containing 3.5 mM of luteolin were completely dissolved in THF at 45 °C. Subsequently, 80 wt% of water was added dropwise to the oil phase with vigorous stirring via a syringe pump (Pump 11Elite, Harvard Apparatus, USA); the flow rate of water was 100 mL/min. During this process, the W/O emulsion formed in low volume of water was readily converted to the O/W emulsion as the volume of water increased in the system. THF was completely removed under reduced pressure on a rotary evaporator at 40 °C for 5min. In order to ensure complete removal of THF, the emulsion sample was left under reduced pressure conditions for 30 min at room temperature. Finally, the nanoscale emulsions were produced using a probe-type sonicator (VCX130, Sonic & Materials Inc., USA). The sonication time was set to 5 min at 60 % amplitude as no further size reduction was observed under these conditions. During sonication, the emulsion container was immersed in a temperature-controlled bath. The temperature was set to 25 °C to prevent overheating during sonication.

Characterization of luteolin-loaded nanoemulsion Size distribution wsas measured by dynamic light scattering (DLS) using a zeta potential & particle size analyzer (ELS-Z, Otsuka electronics, Japan) at 25 °C. To avoid the multiple scattering effect, the nanoemulsion sample was diluted to a droplet concentration of 3.75 wt% using DI water prior to analysis. The optimal dilution concentration was determined by measurement of polydispersity index value, which is less than 0.3. Each measurement was


4

Page 5 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


performed three times and the result was reported as the mean and standard derivation. The morphology of nanoemulsion drops was investigated by using a transmission electron microscope (TEM, Energy-Filtering Transmission Electron Microscope, LIBRA 120, Carl Zeiss, Germany) operating at 120 kV. Before TEM observation, all test samples were negatively stained with 1 wt% uranyl acetate. Nanoemulsion drops were also observed with a bright field microscope and their crystallinity was characterized using a polarized optical microscope (NSB80T, Samwon, Korea).

Evaluation of nanoemulsion stability The stability of nanoemulsions was evaluated by both long-term storage and freeze-thaw cycles. First, 20 mL of nanoemulsion sample was stored at 4 °C for 6 mon, and the drop size changes were monitored using DLS measurement. In addition, the same nanoemulsion sample was kept in a glass vial at -20 °C for 12 h in a freezer and then melted at room temperature for 12 h. This process was repeated 5 times. If the nanoemulsion was stable after the cycle, it was sent back to freezer for another cycle. Between each cycle, besides observation of physical stability of the nanoemulsion by naked eyes, the drop size change was observed by DLS as an indicator of the nanoemulsion stability.

DPPH assay The antioxidant capacity of the nanoemulsions was determined by the scavenging of DPPH radical13. In the assay, 0.1 mL of luteolin-loaded nanoemulsions (0.7 µM) was mixed with 0.1 mL of DPPH solution (2 mM in EtOH). The luteolin in EtOH solution was adjusted to the same concentration as the luteolin-loaded nanoemulsions. Subsequently, the solution was incubated at 37°C in the dark for 10 min. Distilled water was mixed with DPPH solution instead of the sample as a control. Ascorbic acid was used as the standard. The absorbance of the samples after incubation was measured at 517 nm using an ELISA reader (EnSpire Multimode Reader, PerkinElmer Inc, USA). The percentage of DPPH-scavenging ( ) activity was calculated as follows: % =

× 100 , where, !"#$%"& and

!'(&) were the absorbance of the control or sample, respectively.

In vivo hair-growth promotion study


5


Page 6 of 19

Male C57BL/6 mice, weighing 20-23 g, were obtained from YoungBio (Seongnam, Korea). The mice were kept in an environmentally controlled room (temperature 23 ± 3°C, humidity 50 ± 10%, and 12/12 h dark/light cycle) for 1 week prior to experiments. They were kept on a normal diet and had free access to water. The dorsal skin of mice was shaved and depilated to induce synchronization of hair cycle.14 The mice were randomly allocated into four groups: an EtOH group with 200 µl of 50 wt% aqueous EtOH solution administration, a minoxidil group with 200 µl of 3 wt% minoxidil administration, a luteolin group with luteolin (700 µM) in EtOH administration, a luteolin NE group with luteolin (700 µM) loaded nanoemulsion administration. Each solution was topically administered 5 times a week. All animal procedures were conducted in accordance with all appropriate regulatory standards, according to the protocol approved by the ethics committee of the Konkuk University Institutional Animal Care and Use Committee (KU17055).

Histological analysis The mice were sacrificed after 17 days and dorsal skin was harvested for histological examination. The skin tissues was fixed with 10 % neutered formalin (BBC Biochemical, WA, USA). The tissues were then processed and embedded in paraffin. The paraffin embedded samples were cut in sections (4 µm thickness) and stained using hematoxylin and eosin (H&E). The number and diameter of hair follicles were quantified in multiple fields at 100× magnification.

Results and Discussion To facilitate effective follicular delivery of luteolin for enhanced hair growth, we produced highly stable luteolin-loaded nanoemulsions. Prior to the preparation of these nanoemulsions, we examined the solubility of luteolin in a variety of oils at various temperatures. We found that luteolin showed the best solubility in sweet almond oil: 1 mg/mL (at 22 °C), 1 mg/mL (at 50 °C), and 1~2 mg/mL (at 80 °C), respectively. It appears the intermolecular association of luteolin could be minimized in sweet almond oil, because this oil contains lots of unsaturated fatty acid; more than 90% of the fatty acids are unsaturated (oleic and linoleic),15 which presumably blocks π-π stacking. Therefore, we used sweet almond oil as the dispersion oil phase. A feature of this emulsion system is that the O/W interface is composed of PEO-b-PCL and lecithin, thus


6

Page 7 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


providing a mechanically resilient thin polymer membrane (Figure 1A). The phase inversion composition method has been shown to enable effective assembly of such a polymer membrane.16 In fact, coassembly of a critical concentration of lecithin with PEO-b-PCL not only made the interface tightly packed, but also enhanced skin permeability.12 Luteolin-loaded nanoemulsions were characterized by DLS (Figure 1B–C). The hydrodynamic mean size of nanoemulsion drops was ~290 nm, therefore exhibiting a slightly bluish white appearance. We also observed the nanoemulsion drops with TEM (Figure 1D). The average diameter of the emulsion drops obtained from direct image analysis was ~250 nm, which is a reasonable size scale when hydrodynamic thickness is considered. The efficiency of epidermal/dermal drug delivery depends on the size of carriers; the smaller size, the deeper it can be delivered.17 Our emulsion droplet size is already included in the length scale where effective drug delivery takes place, so the particle size effect has not been further investigated.


7


Page 8 of 19

Figure 1. (A) Graphical illustration for a luteolin-loaded nanoemulsion drop stabilized with PEO-b-PCL and lecithin. (B) TEM image and (C) size distribution of luteolin-loaded nanoemulsion drops. (D) Appearance of luteolin-loaded nanoemulsion right after preparation. Oil (sweet almond oil) = 20 wt%, Luteolin = 700 µM. Lecithin/PEO-b-PCL = 1/5 (w/w). Total concentration of lecithin/PEO-b-PCL was 6 wt%. Once we established the mechanism of luteolin-loaded nanoemulsion formation, we tried to confirm the stability of luteolin inside the emulsion drops. Luteolin could be dissolved in the oil at elevated temperatures. However, polarized microscopic observation showed that upon cooling the solution, luteolin was separated from the oil phase to form multiple needle-shaped crystals, as shown in Figure 2A. This phase separation occurs due to the π-π stacking interaction between aromatic rings of neighboring chroman-4-one groups, as well as hydrogen bonding between hydroxy groups and carbonyl groups adjacent luteolin molecules.6 When luteolin was


8

Page 9 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


incorporated into our nanoemulsion drops, interestingly, this molecular crystallization could be completely prevented. From polarized microscopic observations of the luteolin-loaded nanoemulsion after a 6-month storage time, we confirmed that there was no generation of new molecular crystals in the emulsion system (Figure 2B-C). This indicated that nucleation and growth of luteolin crystals were entirely suppressed by encapsulating luteolin in the oil drops in the hundreds of nanometers scale, as their periphery was enveloped with a mechanically robust polymer-lipid membrane with a high curvature, thus geometrically hindering crystal growth.16b,18

Figure 2. (A) Bright-field (upper) and polarized (lower) microscope images 3.5 mM luteolin dissolved in sweet almond oil. Bright-field (upper) and polarized (lower) microscope images of luteolin-loaded nanoemulsions: (B) on preparation and (C) after storage for 6 mon at 10 °C. The same amount of luteolin (3.5 mM) was loaded in sweet almond oil nanoemulsions. Dispersion stability of luteolin-loaded nanoemulsions was examined under two different conditions: (1) long-term storage at 4°C for 6 months (Figure 3A-B) and (2) a repeated freezethaw cycling (Figure 3C). As the nanoemulsions tested in these stability tests did not contain any additional stabilizers or thickeners, the emulsion drops were stably suspended in the water continuous phase, with the aid of both thermal motion and drop-to-drop repulsion. Although the average drop size slightly increased by ~14% after long-term storage compared with the initial


9


Page 10 of 19

drop size, mild homogenization recovered the initial size. This indicates that a few drops flocculate to form aggregates during storage, but they were redispersed easily under the application of shear force. This interpretation was also supported by the TEM image for the nanoemulsion drops after 6-month storage, as shown in the insert of Figure 3A. The morphology and size distribution of drops exactly corresponded to those determined by DLS. It was remarkable that the luteolin-loaded nanoemulsion exhibited neither serious increase in drop size nor oil elution even after repeated freeze-thaw cycles during the repeated freeze-thaw emulsion stability test (Figure 3C). This implies that the emulsion system can retain its own structure against such a high level of mechanical stress, coming from water crystal growth during freezing and from volume change by ~10%. This property presumably stems from the effective assembly of PEO-b-PCL and lecithin at the oil-water interface, thus displaying improved interface robustness.

Figure 3. (A) Long-term dispersion stability of a luteolin-loaded nanoemulsion. Hydrodynamic diameter (DH) changes as a function of storage time. The inset is TEM image of emulsion drops after 6 mon-storage. Scale bar is 200 nm. (B) Size distribution by DLS intensity analysis of luteolin-loaded nanoemulsions on preparation and after storage for 6 mon at 4°C. The average droplet sizes were determined before shaking the emulsion samples. (C) Average hydrodynamic size of a luteolin-loaded nanoemulsion with the freeze-thawing cycle. After establishing a method to physically immobilize luteolin in the nanoscale oil core, we tried to confirm how the biological function of luteolin could be affected. In our study, we evaluated the antioxidation performance of luteolin by using the DPPH radical scavenging method.3d For this experiment, three test samples, (1) ascorbic acid in EtOH, (2) luteolin in EtOH,


10

Page 11 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and (3) luteolin-loaded nanoemulsions, were prepared, and their DPPH radical scavenging capacity was evaluated (Figure 4). First, we compared the radical scavenging activity of luteolin with that of ascorbic acid. Luteolin has an intrinsic yellowish color. As this color can mask the discoloration of the test solution induced by DPPH radicals, we concentrated DPPH to 2 mM, which corresponds to a factor of five compared with the normal test concentration. Under these conditions, ascorbic acid showed only ~10% inhibition activity even at 250 µM. By contrast, luteolin showed a sharp increase in inhibition activity in a concentration-dependent manner below 50 µM, meaning luteolin is a strong antioxidant.19 More interestingly, for the luteolinloaded nanoemulsions, no radical inhibitory activity could be obtained from the DPPH assay. This implies that the phenolic hydroxyl groups of luteolin entrapped in the oil drops could not effectively react with DPPH radicals due to the presence of the robust polymer-lipid membrane at the oil-water interface. Therefore, the entrapped luteolin could not exhibit their own antioxidant activity because the membrane did not allow any influx of hydrophilic DPPH radicals from aqueous continuous phase to hydrophobic oil dispersion phase.20

Figure 4. (A) A well plate showing DPPH free radical scavenging activity: (a) ascorbic acid in EtOH, (b) luteolin in EtOH, and (c) luteolin-loaded nanoemulsion (luteolin NE). For this assay, we concentrated DPPH by 2 mM. (B) DPPH free radical scavenging activity as a function of radical scavenger concentration. Values are given as duplicate and expressed as mean±standard deviation.


11


Page 12 of 19

Using the luteolin-loaded nanoemulsions, we investigated hair growth, in vivo, using C57BL/6 mice. Minoxidil was used as a positive control. Luteolin in EtOH was also used for comparison with the luteolin-loaded nanoemulsion. To identify any contribution of the solvent, an aqueous EtOH solution (50 wt%) was used as a control. Each test solution was topically administered to the depilated skin with synchronized hair follicles. The results are shown in Figure 5. After 10 days, the time taken for melanin to form in mouse skin and for initiation of hair growth, a skin color change from pink to black was observed in all mice groups, except in the EtOH control group, indicating that the telogen-to-anagen transition had occurred. After 17 days of topical application, the skins were covered with fully grown hair, showing excellent growth promotion effect without distinctive disparity in hair growth cycle, which was comparable with the controls. In the cases involving the treatment with minoxidil and luteolin, the amount of hair growth appeared to be similar for both the treatments and to that observed under the treatment with luteolin-loaded nanoemulsion. However, the hair-growth areas were uneven among the compared groups. Moreover, when minoxidil was used for treatment, hair became stiff due to epidermal keratinization, which is a known side effect of minoxidil.20 However, for the mice group treated with the luteolin-loaded nanoemulsion, skin surfaces were more evenly covered with shiny and soft hair.

Figure 5. Hair growth promotion of luteolin loaded nanoemulsion vehicles. Telogen matched, 7 week-aged C57BL/6 mice were shaved and test samples were topically applied with thioglycollic acid (80 wt%). C57BL/6 mice were topically treated with an aqueous EtOH solution (50 wt%), 3 wt% minoxidil (n=7), luteolin (700 µM), and a luteolin nanoemulsion (containing 700 µM). The back skins were photographed with a given period of time: 0 day, 10 day, and 17 day after depilation.


12

Page 13 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For a quantitative evaluation of in vivo hair growth, hair follicles in the anagen phase were characterized by counting the number in deep subcutis and by determining their diameter changes (Figure 6A). In the case of treatment with minoxidil, we could clearly observe hyperkeratosis and thickening of epidermis (Figure 6a), which was comparable with the cases involving treatment with luteolin and luteolin-loaded nanoemulsion (Figure 6b-c). Application of luteolin generated slightly more number of hair follicles compared with that the case of application of minoxidil. The increase in the number of hair follicles observed in this experiment was interpreted to be due to the activation of the reduced hair follicles or the increase in the hair follicle density at a given area rather than the formation of new hair follicles. (Figure 6B). It is noteworthy in our observations that when using the luteolin-loaded nanoemulsion for treatment, the generation of thick hair (diameter ≥ 60 µm), was outstanding (Figure 6C). This can be interpreted as a result of the intact penetration of encapsulated luteolin, which can be attributed to lecithin, a component of our emulsion system. In fact, when drugs are formulated with lecithin, they show enhanced skin permeation, since lecithin lowers the permeability barrier of the skin.13, 21

Moreover, utilization of nanoemulsion vehicles can make it more favorable to deliver luteolin

through the hair follicles. Basically, nanoscale vehicles can readily diffuse along with shunt routes, like hair follicles.21 Consequently, the hair follicle shunt route could be a main pathway of the luteolin-loaded nanoemulsion vehicle and their follicular delivery could be promoted when the size of the nanoemulsion drops is decreased to the nanometer scale.

Conclusions In summary, we reported an O/W nanoemulsion system that can not only provide an improved solubility of luteolin in oil droplets, but also act as vehicles for their enhanced follicular delivery. Luteolin was selected as a model drug for enhanced hair growth. In order to overcome the issue of low solubility of luteolin, we encapsulated it in nanoscale emulsion drops. The luteolin-loaded nanoemulsion was formed by coassembly of PEO-b-PCL and lecithin at the O/W interface. The luteolin-loaded nanoemulsions exhibited extraordinary structural stability against repeated freeze-thaw cycling and long-term storage due to the formation of a mechanically robust thin PEO-b-PCL/lecithin membrane at the interface. Based on in vivo hair growth evaluation studies, we demonstrated that the luteolin-loaded nanoemulsion vehicle exhibited the hair growth promotion at a level similar to that of commercially available minoxidil. From the viewpoint of


13


Page 14 of 19

skin safety, it is significant that the nanoemulsion vehicle system using water as a continuous phase exhibited such follicular delivery efficacy. This was due to the well-designed emulsion composition and hair follicle shunt-mediated delivery route facilitated follicular delivery. The results obtained in this study highlight that our nanoemulsion can be used as a useful vehicle for immobilization of luteolin.

Figure 6. (A) Observation of transverse sections of back skins by hematoxylin-eosin (H&E) staining: (a) 3 wt% minoxidil, (b) 700 µM luteolin, and (c) 700 µM luteolin-loaded nanoemulsion. Scale bars are 200 µm. (B) The number of hair follicles in each hair growth cycle. (C) The diameter of hair follicles in deep subcutis. Values are mean ± standard deviation (n = 7).


14

Page 15 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGMENT This research was supported by National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIP) (2016R1A2B2016148).

REFERENCES 1. (a) Dry, F., THE GOAT OF THE MOUSE (M US MUSOULUS). J. Genet 1926, 16 (3), 287-340, DOI: 10.1007/BF02983004; (b) Alonso, L.; Fuchs, E., The hair cycle. J. Cell Sci. 2006, 119 (3), 391-393, DOI: 10.1242/jcs.02793. 2. Iida, M.; Ihara, S.; Matsuzaki, T., Hair cycle-dependent changes of alkaline phosphatase activity in the mesenchyme and epithelium in mouse vibrissal follicles. Dev. Growth Differ. 2007, 49 (3), 185-195, DOI: 10.1111/j.1440-169X.2007.00907.x. 3. (a) Pandurangan, A. K.; Esa, N. M., Luteolin, a Bioflavonoid Inhibits Colorectal Cancer through Modulation of Multiple Signaling Pathways: A Review. Asian Pac. J. Cancer P. 2014, 15 (14), 5501-5508, DOI: 10.7314/Apjcp.2014.15.14.5501; (b) Seelinger, G.; Merfort, I.; Wölfle, U.; Schempp, C. M., Anti-carcinogenic effects of the flavonoid luteolin. Molecules 2008, 13 (10), 2628-2651, DOI: 10.3390/molecules13102628; (c) Xia, N.; Chen, G.; Liu, M.; Ye, X.; Pan, Y.; Ge, J.; Mao, Y.; Wang, H.; Wang, J.; Xie, S., Anti-inflammatory effects of luteolin on experimental autoimmune thyroiditis in mice. Exp. Ther. Med. 2016, 12 (6), 4049-4054, DOI: 10.3892/etm.2016.3854; (d) Seelinger, G.; Merfort, I.; Schempp, C. M., Anti-oxidant, antiinflammatory and anti-allergic activities of luteolin. Planta Med. 2008, 74 (14), 1667-1677, DOI: 10.1055/s-0028-1088314; (e) Nabavi, S. F.; Braidy, N.; Gortzi, O.; Sobarzo-Sanchez, E.; Daglia, M.; Skalicka-Wozniak, K.; Nabavi, S. M., Luteolin as an anti-inflammatory and neuroprotective agent: A brief review. Brain Res. Bull. 2015, 119, 1-11, DOI: 10.1016/j.brainresbull.2015.09.002; (f) Leopoldini, M.; Pitarch, I. P.; Russo, N.; Toscano, M., Structure, conformation, and electronic properties of apigenin, luteolin, and taxifolin antioxidants. A first principle theoretical study. J. Phys. Chem. A 2004, 108 (1), 92-96, DOI: 10.1021/Jp035901j ; (g) Kwon, S. M.; Kim, S.; Song, N. J.; Chang, S. H.; Hwang, Y. J.; Yang, D. K.; Hong, J. W.; Park, W. J.; Park, K. W., Antiadipogenic and proosteogenic effects of luteolin, a major dietary flavone, are mediated by the induction of DnaJ (Hsp40) Homolog, Subfamily B, Member 1. J. Nutr. Biochem. 2016, 30, 24-32, DOI: 10.1016/j.jnutbio.2015.11.013. 4. (a) Guo, A. J.; Choi, R. C.; Cheung, A. W.; Chen, V. P.; Xu, S. L.; Dong, T. T.; Chen, J. J.; Tsim, K. W., Baicalin, a flavone, induces the differentiation of cultured osteoblasts: an action via the Wnt/beta-catenin signaling pathway. J. Biol. Chem. 2011, 286 (32), 27882-27893, DOI: 10.1074/jbc.M111.236281; (b) Nash, L. A.; Sullivan, P. J.; Peters, S. J.; Ward, W. E., Rooibos flavonoids, orientin and luteolin, stimulate mineralization in human osteoblasts through the Wnt pathway. Mol. Nutr. Food Res. 2015, 59 (3), 443-453, DOI: 10.1002/mnfr.201400592. 5. Sowa, M.; Slepokura, K.; Matczak-Jon, E., Cocrystals of fisetin, luteolin and genistein with pyridinecarboxamide coformers: crystal structures, analysis of intermolecular interactions, spectral and thermal characterization. Crystengcomm 2013, 15 (38), 7696-7708, DOI: 10.1039/c3ce41285g. 6. Dacunha-Marinho, B.; Martinez, A.; Estevez, R. J., 7-hydroxy-5-methoxy-6,8dimethylflavanone: a natural flavonoid. Acta Crystallogr. C. 2008, 64 (Pt 6), o353-356, DOI: 10.1107/S0108270108014959.


15


Page 16 of 19

7. Dang, H.; Meng, M. H. W.; Zhao, H.; Iqbal, J.; Dai, R.; Deng, Y.; Lv, F., Luteolin-loaded solid lipid nanoparticles synthesis, characterization, & improvement of bioavailability, pharmacokinetics in vitro and vivo studies. J. Nanopart. Res. 2014, 16 (4), 2347, DOI: 10.1007/s11051-014-2347-9. 8. (a) Heurtault, B.; Saulnier, P.; Pech, B.; Proust, J. E.; Benoit, J. P., Physico-chemical stability of colloidal lipid particles. Biomaterials 2003, 24 (23), 4283-4300, DOI: 10.1016/S0142-9612(03)00331-4; (b) Triplett, M. D.; Rathman, J. F., Optimization of β-carotene loaded solid lipid nanoparticles preparation using a high shear homogenization technique. J. Nanopart. Res. 2009, 11 (3), 601-614, DOI: 10.1007/s11051-008-9402-3. 9. Liu, B.; Li, W.; Zhao, J.; Liu, Y.; Zhu, X.; Liang, G., Physicochemical characterisation of the supramolecular structure of luteolin/cyclodextrin inclusion complex. Food Chem. 2013, 141 (2), 900-906, DOI: 10.1016/j.foodchem.2013.03.097. 10. (a) Qureshi, W. A.; Zhao, R. F.; Wang, H.; Ji, T. J.; Ding, Y. P.; Ihsan, A.; Mujeeb, A.; Nie, G. J.; Zhao, Y. L., Co-delivery of doxorubicin and quercetin via mPEG-PLGA copolymer assembly for synergistic anti-tumor efficacy and reducing cardio-toxicity. Sci. Bull. 2016, 61 (21), 1689-1698, DOI: 10.1007/s11434-016-1182-z; (b) Curcio, M.; Spizzirri, U. G.; Cirillo, G.; Spataro, T.; Picci, N.; Iemma, F., Tailoring Flavonoids' Antioxidant Properties Through Covalent Immobilization Into Dual Stimuli Responsive Polymers. Int. J. Polym. Mater. Polym. Biomater. 2015, 64 (11), 587-596, DOI: 10.1080/00914037.2014.996708; (c) Khan, A. W.; Kotta, S.; Ansari, S. H.; Sharma, R. K.; Ali, J., Enhanced dissolution and bioavailability of grapefruit flavonoid Naringenin by solid dispersion utilizing fourth generation carrier. Drug Dev. Ind. Pharm. 2015, 41 (5), 772-779, DOI: 10.3109/03639045.2014.902466; (d) Pal, S.; Saha, C., Solvent effect in the synthesis of hydrophobic drug-loaded polymer nanoparticles. IET Nanobiotechnol. 2017, 11 (4), 443-447, DOI: 10.1049/iet-nbt.2016.0116; (e) Caldas Dos Santos, T.; Rescignano, N.; Boff, L.; Reginatto, F. H.; Simoes, C. M. O.; de Campos, A. M.; Mijangos, C., In vitro antiherpes effect of C-glycosyl flavonoid enriched fraction of Cecropia glaziovii encapsulated in PLGA nanoparticles. Mater. Sci. Eng. C Mater. Biol. Appl. 2017, 75, 1214-1220, DOI: 10.1016/j.msec.2017.02.135. 11. (a) Li, Y.; Zheng, J.; Xiao, H.; McClements, D. J., Nanoemulsion-based delivery systems for poorly water-soluble bioactive compounds: Influence of formulation parameters on Polymethoxyflavone crystallization. Food Hydrocoll. 2012, 27 (2), 517-528, DOI: 10.1016/j.foodhyd.2011.08.017; (b) Sutradhar, K. B.; Amin, M., Nanoemulsions: increasing possibilities in drug delivery. Euro. J. Nanomed. 2013, 5 (2), 97-110, DOI: 10.1515/ejnm-20130001. 12. Shin, K.; Gong, G.; Cuadrado, J.; Jeon, S.; Seo, M.; Choi, H. S.; Hwang, J. S.; Lee, Y.; Fernandez-Nieves, A.; Kim, J. W., Structurally Stable Attractive Nanoscale Emulsions with Dipole-Dipole Interaction-Driven Interdrop Percolation. Chem. Eur. J. 2017, 23 (18), 42924297, DOI: 10.1002/chem.201604722. 13. (a) Rancan, F.; Blume-Peytavi, U.; Vogt, A., Utilization of biodegradable polymeric materials as delivery agents in dermatology. Clin. Cosmet. Investig. Dermatol. 2014, 7, 23-34, DOI: 10.2147/CCID.S39559; (b) Li, L.; Hoffman, R. M., Topical liposome delivery of molecules to hair follicles in mice. J. Dermatol. Sci. 1997, 14 (2), 101-108, DOI: 10.1016/S0923-1811(96)00557-9. 14. (a) Slominski, A.; Paus, R., Melanogenesis is coupled to murine anagen: toward new concepts for the role of melanocytes and the regulation of melanogenesis in hair growth. J. Invest. Dermatol. 1993, 101 (s 1), 90-97, DOI: 10.1016/0022-202X(93)90507-E; (b) Stenn, K.; Paus, R.;


16

Page 17 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Dutton, T.; Sarba, B., Glucocorticoid effect on hair growth initiation: a reconsideration. Skin Pharmacol. Physiol. 1993, 6 (2), 125-134, DOI: 10.1159/000211097; (c) Paus, R.; Stenn, K.; Link, R., Telogen skin contains an inhibitor of hair growth. Br. J. Dermatol. 1990, 122 (6), 777784, DOI: 10.1111/j.1365-2133.1990.tb06266.x; (d) Zhang, Y,; Han, L,; Chen, S. S.; Guan, J,; Qu, F. Z.; Zhao, Y. Q., Hair growth promoting activity of cedrol isolated from the leaves of Platycladus orientalis. Biomed. Pharmacother. 2016, 83, 641-647, DOI: 10.1016/j.biopha.2016.07.022; (e) Lee, H. J.; Oh, D. W.; Na, M. J.; Kim, D. W.; Yuk, D. Y.; Choi, H. C.; Lee, Y. B.; Han, K.; Park, C. W., Preparation and in vivo evaluation of lecithinbased microparticles for topical delivery of minoxidil. Arch. Pharm. Res. 2017, 40 (8), 943-951, DOI: 10.1007/s12272-017-0934-x; (f) MuÈller-RoÈver, S.; Handjiski, B.; van der Veen, C.; Èller, S. E.; Foitzik, K.; McKay, I. A.; Stenn, K. S.; Paus, R., A Comprehensive Guide for the Accurate Classification of Murine Hair Follicles in Distinct Hair Cycle Stages, J. Invest. Dermatol. 2001, 117 (1), 3-15, DOI: 10.1046/j.0022-202x.2001.01377.x; (g) Kwack, M. H.; Kim, M. K.; Kim, J. C.; Sung, Y. K., Dickkopf 1 Promotes Regression of Hair Follicles, J. Invest. Dermatol. 2012, 132 (6), 1554-1560, DOI: 10.1038/jid.2012.24. 15. (a) Shim, J.; Seok Kang, H.; Park, W. S.; Han, S. H.; Kim, J.; Chang, I. S., Transdermal delivery of mixnoxidil with block copolymer nanoparticles. J. Control Release 2004, 97 (3), 477-84, DOI: 10.1016/j.jconrel.2004.03.028; (b) Barnes, T. J.; Prestidge, C. A., PEO− PPO− PEO Block Copolymers at the Emulsion Droplet− Water Interface. Langmuir 2000, 16 (9), 4116-4121, DOI: 10.1021/la991217d; (c) Nguyen-Misra, M.; Mattice, W. L., Micellization and gelation of symmetric triblock copolymers with insoluble end blocks. Macromolecules 1995, 28 (5), 1444-1457, DOI: 10.1021/ma00109a015. 16. (a) Shin, K.; Kim, J. W.; Park, H.; Choi, H. S.; Chae, P. S.; Nam, Y. S.; Kim, J. W., Fabrication and stabilization of nanoscale emulsions by formation of a thin polymer membrane at the oil–water interface. RSC Adv. 2015, 5 (57), 46276-46281, DOI: 10.1039/c5ra03872c; (b) Park, H.; Han, D. W.; Kim, J. W., Highly stable phase change material emulsions fabricated by interfacial assembly of amphiphilic block copolymers during phase inversion. Langmuir 2015, 31 (9), 2649-2654, DOI: 10.1021/la504424u. 17. (a) Verma, D. D.; Verma, S.; Blume, G.; Fahr, A., Particle size of liposomes influences dermal delivery of substances into skin, Int. J. Pharm. 2003, 258, 141-151, DOI: 10.1016/S03785173(03)00183-2; (b) Hua, S., Lipid-based nano-delivery systems for skin delivery of drugs and bioactives, Front. Pharmacol. 2015, 6 (219), 1-5, DOI: 10.3389/fphar.2015.00219. 18. Günther, E.; Schmid, T.; Mehling, H.; Hiebler, S.; Huang, L. Subcooling in hexadecane emulsions. Int. J. Refrig. 2010, 33 (8), 1605-1611, DOI: 10.1016/j.ijrefrig.2010.07.022. 19. Mishra, K.; Ojha, H.; Chaudhury, N. K., Estimation of antiradical properties of antioxidants using DPPH assay: A critical review and results. Food Chem. 2012, 130 (4), 10361043, DOI: 10.1016/j.foodchem.2011.07.127. 20. Lee, S. M.; Choi, M. J.; Lee, Y. M.; Jin, B. S., Preparation and Characterization of Ethosome Containing Hydrophobic Flavonoid Luteolin. J. Kor. Ind. Eng. Chem. 2010, 21 (1), 40-45, DOI: 10.14478/ace. 21. (a) Ackerman, B. H.; Townsend, M. E.; Golden, W.; Bryan, A. B., Pruritic rash with actinic keratosis and impending exfoliation in a patient with hypertension managed with minoxidil. Drug Intell. Clin. Pharm. 1988, 22 (9), 702-703, DOI: 10.1177/106002808802200912; (b) Navarini, A. A.; Ziegler, M.; Kolm, I.; Weibel, L.; Huber, C.; Trueb, R. M., Minoxidil-induced trichostasis spinulosa of terminal hair. Arch. Dermatol. 2010, 146 (12), 1434-1435, DOI: 10.1001/archdermatol.2010.363.


17


Page 18 of 19

22. El Maghraby, G. M.; Williams, A. C., Vesicular systems for delivering conventional small organic molecules and larger macromolecules to and through human skin. Expert Opin. Drug Deliv. 2009, 6 (2), 149-163, DOI: 10.1517/17425240802691059.


18

Page 19 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical abstract


19

Nanoemulsion Vehicles as Carriers for Follicular Delivery of Luteolin

Recommend Documents