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Article Type : Original Article
Physical Characterization and In Vitro Skin Permeation of Solid Lipid Nanoparticles for Transdermal Delivery of Quercetin
Saet Byeol Han, Soon Sik Kwon, Yoo Min Jeong, Eun Ryeong Yu and Soo Nam Park*
Department of Fine Chemistry, Nanobiocosmetic Laboratory, and Cosmetic R&D Center, Seoul National University of Science and Technology, 232 Gongneung-ro, Nowon-gu, Seoul 139-743, Republic of Korea * Corresponding author at: Department of Fine Chemistry, Nanobiocosmetic Laboratory, and Cosmetic R&D Center, Seoul National University of Science and Technology, 232 Gongneung-ro, Nowon-gu, Seoul 139-743, Republic of Korea Tel: +82-2-970-6451, Fax: +82-2-972-9585 E-mail address: [email protected] (S.N.Park)
Synopsis OBJECTIVE: Quercetin, a phenolic compound isolated from plants, can act as an antioxidant to protect the skin from oxidative stress induced by ultraviolet rays. The aims of this work were (i) to compare the physical characterization of quercetin loaded solid lipid nanoparticles (QSLNs) and (ii) to investigate the enhanced skin permeation of quercetin using QSLNs. METHODS: QSLNs were prepared with a certain amount lipid (palmitic acid) and the different ratio of surfactant (Tween® 80) by homogenization and ultrasonification method. RESULTS: QSLNs showed mono-dispersed particle size distribution in the ranges of 274.0 to 986.6 nm and zeta potential from –50.4 to −29.4 mV. Entrapment efficiency of QSLN was 15.2–46.2% and their crystallinity index was low (0–18.2%). In vitro occlusion test showed QSLN-2 has the highest
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occlusive effect due to its the smallest particle size (274.0 nm) and through these result, QSLN-2 was selected as the optimum formulation. Transmission electron microscopy (TEM) analysis further confirmed the uniform spherical shape of QSLN-2 particles. Field emission–scanning electron microscope (FE-SEM) analysis and histological observation of hairless rat skin showed that the lipid particles of QSLN-2 formed a fused lipid film and, subsequently, and it hydrated the surface of the rat skin. Franz diffusion cell was used to measure in vitro skin permeation of quercetin dissolved in propylene glycol (QPG), QSLN-2, and QSLN-3. The results showed that QSLN-2 (33.5 µg/cm2, 21.9%) exhibited higher skin permeability than QPG (6.6 µg/cm2, 4.2 %) and QSLN-3 (14.2 µg/cm2, 9.1%), which was visually confirmed by confocal laser scanning microscope (CLSM) image analysis as well. CONCLUSION: The results suggest that QSLN-2, prepared with a surfactant content of 2%, could be used as useful skin delivery system for transdermal delivery of hydrophobic antioxidants such as quercetin.
Keywords: solid lipid nanoparticles (SLNs), quercetin, physical characterization, skin hydration, skin permeation
1. Introduction Skin protects our body from external harmful materials; however, it is aged by various endogenic and environmental factors. Intrinsic aging refers to cutaneous changes occurring not as a result of any environmental factors but as a result of internal factors over the course of time. Extrinsic aging refers to cutaneous changes occurring due to external environmental factors such as ultraviolet (UV) rays, environmental pollution, and microorganisms[1-3]. Among these, consistent exposure to UV rays in daily life generates reactive oxygen species (ROS) which cause photo-oxidative damages to skin and accelerates skin aging. ROS such as superoxide anion radical (O2·-), hydrogen peroxide (H2O2), singlet oxygen (1O2), and hydroxyl radical (·OH) are harmful due to their higher oxidative potential. Further, peroxyl radical (ROO·) and alkoxyl radical (RO·), which are formed by the reaction between the ROS and body composition are also included as ROS. The skin has an antioxidative barrier for self-protection from these ROS. Enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione
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peroxidase (GSPHx), and non-enzymatic antioxidants such as vitamin C (L-ascorbic acid), vitamin E (α-tocopherol), carotenoid, and flavonoids form complementary antioxidative defense network for the skin. However, excessive ROS, formed by extreme exposure to UV rays, collapses the antioxidative barrier. These results in acceleration of skin aging as follows: Lipids, proteins, and DNA that comprise cells are oxidized. Further, degradation of collagen and elastin fibers, which are main components of the skin, occurs and the melanin formation reaction is stimulated[4]. To complement the oxidative imbalance and prevent skin aging, additional antioxidants should be supplemented externally. The known external antioxidants are either synthetic or natural products. Among these, the natural antioxidants gained significant attention in recent years due to the synthetic antioxidant’s instability and undesired side-effects[5]. Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one, Fig. 1) is a flavonoid with phenolic functional groups, and it is found abundantly in plants such as apple and onion[6,7]. Quercetin is known for its excellent anticancer, antiinflammatory, and antioxidation efficacy, and especially for its radical scavenging ability and cellular protective effect against erythrocyte damage induced by 1O2[8-11]. However, quercetin has certain limitations due to its low solubility in water and instability under light and heat[12,13]. Therefore, a way to enhance the practical utilization of quercetin by overcoming the disadvantages has to be identified. Skin can be largely divided into epidermis, dermis, and subcutaneous fat. Stratum corneum, the outermost layer of epidermis, is in direct contact with the external environment. This layer is composed of corneocytes, which are dead keratinocytes, and a lipid matrix surrounding the corneocytes. It forms a sturdy protecting barrier by preventing inflow of external harmful material into the body, and also act as penetration barrier by blocking the delivery of active ingredients into the skin[14-16]. Therefore, development of a robust skin delivery system is required to enhance the delivery of active ingredients. Several skin delivery systems to enhance the absorption of active ingredients have been developed, e.g., oil-in-water (O/W) emulsion, liposomes, and polymer nanoparticles. However, these drug carriers have limitations, such as lack of physical stability and expensive manufacturing process, towards large-scale practical applications[17]. Solid lipid
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nanoparticles (SLNs) have been studied by Müller as an alternative for these carriers since early 1990s[18]. SLNs are prepared by replacing liquid oil of an emulsion with a solid lipid, and are in solid state at room and body temperature. SLNs can effectively encapsulate drug molecules, increase their stability, and control drug release rate. Further, SLNs can be produced without any organic solvent in large quantity through simple manufacturing process[17,19]. In addition, the fine particles of SLNs are highly adhesive to the skin surface. Thus, when they are spread on the skin, they form a lipid film. The lipid film prevents evaporation of water in the skin surface and hydrates the skin, thereby enhancing the skin absorption of active ingredients. In fact, SLN-based cosmetic formulations have been investigated extensively in recent years[16,20,21]. In this study, we intended to compare the physical characterization of quercetin loaded SLNs (QSLNs) and to investigate the enhanced skin permeation of quercetin using QSLNs. QSLNs were prepared using palmitic acid and the non-ionic surfactant, Tween® 80. The physical characterization (e.g., particle size, zeta potential, crystallinity index, occlusive effect, and entrapment efficiency) of the QSLNs were thoroughly investigated. The optimum formulations of QSLNs for enhancing skin delivery of quercetin were identified, by comparing the physical characterizations. Further, its morphological observation using TEM, FE-SEM analysis and histological observation of hairless rat skin on which the formulation was applied were performed. Finally, the enhanced skin permeability of quercetin using QSLNs was evaluated using Franz diffusion cell and CLSM analysis.
2. Materials and methods
2.1.
Materials
Palmitic acid, used as solid lipid for preparing QSLNs, and quercetin were purchased from Sigma (USA) and Tween® 80 (polysorbate 80) surfactant was kindly donated by Saimdang, Co. Ltd. (Korea). Other solvents used such as ethanol, methanol, and ethyl acetate were analytical grade. ;
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2.2.
Preparation of QSLNs
QSLNs were prepared by a homogenization and ultrasonification method[22] and the composition ratio of QSLNs is shown in Table Ⅰ. Palmitic acid was heated to 70 °C using hot plate/magnetic stirrer (MISUNG SCIENTIFIC, Korea) and quercetin (dissolved in ethanol and ethyl acetate 1 : 1, v/v %) was added together, and the molten mixture was stirred (300 rpm, 70 °C, 1 min). Aqueous surfactant solutions were prepared by adding the different ratio of Tween® 80 (0.1, 0.5, 1, 2, 3 and 4%) to 10 mL distilled water and stirred at 70 °C. The aqueous surfactant solution was poured into the lipid phase and the mixture was stirred (300 rpm, 70 °C, 3 min), to form an O/W emulsion and then further homogenized (9000 rpm, 3 min) using a homogenizer (ULTRA TURRAX® T25 Basic). Subsequently, the emulsion was ultrasonicated with 30% amplitude for 10 min using ultrasonicator (BRANSON, USA) and the homogenization and ultrasonification processes were constantly maintained at 70 °C. Finally, QSLNs suspension was prepared by quickly cooling the nanoemulsion at 4 °C.
2.3.
Analysis of particle size and zeta potential
The particle size distribution and zeta potential of QSLNs were measured with a particle size and zeta potential analyzer using OTSUKA ELS-Z series (Japan). QSLNs were diluted with distilled water, and analyzed at 25 °C with 165° scattering angle. All measurements were performed in triplicate.
2.4.
Differential scanning calorimetry (DSC) analysis
The crystallinity index of QSLNs was measured by differential scanning calorimetry (DSC) analysis using a Shimadzu DSC-60 series instrument (Japan). A lipid bulk of approximately 5 mg or QSLNs suspension containing the same amount of lipid was weighed in aluminum pan. An empty aluminum pan was used as reference as well. The analysis was performed from 20 to 100 °C at a heating rate of 5 °C/min. The crystallinity index (CI %) of QSLNs was calculated using equation (1) [23].
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∆HQSLNs aqueous dispersion CI (%)
=
∆Hlipid bulk × Concentrationlipid phase
× 100 %
-
(1)
where ∆HQSLNs aqueous dispersion and ∆Hlipid bulk are the melting enthalpy (J/g) of QSLNs dispersion and lipid bulk, respectively.
2.5.
In vitro occlusive effect test
In vitro occlusive effect test was performed to evaluate the occlusion factor (F) of QSLNs[24]. 50 mL beakers were poured with 20 mL of distilled water, covered with cellulose acetate membrane filter (Advantec, 47 mm, cutoff size: 3 µm, Japan), and sealed. QSLNs suspension (300 µL) was spread evenly on the filter surface (17.3 cm2) and the beakers were stored at 32 °C with a relative humidity of 50 - 55% for 36 h. The beaker covered with filter without QSLNs dispersion was chosen as control. The weight of the water loss was measured at 0, 6, 12, 24, and 36 h. The occlusion factor (F) was calculated using the equation (2) [25].
F = 100 × { ( A - B ) / A }
-
(2)
where A is the water loss in control and B is the water loss with QSLNs dispersion
2.6.
Entrapment efficiency
The prepared QSLNs suspension was filtered through 5 µm syringe filter (Minisart CA, 26 mm) to get rid of any free quercetin crystals. Then methanol was added to the filtered QSLNs dispersion and sonicated for 1 h to extract quercetin from lipid. Methanol was evaporated using rotary evaporator and the residue was re-dissolved in 1 mL methanol. The amount of quercetin incorporated was detected by high-performance liquid chromatography (HPLC). The HPLC (Shimadzu, Japan) system consisted of LC-20AT pump, SPE-M20A UVVis detector and Ship-pack (VP-ODS) C18 analytical column (5 µm, 250×4.6 mm). The mobile phase was a mixture of 2% (v/v %) acetic acid in distilled water and 0.5% (v/v %) acetic acid in 50% (v/v %) acetonitrile. The detection wavelength was 370 nm, and the flow
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rate was 1.0 mL/min at room temperature. The standard calibration curve for quercetin (concentration of 20-200 µM) is shown in Fig. 2. In addition, the entrapment efficiency of quercetin was calculated using equation (3).
Entrapment efficiency (%) = Wentrapped / Winitial × 100
-
(3)
where Wentrapped is the amount of quercetin passing through 5 µm syringe filter and W initial is the initially added amount of quercetin
2.7.
Transmission electron microscopy (TEM) analysis
TEM analysis was performed for the morphological observation of QSLNs using a JEOLJEM1010 instrument (JEOL Ltd., Japan). The QSLNs suspension, diluted with distilled water, was dropped into copper grids and dried. Then, the particle surface was stained with 0.2% (w/v %) phosphotungstic acid and dried. The analysis was performed at an accelerating voltage of 80 kV.
2.8.
Field-emission scanning electron microscopy (FE-SEM) analysis
A FE-SEM instrument, JSM-6700F (JEOL Ltd., Japan), was used to observe the formation of QSLNs lipid film. The dried QSLNs suspension was attached to double-sided carbon tape, subsequently to a metal stub. It was stained with gold using Cressington 208HR (Cressington Scientific Instruments Ltd., UK) at 20 mA for 300 s. The analysis was performed at an accelerating voltage of 20 kV.
2.9.
Histological skin observation
The histological skin observation was performed to visually confirm the occlusive effect of QSLNs and skin hydration. The dorsal skin was obtained from hairless rat (8 weeks, female) and subcutaneous fat and tissue were removed from the skin, and the skin was stored at – 70 °C. The excised hairless rat skin was fixed between donor and receptor chambers of the Franz diffusion cell, exposing the stratum corneum side upwards. QSLNs suspension (0.5
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mL) was applied to the skin, which was drawn from the Franz diffusion cell after 24 h, and the superfluous suspension on the skin was washed with phosphate buffered saline (PBS). The treated skin was stored in 10% formalin to fix the tissue. The skin tissue treated with paraffin was cut to a thickness of about 3 - 5 µm using Rotary microtome HM340 (Microtome, Japan) and stained with hematoxylin and eosin (H&E). The microscopic observation of the skin surface was performed with 200 magnifications using BX41TF (Olympus, Japan)
2.10. In vitro skin permeation study In vitro skin permeation study was performed to investigate the effect of QSLNs on the enhancement of the skin permeation of quercetin using the Franz diffusion cell. The dorsal skin for the skin permeation study was obtained from ICR mice (8 weeks, female) and subcutaneous fat and tissue were removed from the skin and then the skin was stored at – 70 °C. The frozen skin was thawed along with PBS just before the experiment and the receptor phase (5 mL) (HCO-60 (PEG-60 hydrogenated castor oil) : EtOH : PBS = 2 : 20 : 78 ((w/w/w %)) was filled in the receptor chamber. The thawed skin was fixed between the donor and receptor chamber exposing the stratum corneum side upwards and QSLNs suspension (0.2 mL) was applied to the skin surface through the donor. The skin area contacting with the receptor phase was 0.6362 cm2 and the receptor medium was kept at 37 ± 1 °C throughout the experiment using a constant-temperature water bath. At fixed interval of time (3, 6, 9, 12, and 24 h), 0.5 mL receptor phase was taken out and replaced with fresh receptor phase. The amount of quercetin in the sample was analyzed by HPLC. After 24 h, the skin was drawn from the Franz diffusion cell and the superfluous suspension on the skin surface was washed with PBS in triplicate. Subsequently, the area of the skin in contact with the formulation was cutoff, and tape stripping was carried out with the skin surface therein. The stratum corneum of the skin surface was removed in triplicate using the tape; ethanol (10 mL) was added to the tape pieces and ultrasonicated for 1 h to extract quercetin. Subsequently, the ethanol was evaporated using rotary evaporator and the residue was dissolved with receptor phase (0.5 mL). The left-over skin after tape stripping
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was cut into small pieces, and treated in the same way as tape stripping. The amount of quercetin in the samples obtained was detected with HPLC.
2.11. Confocal laser scanning microscopy (CLSM) analysis CSLM analysis was performed to confirm the enhanced skin permeation of quercetin with QSLNs visually using (CLSM Exciter (Carl-Zeiss, Germany)). The newly prepared SLNs were prepared with 0.015% of fluorescein isothiocyanate (FITC), which is a hydrophobic fluorescent probe, instead of quercetin. The dorsal skin was obtained from hairless rat (8 weeks, female) and subcutaneous fat and tissue were removed from the skin to investigate the enhanced skin permeation of FITC loaded SLNs. The Franz diffusion cell was prepared as described earlier using FITC loaded SLNs. The suspension (0.5 mL) was applied to the skin surface through donor, the skin was drawn from the Franz diffusion cell after 24 h, and the superfluous suspension on the skin was washed with PBS. Subsequently, the area of the skin in contact with the formulation were cutoff and the skin surface was frozen in cryomold, filled with OCT embedding matrix (CellPath Ltd., UK) at –70 °C. The frozen skin tissue was cut to a thickness of about 10 µm using cryotome (HM505E, Germany). Subsequently, the cross-sectioned skin tissue was treated with 4',6-diamidino-2phenylindole (DAPI, Sigma) at room temperature to stain the nucleus of skin cells and washed with PBS after 20 min. FITC entrapped in SLNs permeated into the rat skin was processed with the CLSM. The excitation wavelength of DAPI and FITC were 405 and 488 nm, respectively and the emission wavelength range of DAPI and FITC were 420-480 and 505-530 nm, respectively.
2.12. Statistical analysis All the experiments were performed in triplicate and statistical data were analyzed by the Student’s t-test at the level of P = 0.05.
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3. Results and discussion
3.1.
Particle size
We prepared 0.05% of quercetin loaded SLNs with palmitic acid (lipid) and different ratio (0.1, 0.5, 1, 2, 3 and 4%) of Tween® 80 (surfactant), and we aimed to observe the change in the particle size caused by the different surfactant ratios (Fig. 3). With increasing concentration of surfactant from 0.1, 0.5 to 1%, the particle size decreased from 416.9 ± 11.4, 373.7 ± 7.8 to 341.2 ± 1.0 nm, respectively. When the concentration of the surfactant was increased by 2%, the QSLN formulation with smallest particle size of 274.0 ± 14.5 nm was obtained. This is because a higher amount of the surfactant decreases the interfacial tension between lipid and aqueous phase[26,27]. The decreased interfacial tension may restrain the random aggregation of lipid particles and may further facilitate the particle partition, presumably leading to a reduction in the particle size of QSLNs. However, when the concentration of the surfactant was increased by 3 and 4%, the particle sizes also increased by 532.2 ± 9.6 and 986.6 ± 72.2 nm, respectively. This phenomenon is the result of excessive addition of surfactant molecules onto SLNs surfaces. When SLNs are prepared, surfactant molecules cover the surface of lipid molecules via hydrophobic interactions between hydrophobic groups such as alkyl chains of the surfactant and lipid molecules. However, excessive addition of surfactant beyond a certain lipid concentration may lead to continuous accumulation of the surfactant on the surface of the lipid molecules, causing a rapid increase in the particle size [28,29].
3.2.
Zeta potential
Zeta potential is the electrostatic potential in the shear plane of particles and indicates the degree of attraction or repulsion between adjacent and similarly charged particles. For the fine particles or colloids, the absolute value of zeta potential is high, suggesting that the repulsive force between the particles is high, thus leading to stable colloids. On the other hand, when the absolute value of zeta potential is low, the repulsive force between the
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particles decreases, resulting in aggregation of the particles. Therefore, the value of zeta potential can be used for evaluating the stability of the formulation[30]. In this study, we compared zeta potential corresponding to various concentrations of the surfactant (Fig. 4). The zeta potential of QSLN-0.1 was -50.4 mV ± 0.0, which is the highest negative electric charge value among the prepared formulations. However, when the concentration of surfactant was increased with 0.5%, the negative electric charge decreased to -38.7 ± 0.6 mV. When the concentration of the surfactant was further increased (1, 2, 3 and 4%) the negative electric charge value gradually decreased (-31.7 ± 0.2, -31.3 ± 0.3, 30.3 ± 0.3 and -29.4 ± 0.4 mV, respectively). Above mentioned, the surfactant covers lipid particle surfaces due to its amphipathic property. Presumably, when the concentration of the non-ionic surfactant such as Tween® 80 was increased, it might have covered the wider particle surfaces, thereby decreasing the overall negative electric charge of the formulation[31]. The absolute values of the zeta potential of QSLNs prepared in this study were around 30 mV. Such a high absolute value of zeta potential contributes to the stability of the formulation by preventing aggregation between particles[32].
3.3.
DSC analysis
DSC analysis was performed to measure the change in the amount of heat produced from physicochemical change, due to the change in sample temperature. Especially for QSLNs, the phase change or crystallinity of particles can be measured using DSC analysis[32,33]. In this study, we aimed to measure the crystallinity index of particle through melting point and the change in the amount of heat for QSLNs (Table Ⅱ). Melting points of the QSLNs prepared were found to be low in comparison with that of the bulk lipid. The fine particle size of QSLNs, the presence of surfactant, or the addition of drug may result in a decrease in the melting point[32]. We observed that the crystallinity indices of all newly prepared QSLNs are below 20%. Such nanoparticles may exist in a form similar to particles of an O/W emulsion rather than in a dispersion of lipid particles[32-36].
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3.4.
In vitro occlusive effect
The occlusive effect to skin is an unusual property of the fine lipid particles such as SLNs, and it acts as the main cause for skin hydration. In other words, the fine lipid particles spread on the skin may form thin lipid film and restrain the moisture of the skin from evaporating by the occlusive effect of thin lipid film. The hydration of stratum corneum mediated by this occlusive effect not only facilitates distribution of drugs, but also increases the permeation of drugs through its structural transformation. Further, the occlusive effect is influenced by particle size, concentration of lipid, crystallinity of lipid phase, and the amount of sample spread on the skin[16,24, 37,38]. In this study, we aimed to measure the occlusion factor (F) and investigate the effect of particle size and crystallinity index of QSLNs on the occlusive effect (Fig. 5). The early occlusion factor (12 h) of QSLN-0.1 was 28.5 ± 2.0, which is the lowest value among the prepared QSLNs formulations. We observed that when the concentration of surfactant was increased by 0.5, 1, and 2%, the early occlusion factors of the corresponding QSLNs increased by 48.0 ± 1.7, 47.9 ± 0.9, and 62.3 ± 1.9, respectively. However, when the concentration of surfactant was increased by 3 and 4%, the early occlusion factors of QSLN3 and QSLN-4 were found to be decreased by 50.1 ± 0.6 and 37.9 ± 0.8, respectively. The same tendencies were observed after 24 and 36 h of the experiment, as well. This phenomenon can be explained by particle size and crystallinity index of QSLNs. Wissing et al. suggested that the formulation with small particle size have the higher occlusive effect than that with big particle size[37]. In other words, with the smaller particle size, an even lipid film can be formed onto membrane filter which effectively restrain moisture from evaporating. As expected, in QSLN-0.1 and QSLN-2, with decrease in the particle size due to increasing surfactant content, showed rise in occlusion factor. On the other hand, in QSLN-3 and QSLN-4, with higher particle size due to excess surfactant, showed low occlusion effect, due to addition of excess surfactant. Especially, the hourly occlusion factors observed for the QSLN-2, with the smallest particle size were 62.3 ± 1.9, 61.1 ± 1.3 and 60.2 ± 1.5, respectively, which were the highest values observed among the prepared formulations in this study.
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Wissing et al. also suggested crystallinity index of lipid phase as the main factor influencing occlusive effect besides particle size[37]. However, QSLN-0.5 and QSLN-1 with higher crystallinity index of 18.2 and 10.6%, respectively, among the formulations have the lower occlusive effect than that QSLN-2, which has low crystallinity index of 2.3%. We assumed that all QSLNs formulations prepared in this study have low crystallinity of below 20%; thus, any small difference in crystallinity is not influencing the occlusive effect.
3.5.
Entrapment efficiency
SLNs are carrier materials suitable for entrapping hydrophobic active ingredients, such as quercetin, which are otherwise difficult to dissolve in aqueous phase. However, apart from the solubility of drug in lipid phase, entrapment efficiency is also influenced by mode of formulation and the surfactant used[17]. In this study, we compared entrapment efficiency of QSLNs towards quercetin in accordance with the concentration of surfactant (Fig. 6). Entrapment efficiency of QSLN-0.1 is 15.2 ± 1.1%, which is the lowest value in the prepared formulations. When the concentration of surfactant was increased by 0.5 and 1%, the corresponding entrapment efficiency of the SLNs also was found to be increased by 18.0 ± 1.1 and 23.9 ± 3.5%, respectively. Further, QSLN-2, QSLN-3 and QSLN-4, with more than 2% of surfactant, showed 46.2 ± 2.2, 42.9 ± 4.2 and 43.8 ± 2.1% for entrapment efficiency, respectively. No statistically significant differences (p > 0.05) between QSLN-2, QSLN-3, and QSLN-4 were observed. Entrapment efficiency of QSLNs might be affected by the surfactant concentration. Namely, it is thought that the increased concentration of surfactant increases quercetin’s solubility in the lipid, resulting in increased amount of quercetin in formulations [39].
3.6.
TEM analysis
The results showed that QSLN-2 has the smallest particle size, the highest occlusive effect, and the most outstanding entrapment efficiency and through the results, QSLN-2 was selected as the optimum formulations for enhancing skin permeation of quercetin. In this study, TEM analysis was performed to observe the morphology of QSLN-2, which showed best performance among the series (Fig. 7).
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The analysis revealed that QSLN-2 has uniform spherical shape with narrow particle size distribution.
3.7.
FE-SEM analysis
FE-SEM analysis was performed to observe the lipid film formed by QSLN-2 (Fig. 8). FESEM analysis showed the formation of a thin film of QSLN-2 caused by complete moisture evaporation, instead of spherical particles observed the TEM analysis. The moisture in the QSLN-2 suspension on the aluminum foil gets evaporated, and as the result, the uniform spherical particles fuse because of capillary force and then form a thin lipid film. We expected that when this lipid film is formed, hydration of the skin stratum corneum will occur as a result of an occlusive effect by the lipid film.
3.8.
Histological skin observation
The histological skin observation was performed to confirm visually occlusive effect of QSLN-2 and skin hydration by the occlusive effect. Quercetin dissolved in propylene glycol solution (QPG) and QSLN-2, as representative optimal formulation, were selected for this experiment, and their skin surface were compared with skin of non-treated sample group (Fig. 9). The skin surface of non-treated sample group existed in form of flat stratum corneum (Fig. 9a). Meanwhile, we observed that the skin surface treated with QPG was more hydrated and swelled than that of non-treated sample group (Fig. 9b). We further observed that the skin surface treated with QSLN-2 was more hydrated, with higher thickness of stratum corneum than that of the non-treated sample and QPG-treated sample due to outstanding skin hydration of QSLN-2 (Fig. 9c). The results suggest that thin lipid film formed on the skin surface treated with QSLNs induces occlusive effect, which causes skin hydration and it result in structural transformation of the stratum corneum, which in turn may enhance skin permeation of active ingredients.
3.9.
In vitro skin permeation study
In vitro skin permeation study was performed to investigate the effect of QSLNs on enhancement of skin permeation of quercetin using the Franz diffusion cell. QPG and QSLN-
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2 were used for in vitro skin permeation study. QSLN-3, with similar entrapment efficiency to QSLN-2, but with the larger particle size and lower occlusive effect, was also selected in this study to compare the difference of skin permeability caused by the difference of particle size and occlusive effect. All three formulations were prepared with 0.05% quercetin and the content of quercetin permeated in the skin was analyzed by HPLC. The amount of quercetin permeated through skin in a constant area (0.6362 cm2) during 24 h is shown in Fig. 9. QSLN-2 showed the higher amount of skin permeation than others. The amount of quercetin permeated through skin using QPG, QSLN-3 and QSLN-2 were found to be 0.5, 2.5 and 9.0 µg/cm2, respectively, in the initial 12 h. QSLN-2 enabled permeation of quercetin through skin by an amount of 29.4 µg/cm2 after 24 h. However, OPG and QSLN-3 permeated only 1.5 and 10.6 µg/cm2 quercetin through skin during the same duration (Fig. 10). Fig. 11 shows the amount of quercetin present in stratum corneum (Tape), in epidermis and dermis except for stratum corneum (Skin), and in receptor phase passing through skin (Transdermal) by quantitative analysis. The amount of quercetin present in stratum corneum (Tape) permeated using QPG, QSLN-2 and QSLN-3 was found to be 0.5, 0.6 and 0.9 µg/cm2, respectively. When these values were compared with the initially applied amount of quercetin (157.2 µg/cm2), the percentage of quercetin present in stratum corneum (Tape) by QPG, QSLN-2 and QSLN-3 was calculated to be 0.3, 0.4 and 0.6%, respectively. The amount of quercetin present in epidermis and dermis except in stratum corneum (Skin) was found to be 4.6, 3.4 and 2.7 µg/cm2 for QPG, QSLN-2 and QSLN-3, respectively (Fig. 11a). When these values were compared with the initially applied amount of quercetin, it was confirmed that 2.9, 2.2 and 1.7% of quercetin existed in epidermis and dermis except in stratum corneum for QPG, QSLN-2 and QSLN-3, respectively (Fig. 11b). Lastly, the percentage of quercetin present in receptor phase passing through skin (Transdermal) was 1.0, 18.7 and 6.7% for QPG, QSLN2 and QSLN-3, respectively. The experiments indicated that among the three experimental formulations QSLN-2 enabled the highest amount of quercetin to permeate through skin. The total amount of quercetin permeated in skin was 6.6, 33.5 and 14.2 µg/cm2 for QPG, QSLN-2 and QSLN-3, respectively. When these values were compared with initially applied
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amount of quercetin, the percentage for QPG, QSLN-2 and QSLN-3 was 4.2, 21.3 and 9.1%, respectively. Taken together, QSLN-2 is the optimal formulation enabling 5.1 and 2.4 times higher quercetin permeation through skin in comparison to other formulations QPG (control) and QSLN-3, respectively.
3.10. CLSM analysis In this study, CLSM analysis was performed to confirm the enhanced skin permeation of active ingredients by SLNs visually. Instead of quercetin, 0.015% of FITC, which is a hydrophobic fluorescent probe, was used as active ingredients and the degree of skin permeation of FITC was observed (Table Ⅲ). FITC dissolved in propylene glycol solution (FPG) and FITC loaded solid lipid nanoparticles (FSLN-2 and FSLN-3) were selected as experimental formulations for CLSM analysis.(Except quercetin was replaced with FITC, other components of FSLN formulations were identical to QSLN formulations.) After 24 h, FITC dissolved in propylene glycol solution was observed only in the upper layer of epidermis and it did not permeate into stratum basal in which keratinocyte exist. On the other hand, FITC entrapped in FSLN-2 was observed plentifully in the upper layer of epidermis and also in the middle layer of dermis through stratum basal in which keratinocyte exist. FITC entrapped in FSLN-3 was also observed in stratum corneum and stratum basal, but it was visible in the upper layer of dermis, unlike FSLN-2. Those results correspond to the results of in vitro skin permeation study using the Franz diffusion cell and confirm that SLNs can enhance skin permeation of active ingredients.
4. Conclusion In this study, quercetin was selected as antioxidant for protecting skin from oxidative stress induced by ultraviolet rays, and to enhance transdermal delivery of quercetin, QSLNs were prepared with a certain amount of palmitic acid and the different ratio of Tween® 80. By comparing the physical characterizations such as particle size, zeta potential, crystallinity index, in vitro occlusive effect and entrapment efficiency, QSLN-2 was confirmed as optimized formulation. Through morphological observation using TEM analysis, FE-SEM analysis and histological observation, we observed that thin lipid film formed by QSLN-2
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induces skin hydration as a result of an occlusive effect. Finally, the enhanced skin permeability of quercetin using QSLN-2 was confirmed using Franz diffusion cell and CLSM analysis. The results suggest that QSLN-2 which was confirmed as optimized QSLNs could be used as useful skin delivery system for transdermal delivery of hydrophobic antioxidants such as quercetin.
Acknowledgements This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea(Grant No. HN10C0001).
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10. Stewart, L.K., Soileau, J.L., Ribnicky, D., Wang, Z.Q., Raskin, I., Poulev, A., Majewski, M., Cefalu, W.T. and Gettys, T.W. Quercetin transiently increases energy expenditure but persistently decreases circulating markers of inflammation in C57BL/6J mice fed a high-fat diet. Metabolism., 57, 39-46 (2008). 11. Davis, J.M., Murphy, E.A., Carmichael, M.D. and Davis, B. Quercetin increases brain and muscle mitochondrial biogenesis and exercise tolerance. Am. J. Physiol. Regul. Integr. Comp. Physiol., 296, 1071-1077 (2009). 12. Shi, G., Rao, L., Yu, H., Xiang, H., Yang, H. and Ji, R. Stabilization and encapsulation of photosensitive resveratrol within yeast cell. Int. J. Pharm., 349, 83-93 (2008). 13. Thiele, J.J., Scjroeter, C., Hsieh, S.N., Podda, M. and Parker, L. The antioxidant network of the stratum corneum. Curr. Probl. Dermatol., 29, 26-42 (2001). 14. Honeywell-Nguyen, P.L. and Bouwstra, J.A. Vesicles as a tool for transdermal and dermal delivery. Drug Discov. Today., 2, 67-74 (2005). 15. Morrow, D.I.J., McCarron, P.A., Woolfson, A.D. and Donnelly, R.F. Innovative strategies for enhancing topical and transdermal drug delivery. The Open Drug Delivery Journal., 1, 36-59 (2007). 16. Benson, H.A.E. Transdermal drug delivery: Penetration enhancement techniques. Curr. Durg Deliv., 2, 23-33 (2005). 17. Müller, R.H., Mäder, K. and Gohla, S. Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art. Eur. J. Pharm. Biopharm., 50, 161-177 (2000). 18. Pardeike, J., Hommoss, A. and Müller, R.H. Lipid nanoparticles (SLN, NLC) in cosmetic and pharmaceutical dermal products. Int. J.Pharm., 366, 170-184 (2009). 19. Kotikalapudi, L.S., Adepu, L., VijayaRatna, J. and Diwan, P.V. Formulation and invitro characterization of domperidone loaded solid lipid nanoparticles. Int. J. Pharm. Biomed. Res., 3, 22-29 (2012). 20. Tan, Q., Liu, W., Guo, C. and Zhai, G. Preparation and evaluation of quercetin-loaded lecithinchitosan nanoparticles for topical delivery. Int. J. Nanomedicine., 6, 1621-1630 (2011). 21. Bose, S. and Michniak-Kohn, B. Preparation and characterization of lipid based nanosystems for topical delivery of quercetin. Eur. J. Pharm. Sci., 48, 442-452 (2013). 22. Xie, S., Zhu, L., Dong, Z., Wang, Y., Wang, X. and Zhou, W.Z. Preparation and evaluation of ofloxacin-loaded palmitic acid solid lipid nanoparticles. Int. J. Nanomedicine., 6, 547-555 (2011).
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23. Khurana, S. and Bedi, P.M.S. Development of nanostructured lipid carriers for controlled delivery of mefenamic acid. Int. J. Biomedical Nanoscience and Nanotechnology., 2, 232-250 (2012). 24. De Vringer, T. Topical preparation containing a suspension of solid lipid particles. Eurpean Patent No.91200664 (1992). 25. Wissing, S.A. and Müller, R.H. The influence the crystallinity of lipidnanoparticles on their occlusive properties. Int. J. Pharm., 242, 377-379 (2002). 26. Mulla, J.A.S. Hiremath, S.P. and Sharma, N.K. Repaglinide loaded solid lipid nanoparticles: Design and characterization. J. Pharm. Sci., 2, 41-49 (2012). 27. Asasutjarit, R., Sorrachaitawatwong, C., Tipchuwong, N. and Pouthai, S. Effect of formulation compositions on particle size and zeta potential of diclofenac sodium-loaded chitosan nanoparticles. World Academy of Science, Engineering and Technology., 81, 457-459 (2013). 28. Thakkar, H.P., Desai, L.L. and Parmar, M.P. Application of Box-Behnken design for optimization of formulation parameters for nanostructured lipid carriers of candesartan cilexetil. Asian J. Pharm., 8, 8189 (2014). 29. Yoo, H.J. and Kim, K.S. Preparation and drug release profiles of solid lipid nanoparticles(SLN). J. Kor. Pharm. Sci., 26, 125-135 (1996). 30. Kim, B.M. The preparation and stabilization of solid lipid nanoparticles with lipids. Department of Chemical Engineering Graduate School of Soongsil University, Republic of Korea., 1-137 (2011). 31. Wang, Z., Liu, W., Xu, H. and Yang, X. Preparation and in vitro studies of stealth PEGylated PLGA nanoparticles as carriers for arsenic trioxide. Chin. J. Chem. Eng., 15, 795-801 (2007). 32. Han, F., Li, S., Yin, R. Liu, H. and Xu, L. Effect of surfactants on the formation and characterization of a new type of colloidal drug delivery system: Nanostructured lipid carriers. Colloids Surf. A Physicochem. Eng. Asp., 315, 210-216 (2008). 33. Lee, G.S. Properties of Bis-ethylhexyloxyphenolmethoxyphenyl -triazine (BEMT) loaded solid lipid nano-particle (SLN) and cosmetic application. Graduate School of Konkuk University, Republic of Korea., 1-89 (2008). 34. Mehnet, W. and Mäder, K. Solid lipid nanoparticles production, characterization and applications. Adv. Drug Delivery Rev., 47, 165-196 (2001). 35. Souto, E.B., Müller, R.H. and Gohla, S. A novel approach based on lipid nanoparticles (SLN) for topical delivery of α-lipoic acid. J. Microencapsul., 22, 581-592 (2005).
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36. Langevelde, A.V., Malssen, K.V., Hollander, F., Peschar, R. and Schenk, H. Structure of mono-acid even-numbered β-triacylglycerols. Acta. Cryst., B55, 114-122 (1999). 37. Wissing, S.A., Lippacher, A. and Müller, R.H. Investigations on the occlusive properties of solid lipid nanoparticles (SLN). J. Cosmet. Sci., 52, 313-324 (2001). 38. Choi, W.S., Cho, H.I., Lee, H.S., Lee, S.H. and Choi, Y.W. Enhanced occlusiveness of nanostructured lipid carrier (NLC)-based carbogel as a skin moisturizing vehicle. J. Pharm. Investig., 40, 373-378 (2010). 39. Ekambaram, P. and Abdul, H.S.A. Formulation and evaluation of solid lipid nanoparticles of ramipril. J. Young. Pharm., 3, 216-220 (2011).
Figure legends
Figure 1. Structure of quercetin.
Figure 2. Standard calibration curve of quercetin (Equation of line: y = 81780x – 245482, R2 = 0.9992).
Figure 3. Particle size of QSLN formulations (*p < 0.05)
Figure 4. Zeta potential of QSLN formulations.
Figure 5. Occlusion factors (F) of OSLN formulations (*p < 0.05 versus QSLN-2).
Figure 6. Entrapment efficiency of QSLN formulations.
Figure 7. A representative TEM image of QSLN-2.
Figure 8. FE-SEM image of dried QSLN-2 lipid film on aluminum foil.
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Figure 9. Micrograph (200×) of vertical section of hairless rat skin after hematoxylin and eosin staining: (a) non-treated skin, (b) skin treated with QPG and (c) skin treated with QSLN-2.
Figure 10. In vitro permeation profiles of quercetin using QPG, QSLN-2 and QSLN-3 through ICR mice skin.
Figure 11. Proportions of permeated amount of quercetin using QPG, QSLN-2 and QSLN-3 through ICR mice skin after 24 h incubation (Tape: stratum corneum, Skin: epidermis and dermis except stratum corneum, Transdermal: receptor chamber).
Table legends
Table Ⅰ Composition of QSLN formulations
Table Ⅱ Melting point, enthalpy, and crystallinity index of QSLN formulations
Table Ⅲ CLSM images of a cross-section of hairless rat skin incubating with FPG, FSLN-2 and FSLN-3 containing FITC for 24 h (Size bar 50 µm)
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Table Ⅰ Composition of QSLN formulations Formulation code
PAa) (w/v %)
T80b) (w/v %)
Quec) (w/v %)
QSLNd)-0.1
5
0.1
0.5
QSLN-0.5
5
0.5
0.5
QSLN-1
5
1
0.5
QSLN-2
5
2
0.5
QSLN-3
5
3
0.5
QSLN-4
5
4
0.5
PAa) : Palmitic acid, T80b) : Tween® 80, Quec) : Quercetin, QSLNd) : Quercetin-loaded solid lipid nanoparticles formulations.
Table Ⅱ Melting point, enthalpy, and crystallinity index of QSLN formulations Formulation code Melting point (℃) Enthalpy (J/g) Crystallinity index (%) PA bulk
65.3
195.2
100
QSLN-0.1
63.5
4.9
2.5
QSLN-0.5
63.0
35.5
18.2
QSLN-1
61.8
20.7
10.6
QSLN-2
63.5
4.4
2.3
QSLN-3
61.2
5.9
3.0
QSLN-4
N.D.a)
N.D
N.D
N.D.a) : not detectable
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Table Ⅲ CLSM images of a cross-section of hairless rat skin incubating with FPG, FSLN-2 and FSLN-3 containing FITC for 24 h (Size bar 50 µm) DAPI
FITC
FPG
FSLN-2
FSLN-3
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Figure 1. Structure of quercetin.
Figure 2. Standard calibration curve of quercetin (Equation of line: y = 81780x - 245482, R2 = 0.9992).
Figure 3. Particle size of QSLN formulations (*p < 0.05).
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Figure 4. Zeta potential of QSLN formulations.
Figure 5. Occlusion factors (F) of OSLN formulations (*p < 0.05 versus QSLN-2).
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Figure 6. Entrapment efficiency of QSLN formulations.
Figure 7. A representative TEM image of QSLN-2.
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Figure 8. FE-SEM image of dried QSLN-2 lipid film on aluminum foil.
(a)
(b)
(c)
Figure 9. Micrograph (200×) of vertical section of hairless rat skin after hematoxylin and eosin staining: (a) non-treated skin, (b) skin treated with QPG and (c) skin treated with QSLN-2.
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Figure 10. In vitro permeation profiles of quercetin using QPG, QSLN-2 and QSLN-3 through ICR mice skin. (a)
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(b)
Figure 11. Proportions of permeated amount of quercetin using QPG, QSLN-2 and QSLN-3 through ICR mice skin after 24 h incubation (Tape: stratum corneum, Skin: epidermis and dermis except stratum corneum, Transdermal: receptor chamber).
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Article Type : Original Article
Physical Characterization and In Vitro Skin Permeation of Solid Lipid Nanoparticles for Transdermal Delivery of Quercetin
Saet Byeol Han, Soon Sik Kwon, Yoo Min Jeong, Eun Ryeong Yu and Soo Nam Park*
Department of Fine Chemistry, Nanobiocosmetic Laboratory, and Cosmetic R&D Center, Seoul National University of Science and Technology, 232 Gongneung-ro, Nowon-gu, Seoul 139-743, Republic of Korea * Corresponding author at: Department of Fine Chemistry, Nanobiocosmetic Laboratory, and Cosmetic R&D Center, Seoul National University of Science and Technology, 232 Gongneung-ro, Nowon-gu, Seoul 139-743, Republic of Korea Tel: +82-2-970-6451, Fax: +82-2-972-9585 E-mail address: [email protected] (S.N.Park)
Synopsis OBJECTIVE: Quercetin, a phenolic compound isolated from plants, can act as an antioxidant to protect the skin from oxidative stress induced by ultraviolet rays. The aims of this work were (i) to compare the physical characterization of quercetin loaded solid lipid nanoparticles (QSLNs) and (ii) to investigate the enhanced skin permeation of quercetin using QSLNs. METHODS: QSLNs were prepared with a certain amount lipid (palmitic acid) and the different ratio of surfactant (Tween® 80) by homogenization and ultrasonification method. RESULTS: QSLNs showed mono-dispersed particle size distribution in the ranges of 274.0 to 986.6 nm and zeta potential from –50.4 to −29.4 mV. Entrapment efficiency of QSLN was 15.2–46.2% and their crystallinity index was low (0–18.2%). In vitro occlusion test showed QSLN-2 has the highest
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/ics.12160 This article is protected by copyright. All rights reserved.
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occlusive effect due to its the smallest particle size (274.0 nm) and through these result, QSLN-2 was selected as the optimum formulation. Transmission electron microscopy (TEM) analysis further confirmed the uniform spherical shape of QSLN-2 particles. Field emission–scanning electron microscope (FE-SEM) analysis and histological observation of hairless rat skin showed that the lipid particles of QSLN-2 formed a fused lipid film and, subsequently, and it hydrated the surface of the rat skin. Franz diffusion cell was used to measure in vitro skin permeation of quercetin dissolved in propylene glycol (QPG), QSLN-2, and QSLN-3. The results showed that QSLN-2 (33.5 µg/cm2, 21.9%) exhibited higher skin permeability than QPG (6.6 µg/cm2, 4.2 %) and QSLN-3 (14.2 µg/cm2, 9.1%), which was visually confirmed by confocal laser scanning microscope (CLSM) image analysis as well. CONCLUSION: The results suggest that QSLN-2, prepared with a surfactant content of 2%, could be used as useful skin delivery system for transdermal delivery of hydrophobic antioxidants such as quercetin.
Keywords: solid lipid nanoparticles (SLNs), quercetin, physical characterization, skin hydration, skin permeation
1. Introduction Skin protects our body from external harmful materials; however, it is aged by various endogenic and environmental factors. Intrinsic aging refers to cutaneous changes occurring not as a result of any environmental factors but as a result of internal factors over the course of time. Extrinsic aging refers to cutaneous changes occurring due to external environmental factors such as ultraviolet (UV) rays, environmental pollution, and microorganisms[1-3]. Among these, consistent exposure to UV rays in daily life generates reactive oxygen species (ROS) which cause photo-oxidative damages to skin and accelerates skin aging. ROS such as superoxide anion radical (O2·-), hydrogen peroxide (H2O2), singlet oxygen (1O2), and hydroxyl radical (·OH) are harmful due to their higher oxidative potential. Further, peroxyl radical (ROO·) and alkoxyl radical (RO·), which are formed by the reaction between the ROS and body composition are also included as ROS. The skin has an antioxidative barrier for self-protection from these ROS. Enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione
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peroxidase (GSPHx), and non-enzymatic antioxidants such as vitamin C (L-ascorbic acid), vitamin E (α-tocopherol), carotenoid, and flavonoids form complementary antioxidative defense network for the skin. However, excessive ROS, formed by extreme exposure to UV rays, collapses the antioxidative barrier. These results in acceleration of skin aging as follows: Lipids, proteins, and DNA that comprise cells are oxidized. Further, degradation of collagen and elastin fibers, which are main components of the skin, occurs and the melanin formation reaction is stimulated[4]. To complement the oxidative imbalance and prevent skin aging, additional antioxidants should be supplemented externally. The known external antioxidants are either synthetic or natural products. Among these, the natural antioxidants gained significant attention in recent years due to the synthetic antioxidant’s instability and undesired side-effects[5]. Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one, Fig. 1) is a flavonoid with phenolic functional groups, and it is found abundantly in plants such as apple and onion[6,7]. Quercetin is known for its excellent anticancer, antiinflammatory, and antioxidation efficacy, and especially for its radical scavenging ability and cellular protective effect against erythrocyte damage induced by 1O2[8-11]. However, quercetin has certain limitations due to its low solubility in water and instability under light and heat[12,13]. Therefore, a way to enhance the practical utilization of quercetin by overcoming the disadvantages has to be identified. Skin can be largely divided into epidermis, dermis, and subcutaneous fat. Stratum corneum, the outermost layer of epidermis, is in direct contact with the external environment. This layer is composed of corneocytes, which are dead keratinocytes, and a lipid matrix surrounding the corneocytes. It forms a sturdy protecting barrier by preventing inflow of external harmful material into the body, and also act as penetration barrier by blocking the delivery of active ingredients into the skin[14-16]. Therefore, development of a robust skin delivery system is required to enhance the delivery of active ingredients. Several skin delivery systems to enhance the absorption of active ingredients have been developed, e.g., oil-in-water (O/W) emulsion, liposomes, and polymer nanoparticles. However, these drug carriers have limitations, such as lack of physical stability and expensive manufacturing process, towards large-scale practical applications[17]. Solid lipid
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nanoparticles (SLNs) have been studied by Müller as an alternative for these carriers since early 1990s[18]. SLNs are prepared by replacing liquid oil of an emulsion with a solid lipid, and are in solid state at room and body temperature. SLNs can effectively encapsulate drug molecules, increase their stability, and control drug release rate. Further, SLNs can be produced without any organic solvent in large quantity through simple manufacturing process[17,19]. In addition, the fine particles of SLNs are highly adhesive to the skin surface. Thus, when they are spread on the skin, they form a lipid film. The lipid film prevents evaporation of water in the skin surface and hydrates the skin, thereby enhancing the skin absorption of active ingredients. In fact, SLN-based cosmetic formulations have been investigated extensively in recent years[16,20,21]. In this study, we intended to compare the physical characterization of quercetin loaded SLNs (QSLNs) and to investigate the enhanced skin permeation of quercetin using QSLNs. QSLNs were prepared using palmitic acid and the non-ionic surfactant, Tween® 80. The physical characterization (e.g., particle size, zeta potential, crystallinity index, occlusive effect, and entrapment efficiency) of the QSLNs were thoroughly investigated. The optimum formulations of QSLNs for enhancing skin delivery of quercetin were identified, by comparing the physical characterizations. Further, its morphological observation using TEM, FE-SEM analysis and histological observation of hairless rat skin on which the formulation was applied were performed. Finally, the enhanced skin permeability of quercetin using QSLNs was evaluated using Franz diffusion cell and CLSM analysis.
2. Materials and methods
2.1.
Materials
Palmitic acid, used as solid lipid for preparing QSLNs, and quercetin were purchased from Sigma (USA) and Tween® 80 (polysorbate 80) surfactant was kindly donated by Saimdang, Co. Ltd. (Korea). Other solvents used such as ethanol, methanol, and ethyl acetate were analytical grade. ;
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2.2.
Preparation of QSLNs
QSLNs were prepared by a homogenization and ultrasonification method[22] and the composition ratio of QSLNs is shown in Table Ⅰ. Palmitic acid was heated to 70 °C using hot plate/magnetic stirrer (MISUNG SCIENTIFIC, Korea) and quercetin (dissolved in ethanol and ethyl acetate 1 : 1, v/v %) was added together, and the molten mixture was stirred (300 rpm, 70 °C, 1 min). Aqueous surfactant solutions were prepared by adding the different ratio of Tween® 80 (0.1, 0.5, 1, 2, 3 and 4%) to 10 mL distilled water and stirred at 70 °C. The aqueous surfactant solution was poured into the lipid phase and the mixture was stirred (300 rpm, 70 °C, 3 min), to form an O/W emulsion and then further homogenized (9000 rpm, 3 min) using a homogenizer (ULTRA TURRAX® T25 Basic). Subsequently, the emulsion was ultrasonicated with 30% amplitude for 10 min using ultrasonicator (BRANSON, USA) and the homogenization and ultrasonification processes were constantly maintained at 70 °C. Finally, QSLNs suspension was prepared by quickly cooling the nanoemulsion at 4 °C.
2.3.
Analysis of particle size and zeta potential
The particle size distribution and zeta potential of QSLNs were measured with a particle size and zeta potential analyzer using OTSUKA ELS-Z series (Japan). QSLNs were diluted with distilled water, and analyzed at 25 °C with 165° scattering angle. All measurements were performed in triplicate.
2.4.
Differential scanning calorimetry (DSC) analysis
The crystallinity index of QSLNs was measured by differential scanning calorimetry (DSC) analysis using a Shimadzu DSC-60 series instrument (Japan). A lipid bulk of approximately 5 mg or QSLNs suspension containing the same amount of lipid was weighed in aluminum pan. An empty aluminum pan was used as reference as well. The analysis was performed from 20 to 100 °C at a heating rate of 5 °C/min. The crystallinity index (CI %) of QSLNs was calculated using equation (1) [23].
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∆HQSLNs aqueous dispersion CI (%)
=
∆Hlipid bulk × Concentrationlipid phase
× 100 %
-
(1)
where ∆HQSLNs aqueous dispersion and ∆Hlipid bulk are the melting enthalpy (J/g) of QSLNs dispersion and lipid bulk, respectively.
2.5.
In vitro occlusive effect test
In vitro occlusive effect test was performed to evaluate the occlusion factor (F) of QSLNs[24]. 50 mL beakers were poured with 20 mL of distilled water, covered with cellulose acetate membrane filter (Advantec, 47 mm, cutoff size: 3 µm, Japan), and sealed. QSLNs suspension (300 µL) was spread evenly on the filter surface (17.3 cm2) and the beakers were stored at 32 °C with a relative humidity of 50 - 55% for 36 h. The beaker covered with filter without QSLNs dispersion was chosen as control. The weight of the water loss was measured at 0, 6, 12, 24, and 36 h. The occlusion factor (F) was calculated using the equation (2) [25].
F = 100 × { ( A - B ) / A }
-
(2)
where A is the water loss in control and B is the water loss with QSLNs dispersion
2.6.
Entrapment efficiency
The prepared QSLNs suspension was filtered through 5 µm syringe filter (Minisart CA, 26 mm) to get rid of any free quercetin crystals. Then methanol was added to the filtered QSLNs dispersion and sonicated for 1 h to extract quercetin from lipid. Methanol was evaporated using rotary evaporator and the residue was re-dissolved in 1 mL methanol. The amount of quercetin incorporated was detected by high-performance liquid chromatography (HPLC). The HPLC (Shimadzu, Japan) system consisted of LC-20AT pump, SPE-M20A UVVis detector and Ship-pack (VP-ODS) C18 analytical column (5 µm, 250×4.6 mm). The mobile phase was a mixture of 2% (v/v %) acetic acid in distilled water and 0.5% (v/v %) acetic acid in 50% (v/v %) acetonitrile. The detection wavelength was 370 nm, and the flow
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rate was 1.0 mL/min at room temperature. The standard calibration curve for quercetin (concentration of 20-200 µM) is shown in Fig. 2. In addition, the entrapment efficiency of quercetin was calculated using equation (3).
Entrapment efficiency (%) = Wentrapped / Winitial × 100
-
(3)
where Wentrapped is the amount of quercetin passing through 5 µm syringe filter and W initial is the initially added amount of quercetin
2.7.
Transmission electron microscopy (TEM) analysis
TEM analysis was performed for the morphological observation of QSLNs using a JEOLJEM1010 instrument (JEOL Ltd., Japan). The QSLNs suspension, diluted with distilled water, was dropped into copper grids and dried. Then, the particle surface was stained with 0.2% (w/v %) phosphotungstic acid and dried. The analysis was performed at an accelerating voltage of 80 kV.
2.8.
Field-emission scanning electron microscopy (FE-SEM) analysis
A FE-SEM instrument, JSM-6700F (JEOL Ltd., Japan), was used to observe the formation of QSLNs lipid film. The dried QSLNs suspension was attached to double-sided carbon tape, subsequently to a metal stub. It was stained with gold using Cressington 208HR (Cressington Scientific Instruments Ltd., UK) at 20 mA for 300 s. The analysis was performed at an accelerating voltage of 20 kV.
2.9.
Histological skin observation
The histological skin observation was performed to visually confirm the occlusive effect of QSLNs and skin hydration. The dorsal skin was obtained from hairless rat (8 weeks, female) and subcutaneous fat and tissue were removed from the skin, and the skin was stored at – 70 °C. The excised hairless rat skin was fixed between donor and receptor chambers of the Franz diffusion cell, exposing the stratum corneum side upwards. QSLNs suspension (0.5
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mL) was applied to the skin, which was drawn from the Franz diffusion cell after 24 h, and the superfluous suspension on the skin was washed with phosphate buffered saline (PBS). The treated skin was stored in 10% formalin to fix the tissue. The skin tissue treated with paraffin was cut to a thickness of about 3 - 5 µm using Rotary microtome HM340 (Microtome, Japan) and stained with hematoxylin and eosin (H&E). The microscopic observation of the skin surface was performed with 200 magnifications using BX41TF (Olympus, Japan)
2.10. In vitro skin permeation study In vitro skin permeation study was performed to investigate the effect of QSLNs on the enhancement of the skin permeation of quercetin using the Franz diffusion cell. The dorsal skin for the skin permeation study was obtained from ICR mice (8 weeks, female) and subcutaneous fat and tissue were removed from the skin and then the skin was stored at – 70 °C. The frozen skin was thawed along with PBS just before the experiment and the receptor phase (5 mL) (HCO-60 (PEG-60 hydrogenated castor oil) : EtOH : PBS = 2 : 20 : 78 ((w/w/w %)) was filled in the receptor chamber. The thawed skin was fixed between the donor and receptor chamber exposing the stratum corneum side upwards and QSLNs suspension (0.2 mL) was applied to the skin surface through the donor. The skin area contacting with the receptor phase was 0.6362 cm2 and the receptor medium was kept at 37 ± 1 °C throughout the experiment using a constant-temperature water bath. At fixed interval of time (3, 6, 9, 12, and 24 h), 0.5 mL receptor phase was taken out and replaced with fresh receptor phase. The amount of quercetin in the sample was analyzed by HPLC. After 24 h, the skin was drawn from the Franz diffusion cell and the superfluous suspension on the skin surface was washed with PBS in triplicate. Subsequently, the area of the skin in contact with the formulation was cutoff, and tape stripping was carried out with the skin surface therein. The stratum corneum of the skin surface was removed in triplicate using the tape; ethanol (10 mL) was added to the tape pieces and ultrasonicated for 1 h to extract quercetin. Subsequently, the ethanol was evaporated using rotary evaporator and the residue was dissolved with receptor phase (0.5 mL). The left-over skin after tape stripping
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was cut into small pieces, and treated in the same way as tape stripping. The amount of quercetin in the samples obtained was detected with HPLC.
2.11. Confocal laser scanning microscopy (CLSM) analysis CSLM analysis was performed to confirm the enhanced skin permeation of quercetin with QSLNs visually using (CLSM Exciter (Carl-Zeiss, Germany)). The newly prepared SLNs were prepared with 0.015% of fluorescein isothiocyanate (FITC), which is a hydrophobic fluorescent probe, instead of quercetin. The dorsal skin was obtained from hairless rat (8 weeks, female) and subcutaneous fat and tissue were removed from the skin to investigate the enhanced skin permeation of FITC loaded SLNs. The Franz diffusion cell was prepared as described earlier using FITC loaded SLNs. The suspension (0.5 mL) was applied to the skin surface through donor, the skin was drawn from the Franz diffusion cell after 24 h, and the superfluous suspension on the skin was washed with PBS. Subsequently, the area of the skin in contact with the formulation were cutoff and the skin surface was frozen in cryomold, filled with OCT embedding matrix (CellPath Ltd., UK) at –70 °C. The frozen skin tissue was cut to a thickness of about 10 µm using cryotome (HM505E, Germany). Subsequently, the cross-sectioned skin tissue was treated with 4',6-diamidino-2phenylindole (DAPI, Sigma) at room temperature to stain the nucleus of skin cells and washed with PBS after 20 min. FITC entrapped in SLNs permeated into the rat skin was processed with the CLSM. The excitation wavelength of DAPI and FITC were 405 and 488 nm, respectively and the emission wavelength range of DAPI and FITC were 420-480 and 505-530 nm, respectively.
2.12. Statistical analysis All the experiments were performed in triplicate and statistical data were analyzed by the Student’s t-test at the level of P = 0.05.
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3. Results and discussion
3.1.
Particle size
We prepared 0.05% of quercetin loaded SLNs with palmitic acid (lipid) and different ratio (0.1, 0.5, 1, 2, 3 and 4%) of Tween® 80 (surfactant), and we aimed to observe the change in the particle size caused by the different surfactant ratios (Fig. 3). With increasing concentration of surfactant from 0.1, 0.5 to 1%, the particle size decreased from 416.9 ± 11.4, 373.7 ± 7.8 to 341.2 ± 1.0 nm, respectively. When the concentration of the surfactant was increased by 2%, the QSLN formulation with smallest particle size of 274.0 ± 14.5 nm was obtained. This is because a higher amount of the surfactant decreases the interfacial tension between lipid and aqueous phase[26,27]. The decreased interfacial tension may restrain the random aggregation of lipid particles and may further facilitate the particle partition, presumably leading to a reduction in the particle size of QSLNs. However, when the concentration of the surfactant was increased by 3 and 4%, the particle sizes also increased by 532.2 ± 9.6 and 986.6 ± 72.2 nm, respectively. This phenomenon is the result of excessive addition of surfactant molecules onto SLNs surfaces. When SLNs are prepared, surfactant molecules cover the surface of lipid molecules via hydrophobic interactions between hydrophobic groups such as alkyl chains of the surfactant and lipid molecules. However, excessive addition of surfactant beyond a certain lipid concentration may lead to continuous accumulation of the surfactant on the surface of the lipid molecules, causing a rapid increase in the particle size [28,29].
3.2.
Zeta potential
Zeta potential is the electrostatic potential in the shear plane of particles and indicates the degree of attraction or repulsion between adjacent and similarly charged particles. For the fine particles or colloids, the absolute value of zeta potential is high, suggesting that the repulsive force between the particles is high, thus leading to stable colloids. On the other hand, when the absolute value of zeta potential is low, the repulsive force between the
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particles decreases, resulting in aggregation of the particles. Therefore, the value of zeta potential can be used for evaluating the stability of the formulation[30]. In this study, we compared zeta potential corresponding to various concentrations of the surfactant (Fig. 4). The zeta potential of QSLN-0.1 was -50.4 mV ± 0.0, which is the highest negative electric charge value among the prepared formulations. However, when the concentration of surfactant was increased with 0.5%, the negative electric charge decreased to -38.7 ± 0.6 mV. When the concentration of the surfactant was further increased (1, 2, 3 and 4%) the negative electric charge value gradually decreased (-31.7 ± 0.2, -31.3 ± 0.3, 30.3 ± 0.3 and -29.4 ± 0.4 mV, respectively). Above mentioned, the surfactant covers lipid particle surfaces due to its amphipathic property. Presumably, when the concentration of the non-ionic surfactant such as Tween® 80 was increased, it might have covered the wider particle surfaces, thereby decreasing the overall negative electric charge of the formulation[31]. The absolute values of the zeta potential of QSLNs prepared in this study were around 30 mV. Such a high absolute value of zeta potential contributes to the stability of the formulation by preventing aggregation between particles[32].
3.3.
DSC analysis
DSC analysis was performed to measure the change in the amount of heat produced from physicochemical change, due to the change in sample temperature. Especially for QSLNs, the phase change or crystallinity of particles can be measured using DSC analysis[32,33]. In this study, we aimed to measure the crystallinity index of particle through melting point and the change in the amount of heat for QSLNs (Table Ⅱ). Melting points of the QSLNs prepared were found to be low in comparison with that of the bulk lipid. The fine particle size of QSLNs, the presence of surfactant, or the addition of drug may result in a decrease in the melting point[32]. We observed that the crystallinity indices of all newly prepared QSLNs are below 20%. Such nanoparticles may exist in a form similar to particles of an O/W emulsion rather than in a dispersion of lipid particles[32-36].
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3.4.
In vitro occlusive effect
The occlusive effect to skin is an unusual property of the fine lipid particles such as SLNs, and it acts as the main cause for skin hydration. In other words, the fine lipid particles spread on the skin may form thin lipid film and restrain the moisture of the skin from evaporating by the occlusive effect of thin lipid film. The hydration of stratum corneum mediated by this occlusive effect not only facilitates distribution of drugs, but also increases the permeation of drugs through its structural transformation. Further, the occlusive effect is influenced by particle size, concentration of lipid, crystallinity of lipid phase, and the amount of sample spread on the skin[16,24, 37,38]. In this study, we aimed to measure the occlusion factor (F) and investigate the effect of particle size and crystallinity index of QSLNs on the occlusive effect (Fig. 5). The early occlusion factor (12 h) of QSLN-0.1 was 28.5 ± 2.0, which is the lowest value among the prepared QSLNs formulations. We observed that when the concentration of surfactant was increased by 0.5, 1, and 2%, the early occlusion factors of the corresponding QSLNs increased by 48.0 ± 1.7, 47.9 ± 0.9, and 62.3 ± 1.9, respectively. However, when the concentration of surfactant was increased by 3 and 4%, the early occlusion factors of QSLN3 and QSLN-4 were found to be decreased by 50.1 ± 0.6 and 37.9 ± 0.8, respectively. The same tendencies were observed after 24 and 36 h of the experiment, as well. This phenomenon can be explained by particle size and crystallinity index of QSLNs. Wissing et al. suggested that the formulation with small particle size have the higher occlusive effect than that with big particle size[37]. In other words, with the smaller particle size, an even lipid film can be formed onto membrane filter which effectively restrain moisture from evaporating. As expected, in QSLN-0.1 and QSLN-2, with decrease in the particle size due to increasing surfactant content, showed rise in occlusion factor. On the other hand, in QSLN-3 and QSLN-4, with higher particle size due to excess surfactant, showed low occlusion effect, due to addition of excess surfactant. Especially, the hourly occlusion factors observed for the QSLN-2, with the smallest particle size were 62.3 ± 1.9, 61.1 ± 1.3 and 60.2 ± 1.5, respectively, which were the highest values observed among the prepared formulations in this study.
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Wissing et al. also suggested crystallinity index of lipid phase as the main factor influencing occlusive effect besides particle size[37]. However, QSLN-0.5 and QSLN-1 with higher crystallinity index of 18.2 and 10.6%, respectively, among the formulations have the lower occlusive effect than that QSLN-2, which has low crystallinity index of 2.3%. We assumed that all QSLNs formulations prepared in this study have low crystallinity of below 20%; thus, any small difference in crystallinity is not influencing the occlusive effect.
3.5.
Entrapment efficiency
SLNs are carrier materials suitable for entrapping hydrophobic active ingredients, such as quercetin, which are otherwise difficult to dissolve in aqueous phase. However, apart from the solubility of drug in lipid phase, entrapment efficiency is also influenced by mode of formulation and the surfactant used[17]. In this study, we compared entrapment efficiency of QSLNs towards quercetin in accordance with the concentration of surfactant (Fig. 6). Entrapment efficiency of QSLN-0.1 is 15.2 ± 1.1%, which is the lowest value in the prepared formulations. When the concentration of surfactant was increased by 0.5 and 1%, the corresponding entrapment efficiency of the SLNs also was found to be increased by 18.0 ± 1.1 and 23.9 ± 3.5%, respectively. Further, QSLN-2, QSLN-3 and QSLN-4, with more than 2% of surfactant, showed 46.2 ± 2.2, 42.9 ± 4.2 and 43.8 ± 2.1% for entrapment efficiency, respectively. No statistically significant differences (p > 0.05) between QSLN-2, QSLN-3, and QSLN-4 were observed. Entrapment efficiency of QSLNs might be affected by the surfactant concentration. Namely, it is thought that the increased concentration of surfactant increases quercetin’s solubility in the lipid, resulting in increased amount of quercetin in formulations [39].
3.6.
TEM analysis
The results showed that QSLN-2 has the smallest particle size, the highest occlusive effect, and the most outstanding entrapment efficiency and through the results, QSLN-2 was selected as the optimum formulations for enhancing skin permeation of quercetin. In this study, TEM analysis was performed to observe the morphology of QSLN-2, which showed best performance among the series (Fig. 7).
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The analysis revealed that QSLN-2 has uniform spherical shape with narrow particle size distribution.
3.7.
FE-SEM analysis
FE-SEM analysis was performed to observe the lipid film formed by QSLN-2 (Fig. 8). FESEM analysis showed the formation of a thin film of QSLN-2 caused by complete moisture evaporation, instead of spherical particles observed the TEM analysis. The moisture in the QSLN-2 suspension on the aluminum foil gets evaporated, and as the result, the uniform spherical particles fuse because of capillary force and then form a thin lipid film. We expected that when this lipid film is formed, hydration of the skin stratum corneum will occur as a result of an occlusive effect by the lipid film.
3.8.
Histological skin observation
The histological skin observation was performed to confirm visually occlusive effect of QSLN-2 and skin hydration by the occlusive effect. Quercetin dissolved in propylene glycol solution (QPG) and QSLN-2, as representative optimal formulation, were selected for this experiment, and their skin surface were compared with skin of non-treated sample group (Fig. 9). The skin surface of non-treated sample group existed in form of flat stratum corneum (Fig. 9a). Meanwhile, we observed that the skin surface treated with QPG was more hydrated and swelled than that of non-treated sample group (Fig. 9b). We further observed that the skin surface treated with QSLN-2 was more hydrated, with higher thickness of stratum corneum than that of the non-treated sample and QPG-treated sample due to outstanding skin hydration of QSLN-2 (Fig. 9c). The results suggest that thin lipid film formed on the skin surface treated with QSLNs induces occlusive effect, which causes skin hydration and it result in structural transformation of the stratum corneum, which in turn may enhance skin permeation of active ingredients.
3.9.
In vitro skin permeation study
In vitro skin permeation study was performed to investigate the effect of QSLNs on enhancement of skin permeation of quercetin using the Franz diffusion cell. QPG and QSLN-
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2 were used for in vitro skin permeation study. QSLN-3, with similar entrapment efficiency to QSLN-2, but with the larger particle size and lower occlusive effect, was also selected in this study to compare the difference of skin permeability caused by the difference of particle size and occlusive effect. All three formulations were prepared with 0.05% quercetin and the content of quercetin permeated in the skin was analyzed by HPLC. The amount of quercetin permeated through skin in a constant area (0.6362 cm2) during 24 h is shown in Fig. 9. QSLN-2 showed the higher amount of skin permeation than others. The amount of quercetin permeated through skin using QPG, QSLN-3 and QSLN-2 were found to be 0.5, 2.5 and 9.0 µg/cm2, respectively, in the initial 12 h. QSLN-2 enabled permeation of quercetin through skin by an amount of 29.4 µg/cm2 after 24 h. However, OPG and QSLN-3 permeated only 1.5 and 10.6 µg/cm2 quercetin through skin during the same duration (Fig. 10). Fig. 11 shows the amount of quercetin present in stratum corneum (Tape), in epidermis and dermis except for stratum corneum (Skin), and in receptor phase passing through skin (Transdermal) by quantitative analysis. The amount of quercetin present in stratum corneum (Tape) permeated using QPG, QSLN-2 and QSLN-3 was found to be 0.5, 0.6 and 0.9 µg/cm2, respectively. When these values were compared with the initially applied amount of quercetin (157.2 µg/cm2), the percentage of quercetin present in stratum corneum (Tape) by QPG, QSLN-2 and QSLN-3 was calculated to be 0.3, 0.4 and 0.6%, respectively. The amount of quercetin present in epidermis and dermis except in stratum corneum (Skin) was found to be 4.6, 3.4 and 2.7 µg/cm2 for QPG, QSLN-2 and QSLN-3, respectively (Fig. 11a). When these values were compared with the initially applied amount of quercetin, it was confirmed that 2.9, 2.2 and 1.7% of quercetin existed in epidermis and dermis except in stratum corneum for QPG, QSLN-2 and QSLN-3, respectively (Fig. 11b). Lastly, the percentage of quercetin present in receptor phase passing through skin (Transdermal) was 1.0, 18.7 and 6.7% for QPG, QSLN2 and QSLN-3, respectively. The experiments indicated that among the three experimental formulations QSLN-2 enabled the highest amount of quercetin to permeate through skin. The total amount of quercetin permeated in skin was 6.6, 33.5 and 14.2 µg/cm2 for QPG, QSLN-2 and QSLN-3, respectively. When these values were compared with initially applied
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amount of quercetin, the percentage for QPG, QSLN-2 and QSLN-3 was 4.2, 21.3 and 9.1%, respectively. Taken together, QSLN-2 is the optimal formulation enabling 5.1 and 2.4 times higher quercetin permeation through skin in comparison to other formulations QPG (control) and QSLN-3, respectively.
3.10. CLSM analysis In this study, CLSM analysis was performed to confirm the enhanced skin permeation of active ingredients by SLNs visually. Instead of quercetin, 0.015% of FITC, which is a hydrophobic fluorescent probe, was used as active ingredients and the degree of skin permeation of FITC was observed (Table Ⅲ). FITC dissolved in propylene glycol solution (FPG) and FITC loaded solid lipid nanoparticles (FSLN-2 and FSLN-3) were selected as experimental formulations for CLSM analysis.(Except quercetin was replaced with FITC, other components of FSLN formulations were identical to QSLN formulations.) After 24 h, FITC dissolved in propylene glycol solution was observed only in the upper layer of epidermis and it did not permeate into stratum basal in which keratinocyte exist. On the other hand, FITC entrapped in FSLN-2 was observed plentifully in the upper layer of epidermis and also in the middle layer of dermis through stratum basal in which keratinocyte exist. FITC entrapped in FSLN-3 was also observed in stratum corneum and stratum basal, but it was visible in the upper layer of dermis, unlike FSLN-2. Those results correspond to the results of in vitro skin permeation study using the Franz diffusion cell and confirm that SLNs can enhance skin permeation of active ingredients.
4. Conclusion In this study, quercetin was selected as antioxidant for protecting skin from oxidative stress induced by ultraviolet rays, and to enhance transdermal delivery of quercetin, QSLNs were prepared with a certain amount of palmitic acid and the different ratio of Tween® 80. By comparing the physical characterizations such as particle size, zeta potential, crystallinity index, in vitro occlusive effect and entrapment efficiency, QSLN-2 was confirmed as optimized formulation. Through morphological observation using TEM analysis, FE-SEM analysis and histological observation, we observed that thin lipid film formed by QSLN-2
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induces skin hydration as a result of an occlusive effect. Finally, the enhanced skin permeability of quercetin using QSLN-2 was confirmed using Franz diffusion cell and CLSM analysis. The results suggest that QSLN-2 which was confirmed as optimized QSLNs could be used as useful skin delivery system for transdermal delivery of hydrophobic antioxidants such as quercetin.
Acknowledgements This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea(Grant No. HN10C0001).
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23. Khurana, S. and Bedi, P.M.S. Development of nanostructured lipid carriers for controlled delivery of mefenamic acid. Int. J. Biomedical Nanoscience and Nanotechnology., 2, 232-250 (2012). 24. De Vringer, T. Topical preparation containing a suspension of solid lipid particles. Eurpean Patent No.91200664 (1992). 25. Wissing, S.A. and Müller, R.H. The influence the crystallinity of lipidnanoparticles on their occlusive properties. Int. J. Pharm., 242, 377-379 (2002). 26. Mulla, J.A.S. Hiremath, S.P. and Sharma, N.K. Repaglinide loaded solid lipid nanoparticles: Design and characterization. J. Pharm. Sci., 2, 41-49 (2012). 27. Asasutjarit, R., Sorrachaitawatwong, C., Tipchuwong, N. and Pouthai, S. Effect of formulation compositions on particle size and zeta potential of diclofenac sodium-loaded chitosan nanoparticles. World Academy of Science, Engineering and Technology., 81, 457-459 (2013). 28. Thakkar, H.P., Desai, L.L. and Parmar, M.P. Application of Box-Behnken design for optimization of formulation parameters for nanostructured lipid carriers of candesartan cilexetil. Asian J. Pharm., 8, 8189 (2014). 29. Yoo, H.J. and Kim, K.S. Preparation and drug release profiles of solid lipid nanoparticles(SLN). J. Kor. Pharm. Sci., 26, 125-135 (1996). 30. Kim, B.M. The preparation and stabilization of solid lipid nanoparticles with lipids. Department of Chemical Engineering Graduate School of Soongsil University, Republic of Korea., 1-137 (2011). 31. Wang, Z., Liu, W., Xu, H. and Yang, X. Preparation and in vitro studies of stealth PEGylated PLGA nanoparticles as carriers for arsenic trioxide. Chin. J. Chem. Eng., 15, 795-801 (2007). 32. Han, F., Li, S., Yin, R. Liu, H. and Xu, L. Effect of surfactants on the formation and characterization of a new type of colloidal drug delivery system: Nanostructured lipid carriers. Colloids Surf. A Physicochem. Eng. Asp., 315, 210-216 (2008). 33. Lee, G.S. Properties of Bis-ethylhexyloxyphenolmethoxyphenyl -triazine (BEMT) loaded solid lipid nano-particle (SLN) and cosmetic application. Graduate School of Konkuk University, Republic of Korea., 1-89 (2008). 34. Mehnet, W. and Mäder, K. Solid lipid nanoparticles production, characterization and applications. Adv. Drug Delivery Rev., 47, 165-196 (2001). 35. Souto, E.B., Müller, R.H. and Gohla, S. A novel approach based on lipid nanoparticles (SLN) for topical delivery of α-lipoic acid. J. Microencapsul., 22, 581-592 (2005).
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Figure legends
Figure 1. Structure of quercetin.
Figure 2. Standard calibration curve of quercetin (Equation of line: y = 81780x – 245482, R2 = 0.9992).
Figure 3. Particle size of QSLN formulations (*p < 0.05)
Figure 4. Zeta potential of QSLN formulations.
Figure 5. Occlusion factors (F) of OSLN formulations (*p < 0.05 versus QSLN-2).
Figure 6. Entrapment efficiency of QSLN formulations.
Figure 7. A representative TEM image of QSLN-2.
Figure 8. FE-SEM image of dried QSLN-2 lipid film on aluminum foil.
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Figure 9. Micrograph (200×) of vertical section of hairless rat skin after hematoxylin and eosin staining: (a) non-treated skin, (b) skin treated with QPG and (c) skin treated with QSLN-2.
Figure 10. In vitro permeation profiles of quercetin using QPG, QSLN-2 and QSLN-3 through ICR mice skin.
Figure 11. Proportions of permeated amount of quercetin using QPG, QSLN-2 and QSLN-3 through ICR mice skin after 24 h incubation (Tape: stratum corneum, Skin: epidermis and dermis except stratum corneum, Transdermal: receptor chamber).
Table legends
Table Ⅰ Composition of QSLN formulations
Table Ⅱ Melting point, enthalpy, and crystallinity index of QSLN formulations
Table Ⅲ CLSM images of a cross-section of hairless rat skin incubating with FPG, FSLN-2 and FSLN-3 containing FITC for 24 h (Size bar 50 µm)
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Table Ⅰ Composition of QSLN formulations Formulation code
PAa) (w/v %)
T80b) (w/v %)
Quec) (w/v %)
QSLNd)-0.1
5
0.1
0.5
QSLN-0.5
5
0.5
0.5
QSLN-1
5
1
0.5
QSLN-2
5
2
0.5
QSLN-3
5
3
0.5
QSLN-4
5
4
0.5
PAa) : Palmitic acid, T80b) : Tween® 80, Quec) : Quercetin, QSLNd) : Quercetin-loaded solid lipid nanoparticles formulations.
Table Ⅱ Melting point, enthalpy, and crystallinity index of QSLN formulations Formulation code Melting point (℃) Enthalpy (J/g) Crystallinity index (%) PA bulk
65.3
195.2
100
QSLN-0.1
63.5
4.9
2.5
QSLN-0.5
63.0
35.5
18.2
QSLN-1
61.8
20.7
10.6
QSLN-2
63.5
4.4
2.3
QSLN-3
61.2
5.9
3.0
QSLN-4
N.D.a)
N.D
N.D
N.D.a) : not detectable
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Table Ⅲ CLSM images of a cross-section of hairless rat skin incubating with FPG, FSLN-2 and FSLN-3 containing FITC for 24 h (Size bar 50 µm) DAPI
FITC
FPG
FSLN-2
FSLN-3
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Figure 1. Structure of quercetin.
Figure 2. Standard calibration curve of quercetin (Equation of line: y = 81780x - 245482, R2 = 0.9992).
Figure 3. Particle size of QSLN formulations (*p < 0.05).
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Figure 4. Zeta potential of QSLN formulations.
Figure 5. Occlusion factors (F) of OSLN formulations (*p < 0.05 versus QSLN-2).
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Figure 6. Entrapment efficiency of QSLN formulations.
Figure 7. A representative TEM image of QSLN-2.
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Figure 8. FE-SEM image of dried QSLN-2 lipid film on aluminum foil.
(a)
(b)
(c)
Figure 9. Micrograph (200×) of vertical section of hairless rat skin after hematoxylin and eosin staining: (a) non-treated skin, (b) skin treated with QPG and (c) skin treated with QSLN-2.
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Figure 10. In vitro permeation profiles of quercetin using QPG, QSLN-2 and QSLN-3 through ICR mice skin. (a)
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(b)
Figure 11. Proportions of permeated amount of quercetin using QPG, QSLN-2 and QSLN-3 through ICR mice skin after 24 h incubation (Tape: stratum corneum, Skin: epidermis and dermis except stratum corneum, Transdermal: receptor chamber).
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