Skip to main content
Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Preparation and Characterization of Naringenin-Loaded Elastic Liposomes for Topical Application

  • Ming-Jun Tsai ,

    Contributed equally to this work with: Ming-Jun Tsai, Yaw-Syan Fu

    Affiliations Department of Neurology, China Medical University Hospital, Taichung, Taiwan, ROC, School of Medicine, Medical College, China Medical University, Taichung, Taiwan, ROC

  • Yaw-Bin Huang,

    Affiliation School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC

  • Jhih-Wun Fang,

    Affiliation School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC

  • Yaw-Syan Fu ,

    Contributed equally to this work with: Ming-Jun Tsai, Yaw-Syan Fu

    Affiliation Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC

  • Pao-Chu Wu

    pachwu@kmu.edu.tw

    Affiliation School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC

Abstract

Excessive production of radical oxygen species in skin is a contributor to a variety of skin pathologies. Naringenin is a potent antioxidant. The purpose of the present study was to develop elastic liposomes for naringenin topical application. Naringenin-loaded elastic liposomes containing different amounts of Tween 80 and cholesterol were prepared. The physicochemical properties including vesicle size, surface charge, encapsulation efficiency, and permeability capacity were determined to evaluate the effect of components. The stability of formulation and skin irritation caused by drug-loaded elastic liposomes were also evaluated for assessment of the clinical utility of elastic liposomes. Saturated aqueous solution of naringenin and naringenin dissolved in 10% Tween 80 solution (5 mg/mL) were used as the control group. The result showed that in using elastic liposomes as carrier, the deposition amounts in the skin of naringenin were significantly increased about 7.3~11.8-fold and 1.2~1.9-fold respectively, when compared with the saturated aqueous solution and Tween 80 solution-treated groups. The level of drug was more than 98.89±3.90% after 3 months of storage at 4℃. In a skin irritation test, the result showed experimental formulation exhibit considerably less irritating than the positive control (paraformaldehyde-treated) group, suggesting its potential therapeutic application.

Introduction

Reactive oxygen species including hydrogen peroxide, superoxide anion, and singlet oxygen are generated as by-products of cellular metabolism primarily in mitochondria, and play a predominant role in many pathological conditions, including immune suppression, photo-carcinogenesis, and photo-aging [14]. Excessive generation of reactive oxygen species in the skin is a major contributor for various cutaneous pathologies [5]. Using antioxidants to prevent oxidative skin damage appears to be a promising approach. Naringenin (5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one, C15H12O5, MW 272.3) a flavanone found in many citrus fruits, has been proven to possess anti-inflammatory, antioxidant, and free radical scavenger properties [6,7]. Furthermore, previous studies [8,9] reported that naringenin can increase the tyrosinase activity and melanin content, demonstrating naringenin can be used to prevent oxidative skin damage. Nevertheless, naringenin is a poor water-soluble compound and has minimal oral bioavailability (approximately 5.8%) owing to its largely hydrophobic ring structure [1012]. Therefore, the purpose of this study was to design a naringenin formulation for topical administration.

In recent years, nano-scale structures such as microemulsions, ethosomes, liposomes and solid lipid nanoparticles have attracted increasing attention because they can provide a better chance for adhesion to biological membranes while delivering therapeutic drugs in a controlled manner. Moreover, nano-scale structures are capable of increased drug loading, sustained release, and the promise of tissue-specific targeting [1322]. Liposomes are microscopic vesicles with an aqueous core surrounded by one or more outer shell(s) composed of phospholipids in a bilayer. They can incorporate a variety of hydrophilic and hydrophobic drugs, improve the accumulation of the drug at the administration site, and reduce side effects [2326]. Hence, liposomes have been widely used as safe and effective drug vehicles in topical treatment of disease [2730]. Modified liposomes such as elastic liposomes were first described by Cevc and Blume [31]. They consist of phospholipids and a single chain surfactant such as deoxycholate, sodium cholate, Tween 80 or Span 80, which can destabilize the lipid bilayers and provide greater flexibility compared to the liposome itself [3234]. Numerous studies have demonstrated that elastic liposomes could provide potentially deeper permeation of drugs compared to conventional liposomes [15,35,36]. Thus, the present work was aimed at the development of an effective elastic liposome for naringenin topical application. With this purpose, different elastic liposome formulations were prepared. The vesicle size, surface charge and encapsulation efficiency were determined. The permeation properties of drug from these delivery systems through rat-excised skin were evaluated and compared with those of a saturated drug aqueous solution. The stability of formulation and skin irritation caused by drug-loaded elastic liposomes were also evaluated for assessing the clinical utility of elastic liposomes.

Materials and Methods

Materials

Naringenin and hesperetin were purchased from Tokyo Chemical Industry (Tokyo, Japan). Polyoxyethylene sorbitan monooleate (Tween 80) and propylene glycol (PG) was from J. T. Baker (Phillipsburg, USA). Epikuron-200 (containing more than 92% of phosphatidylcholine and others of lysoPC, phosphatidic acids, and triglycerides.) was acquired from Cargill, Inc. (Minnetonka, Minnesota, U.S.). Cholesterol and paraformaldehyde were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). All other chemicals and solvents were of analytical reagent grade.

Naringenin-loaded elastic liposomes preparation

In order to easily evaluate the effect of components, a two-factor three-level factorial design [37] was used to prepare different naringenin-loaded elastic liposome. Each formulation (4 mL) contained 20 mg naringenin and 340 mg other ingredients of cholesterol of 5~15%, Tween 80 of 10~20% and Epikuron-200. Cholesterol and Tween 80 were set as formulation factors. The compositions of all different elastic liposome are listed in Table 1.

thumbnail
Table 1. The composition and physicochemical characteristics of naringenin-loaded elastic liposomes.

https://doi.org/10.1371/journal.pone.0131026.t001

The accurately weighed amounts of naringenin, epikuron, Tween 80 and cholesterol were placed in a round-bottom flask, and the mixture was dissolved in 4 mL mixture solvent of chloroform-methanol at a ratio of 1: 1. The organic solvent was removed by rotary evaporation under reduced pressure at 65°C, and then the solvent traces were removed by maintaining the lipid film under a vacuum overnight. The deposited lipid film was hydrated with 4 mL aqueous solution by a probe-type sonicator (UP50H,Hielscher Ultrasonics, Teltow, Germany) for 10 min at 50 W [34]. The final concentration of drug-loaded liposomes was 5 mg/mL.

Physicochemical characterizes of elastic liposomes determination

The mean vesicle size and zeta potential (surface charge) of the naringenin-loaded elastic liposomes were measured using Zetasizer 3000HSA (Malvern Instruments, Malvern, UK) with a helium-neon laser with a wavelength of 633 nm at room temperature. A 1:80 dilution of the drug-loaded liposome was made using double-distilled water for the measurements. The size values were given as a volume distribution. Analysis time was kept at 60 sec, and average size and zeta potential of the vesicles were determined.

Encapsulation efficiency determination

The entrapment percentage of naringenin loaded in elastic liposomes was determined by an ultracentrifugation method. The sample was centrifuged at 120,000 rpm for 1 h at 4°C in a Hitachi CS150GXL ultracentrifuge (Tokyo, Japan) to separate the incorporated drug from the free form. There were no vesicles in the supernatant after Zetasizer examination. The supernatant was analyzed by high-performance liquid chromatography (HPLC) [38] to determine the drug encapsulation percentage of the total naringenin load. The percentage encapsulation efficiency of naringenin in elastic liposome was calculated as: Each experimental was performed in triplicate, and the data reported is the mean value.

Skin permeation and drug deposition studies

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University (Kaohsiung, Taiwan). The Committee confirmed that the permeation experiment followed the guidelines as set forth by the Guide for Laboratory Fact lines and Care. The in vitro skin permeation and skin deposition studies of naringenin-loaded elastic liposomes (5 mg/ mL) and control group (the saturated aqueous solution of drug and drug dissolved in 10% Tween 80 of 5 mg/ mL) were conducted by using a modified transdermal Franz diffusion cell. The effective diffusion area of the diffusion cell and receptor cell volume were 3.46 cm2 and 20 mL respectively. The abdominal skin was excised from a Sprague-Dawley rat weighing 275–300 g, and then mounted on the receptor chamber with the stratum corneum side facing upward to the donor chamber. Samples of 1 mL were placed in the donor chamber and occluded by parafilm. Twenty mL of pH 7.4 phosphate buffer saline containing 40% PG (drug solubility of 941.50 ±3.54 mg/mL) was placed in the receiver chamber. The temperature of receiver medium was maintained at 37±0.5°C by thermostatic pump and was constantly stirred at 600 rpm by a magnetic stirrer during the experiment. At determined intervals, i.e., 1, 2, 3, 4, 6, 8, 10, and 24 h, one milliliter of receptor medium was withdrawn via the sampling port and was quantified for naringenin level by a modified HPLC method [38].

At the end of the skin permeation experiments, the donor phase was removed and the rat skin was washed with deionized water to remove the residual naringenin on the skin surface. Then, the skin was dried with cotton wool and the stratum corneum (SC) was removed from the rest of the skin by tape-stripping the skin with 11 adhesive cellophane tapes (Scotch Book Tape no. 845, 3M, St Paul, MN) [39,40]. The first tape was discarded. The other stripping tapes were put in glass tubes containing 2.0 mL of methanol and then shaken horizontally for 1 h. The solution was filtered through a 0.45 mm membrane (Sartorius, Goettingen, Germany). Naringenin in filtrate was determined by HPLC. The epidermis was separated from the dermis with heat application at 80°C for 3 min and the help of forceps [41]. Then, the epidermis and dermis were separately cut into small pieces to extract the drug content present in the skin with methanol. The resulting solution was centrifuged for 10 min at 8533 g, and filtered through a 0.45 mm membrane. The filtrate was analyzed for naringenin by HPLC.

Chromatographic condition

Hitachi L-7100 series HPLC system and a LiChroCART RP-18e column (125×4 mm I.D., particle size 5 μm) were used in this study. The detection wave was set at 281 nm. The mobile phase of 0.5% triethylamine (adjusted to pH 3.0 by acetic acid) containing 28% acetonitrile was delivered at a rate of 1.0 mL/min. Hesperetin of 200 μg/mL was used as internal standard. The method was successfully validated with coefficient of variation (CV, %) of 3.72%, relative error (RE, %) of 7.57% and a determination coefficient (r) of 0.9998. The limit of quantitation was 0.1 μg/mL.

Skin irritation determination

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University (Kaohsiung, Taiwan). The committee confirmed that the permeation experiment followed the guidelines as set forth by the Guide for Laboratory Fact Lines and Care. The hair on the abdomen of the rat was shaved before the skin was randomly divided into three study groups. A glass ring with 2.54 cm2 was adhered to the abdomen skin. The experimental naringenin-loaded elastic liposome (F4), aqueous water (normal control group) and 0.8% paraformaldehyde (positive control group) of 0.5 mL were loaded into the glass ring and left for 24 h [28,42]. Then, the tested skin regions were excised and fixed in 4% buffered formaldehyde solution for 24 h. The skins were then embedded in paraffin and sliced transversely. The sections were rehydrated stepwise, stained with hematoxylin and eosin, and observed using an optical microscope (Nikon Eclipse Ci, Tokyo, Japan).

Stability determination

The naringenin-loaded elastic liposome was stored in dark-brown bottles for protection from light. The stability of drug-loaded liposome formulation (F4) was evaluated via physicochemical properties and drug content at 4°C. The physical stability was evaluated by mean vesicle size and zeta potential measurement over a three-month period.

Data analysis

All experimental measurements were performed in triplicate. Result values were expressed as the mean value ±standard deviation. Statistical analysis of differences between the experimental formulations was performed using ANOVA test provided by Winks SDA 6.0 software (Texasoft, Cedar Hill, TX, USA). The post hoc Newman—Keuls test was used to check individual differences between groups. A 0.05 level of probability (p < 0.05) was taken as the level of significance.

Results and Discussion

Typically, liposomes are composed of neutral phospholipids, which are biocompatible molecules. Cholesterol is often added to improve mechanical stability of the bilayer and decrease leakage of the encapsulated materials. Cholesterol has been shown to increase mechanical strength of membrane, affect its elasticity, and increase the packing density of lipid via the “ordering and condensing” effect [4346]. Some studies have indicated that the cholesterol content might be the crucial factor for the effective delivery of liposome-entrapped drugs into the skin [47,48].

Conventional liposomes are reported to remain confined to the upper layer of the SC and to accumulate in the skin appendages, with minimal penetration to deeper tissues, due to their large vesicle size and lower flexibility of membrane [14,4951]. Tween 80 is a single chain surfactant. It can act as an “edge activator” and destabilize the lipid bilayers of traditional liposomes, and this then provides greater flexibility of membrane [36,52,53]. However, liposomes in the present high concentration of Tween 80 are unstable; hence, the effect of concentration of cholesterol and Tween 80 on the physicochemical characteristic and skin permeation capacity of liposome was investigated in this study. Naringenin-loaded elastic liposomes containing different amounts of cholesterol and Tween 80 were prepared.

Vesicle size, zeta potential and encapsulation efficiency

The average vesicle size, zeta potential (surface charge) and encapsulation efficiency of experimental formulations are listed in Table 1. Except for formulations F3 (cholesterol was at a high level and Tween 80 was at a low level) and F7 (cholesterol at low levels and Tween 80 at high levels), the average vesicle size of all formulations ranged from 123.7 to 177.7 nm. The polydispersity index values of the elastic liposomes were obtained in a range of 0.11–0.22, showing homogenous size distribution in all formulations. From Table 1, it can be found that the average size of elastic liposomes tended to become large when formulated with higher levels of cholesterol. On the contrary, the average size tended to diminish, when formulated with higher levels of Tween 80. A possible explanation may be that the edge activator, Tween 80, destabilizes the lipid bilayers of liposomes, thus resulting in smaller vesicles [14]. However, the smallest size was obtained at elastic liposomes containing a medium level of cholesterol and a high level of Tween 80.

The surface charges of experimental formulations ranged from -2.2 to -16.1 mV. The drug-loaded elastic liposomes had lower surface charge when a high level of cholesterol was incorporated. The results agreed with a previous study, which reported that increasing the level of cholesterol in a phospholipid membrane decreases surface charge in the physiological environment [54].

Numerous studies have reported that the elastic liposome could significantly increase solubility of hydrophobic and hydrophilic compounds [15,55]. Naringenin is a poor water-soluble compound; its solubility in water was 41.76 ±0.51 μg/mL. In this study, 5 mg/mL of drug was loaded into the elastic liposomes. The encapsulation efficiency of all experimental formulations was larger than 99%, indicating that elastic liposomes should be a good carrier for naringenin.

Skin permeation and drug deposition

The cumulative amount transported through rat skin was plotted as a function of time, and the linear regression analysis was used to determine the permeation rate (flux) and permeation mechanism of drug. The result showed that the permeation profiles followed a zero-order model (R2 > 0.9915). The permeation rate, cumulative amount at 24 h and deposition amount in three skin layers including SC, epidermis and dermis layer after 24 h treated with naringenin-loaded elastic liposomes with different levels of cholesterol and Tween 80 are presented in Figs 1 and 2. The saturated aqueous solution and 5 mg/mL of drug dissolved in 10% Tween 80 solution were used as control groups to evaluate the enhancement effect of formulations. The permeation rate and cumulative amounts at 24 h were 0.25±0.1 μg/h/cm3 and 4.8±2.6 μg/cm3 for saturated aqueous solution, 0.37±0.15 μg/h/cm3 and 14.4±3.1 μg/cm3 for 10% Tween 80 solution, and 0.25±0.05~0.76±0.21 μg/h/cm3 and 6.4±0.8 ~16.5±3.4 μg/cm3 for elastic liposomes (Fig 1A and 1B). The permeation rate and cumulative amount were increased 1.5-fold and 3.0-fold by using permeation enhancer (Tween 80), indicating Tween 80 was an effective penetration enhancer (p<0.05) [56,57]. When elastic liposomes were used as carrier vehicles, the enhancement ratios were 1.0~3.0-fold for permeation rate and 1.3~3.5-fold for cumulative amount. The result showed that the composition proportions of elastic liposomes would affect the enhancement degree of drug transportation through skin. An appropriate composition proportion of formulation of F1 with high-level cholesterol and Tween 80 could obtain the highest enhancement effect. Its enhancement effect was similar to that of 10% Tween 80 solution. The result agreed with previous studies, which reported that liposome-like vesicles and/or penetration enhancer-containing vesicles could improve the transportation through skin of the drug [2729].

thumbnail
Fig 1. The permeation rate and transdermal amount at 24 h of naringenin-loaded elastic liposomes through rat skin.

https://doi.org/10.1371/journal.pone.0131026.g001

thumbnail
Fig 2. The skin deposition amount of naringenin-loaded elastic liposomes after 24 h treatment.

https://doi.org/10.1371/journal.pone.0131026.g002

The deposition amounts of drug in SC, epidermis, dermis layers and total deposition amounts were 0.5±0.1, 1.8±0.5, 0.3± 0.1, and 2.6± 0.7 μg/cm3 for the saturated aqueous solution-treated group and 4.4±1.2, 9.3±2.7, 2.2±1.6, and 15.9±2.5 μg/cm3 for the 10% Tween 80 solution-treated group. The result showed that naringenin was deposited in different skin layers, particularly in the epidermis layer. Using elastic liposomes as carrier also showed similar results (Fig 2). Hence, the value of total deposition amount was used to evaluate the efficacy of formulations. In comparison with the two control groups, the total deposition amount increased about 6.1-fold when 10% of Tween 80 was used as permeation enhancer (p<0.05) [56,57]. When elastic liposomes were used as drug carrier vehicles, the total deposition amounts (19.0±3.7 ~30.7±13. μg/cm3) further increased 1.2~1.9-fold compared with the Tween 80-treated group, indicating that the elastic liposomes had even more potential for naringenin skin transportation (p<0.05). Furthermore, the enhancement efficiency of these elastic liposomes on the deposition amount was more than that on the permeation amount, indicating that the present liposomes were more suitable for topical skin target of naringenin. This finding is in agreement with published studies that reported use of liposomes as carrier may produce higher drug concentrations in the skin layers and lower systemic concentration [14,15,36,58]. The enhancement mechanism of skin delivery includes but is not limited to: the small size of vesicles; the cell membrane-like structure of the vesicles having good biocompatibility; formation of drug reservoirs in the skin; specific liposome-skin interactions; and the elastic vesicles being able to squeeze through intercellular regions of the SC under the influence of the transepidermal water-activity gradient. [14,51,59].

In evaluation of the effect of physicochemical characteristics and compositions of liposomes on the permeation parameters, a non-significant relationship was found between the average size vs permeation rate and cumulative amount (p>0.05). A possible explanation may be that elastic liposomes being flexible should be able to more easily squeeze through the skin without being affected in size. On the contrary, the total deposition amount in skin increased by increasing the amount of Tween 80 (Fig 2). The result is consistent with previous studies [14,55,58,60], which reported surfactant “Tween 80” can be inserted into the lipid bilayers of liposome thereby creating a “softened” and “flexible” bilayer membrane, thus facilitating elastic liposome rapid distribution into the skin retaining the drug on, in, and below the skin barrier. In cases of elastic liposomes with different added amounts of cholesterol, it was found that the formulation with medium level(s) of cholesterol showed highest skin deposition. It is possible that excess cholesterol resulted in a rigid membrane, and insufficient cholesterol resulted in a looser membrane, both making less-permeable conditions [46,47]. However, the naringenin-loaded elastic liposome with high-level Tween 80 and medium-level cholesterol showed highest total deposition of 30.7±13.7 μg/cm3, which was 11.8-times that of saturated aqueous solution.

Skin irritation

Formulations might elicit primary skin irritation. Rat skin irritation experiments were conducted to assess the potential irritant effects of the developed elastic liposome formulation. The 0.8% v/v aqueous solution of paraformaldehyde was used as a standard irritant [28,42]. As shown in Fig 3B, a slight edema exfoliation of the stratum corneum, and collagen dissociate was caused by application with paraformaldehyde. Non-significant edema and erythema was found in tested elastic liposome (Fig 3C) and aqueous solution control (Fig 3A), when compared to the positive control group, indicating that the experimental elastic liposome formulation appeared to be safe for transdermal delivery.

thumbnail
Fig 3. The microstructure of a rat abdominal skin section, viewed under a light microscope.

https://doi.org/10.1371/journal.pone.0131026.g003

Stability

After three months of storage at 4°C, non-aggregation and creaming were observed. The size and zeta potential of drug-loaded liposome had slight change from 123.7 to 128.1 nm and -11.0 to 12.7 mV respectively, showing non-significant difference. The residual drug content of tested drug-loaded elastic liposome was 98.89±3.90%, indicating that the formulation was stable.

Conclusions

The naringenin deposition amounts in SC, epidermis, dermis and total skin were significantly increased by using elastic liposomes as drug carrier when compared to the saturated aqueous solution and Tween 80-treated groups. The added contents of cholesterol and Tween 80 showed significant influence on the physicochemical properties and permeation capacity of naringenin from elastic liposomes. The naringenin-loaded elastic liposome with high-level Tween 80 and medium-level cholesterol showed highest deposition of the drug in skin, which was 11.8-times that of the saturated aqueous solution. It has also been demonstrated that naringenin-loaded elastic liposome was stable after three months of storage and produced less skin irritation than that of the standard irritant group. The result suggests that elastic liposome is a promising carrier for naringenin topical application.

Acknowledgments

This work was supported by grants from the Ministry of Science and Technology of Taiwan (NSC 102-2320-B-037-006-MY2 and NSC101-2320-B-037-33).

Author Contributions

Conceived and designed the experiments: MJT YBH PCW. Performed the experiments: JWF YSF. Analyzed the data: MJT YBH PCW. Contributed reagents/materials/analysis tools: MJT PCW. Wrote the paper: YSF PCW.

References

  1. 1. Montesano Gesualdi N, Chirico G, Catanese MT, Pirozzi G, Esposito F (2006) AROS-29 is involved in adaptive response to oxidative stress. Free Radic Res 40: 467–476. pmid:16551573
  2. 2. Portugal M, Barak V, Ginsburg I, Kohen R (2007) Interplay among oxidants, antioxidants, and cytokines in skin disorders: present status and future considerations. Biomed Pharmacother 61: 412–422. pmid:17604942
  3. 3. Scharffetter-Kochanek K, Wlaschek M, Brenneisen P, Schauen M, Blaudschun R, Wenk J (1997) UV-induced reactive oxygen species in photocarcinogenesis and photoaging. Biol Chem 378: 1247–1257. pmid:9426184
  4. 4. Trenam CW, Blake DR, Morris CJ (1992) Skin inflammation: reactive oxygen species and the role of iron. J Invest Dermatol 99: 675–682. pmid:1469283
  5. 5. Black HS, deGruijl FR, Forbes PD, Cleaver JE, Ananthaswamy HN, deFabo EC, et al. (1997) Photocarcinogenesis: an overview. J Photochem Photobiol B 40: 29–47. pmid:9301042
  6. 6. Cavia-Saiz M, Busto MD, Pilar-Izquierdo MC, Ortega N, Perez-Mateos M, Muniz P (2010) Antioxidant properties, radical scavenging activity and biomolecule protection capacity of flavonoid naringenin and its glycoside naringin: a comparative study. J Sci Food Agric 90: 1238–1244. pmid:20394007
  7. 7. Renugadevi J, Prabu SM (2009) Naringenin protects against cadmium-induced oxidative renal dysfunction in rats. Toxicology 256: 128–134. pmid:19063931
  8. 8. Huang YC, Yang CH, Chiou YL (2011) Citrus flavanone naringenin enhances melanogenesis through the activation of Wnt/beta-catenin signalling in mouse melanoma cells. Phytomedicine 18: 1244–1249. pmid:21802267
  9. 9. Ohguchi K, Akao Y, Nozawa Y (2006) Stimulation of melanogenesis by the citrus flavonoid naringenin in mouse B16 melanoma cells. Biosci Biotechnol Biochem 70: 1499–1501. pmid:16794334
  10. 10. Felgines C, Texier O, Morand C, Manach C, Scalbert A, Regerat F, et al. (2000) Bioavailability of the flavanone naringenin and its glycosides in rats. Am J Physiol Gastrointest Liver Physiol 279: G1148–1154. pmid:11093936
  11. 11. Shulman M, Cohen M, Soto-Gutierrez A, Yagi H, Wang H, Goldwasser J, et al. (2011) Enhancement of naringenin bioavailability by complexation with hydroxypropyl-beta-cyclodextrin. [corrected]. PLoS One 6: e18033. pmid:21494673
  12. 12. Hsiu SL, Huang TY, Hou YC, Chin DH, Chao PD (2002) Comparison of metabolic pharmacokinetics of naringin and naringenin in rabbits. Life Sci 70: 1481–1489. pmid:11895099
  13. 13. Mura P, Bragagni M, Mennini N, Cirri M, Maestrelli F (2014) Development of liposomal and microemulsion formulations for transdermal delivery of clonazepam: Effect of randomly methylated beta-cyclodextrin. Int J Pharm 475: 306–314. pmid:25194352
  14. 14. El Zaafarany GM, Awad GA, Holayel SM, Mortada ND (2010) Role of edge activators and surface charge in developing ultradeformable vesicles with enhanced skin delivery. Int J Pharm 397: 164–172. pmid:20599487
  15. 15. Zhao YZ, Lu CT, Zhang Y, Xiao J, Zhao YP, Tian JL, et al. (2013) Selection of high efficient transdermal lipid vesicle for curcumin skin delivery. Int J Pharm 454: 302–309. pmid:23830940
  16. 16. Tsai MJ, Huang YB, Fang JW, Fu YS, Wu PC (2015) Preparation and evaluation of submicron-carriers for naringenin topical application. Int J Pharm 481: 84–90. pmid:25615985
  17. 17. Marianecci C, Paolino D, Celia C, Fresta M, Carafa M, Alhaique F (2010) Non-ionic surfactant vesicles in pulmonary glucocorticoid delivery: characterization and interaction with human lung fibroblasts. J Control Release 147: 127–135. pmid:20603167
  18. 18. Paolino D, Lucania G, Mardente D, Alhaique F, Fresta M (2005) Ethosomes for skin delivery of ammonium glycyrrhizinate: in vitro percutaneous permeation through human skin and in vivo anti-inflammatory activity on human volunteers. J Control Release 106: 99–110. pmid:15935505
  19. 19. Celia C, Cilurzo F, Trapasso E, Cosco D, Fresta M, Paolino D (2012) Ethosomes(R) and transfersomes(R) containing linoleic acid: physicochemical and technological features of topical drug delivery carriers for the potential treatment of melasma disorders. Biomed Microdevices 14: 119–130. pmid:21960035
  20. 20. Paolino D, Celia C, Trapasso E, Cilurzo F, Fresta M (2012) Paclitaxel-loaded ethosomes(R): potential treatment of squamous cell carcinoma, a malignant transformation of actinic keratoses. Eur J Pharm Biopharm 81: 102–112. pmid:22414731
  21. 21. Paolino D, Ventura CA, Nistico S, Puglisi G, Fresta M (2002) Lecithin microemulsions for the topical administration of ketoprofen: percutaneous adsorption through human skin and in vivo human skin tolerability. Int J Pharm 244: 21–31. pmid:12204562
  22. 22. Montenegro L, Carbone C, Paolino D, Drago R, Stancampiano AH, Puglisi G (2008) In vitro skin permeation of sunscreen agents from O/W emulsions. Int J Cosmet Sci 30: 57–65. pmid:18377631
  23. 23. Pasut G, Paolino D, Celia C, Mero A, Joseph AS, Wolfram J, et al. (2015) Polyethylene glycol (PEG)-dendron phospholipids as innovative constructs for the preparation of super stealth liposomes for anticancer therapy. J Control Release 199: 106–113. pmid:25499917
  24. 24. Paolino D, Cosco D, Gaspari M, Celano M, Wolfram J, Voce P, et al. (2014) Targeting the thyroid gland with thyroid-stimulating hormone (TSH)-nanoliposomes. Biomaterials 35: 7101–7109. pmid:24836306
  25. 25. Cosco D, Paolino D, Cilurzo F, Casale F, Fresta M (2012) Gemcitabine and tamoxifen-loaded liposomes as multidrug carriers for the treatment of breast cancer diseases. Int J Pharm 422: 229–237. pmid:22093954
  26. 26. Paolino D, Cosco D, Racanicchi L, Trapasso E, Celia C, Iannone M, et al. (2010) Gemcitabine-loaded PEGylated unilamellar liposomes vs GEMZAR: biodistribution, pharmacokinetic features and in vivo antitumor activity. J Control Release 144: 144–150. pmid:20184929
  27. 27. Mura P, Maestrelli F, Gonzalez-Rodriguez ML, Michelacci I, Ghelardini C, Rabasco AM (2007) Development, characterization and in vivo evaluation of benzocaine-loaded liposomes. Eur J Pharm Biopharm 67: 86–95. pmid:17350813
  28. 28. Azeem A, Ahmad FJ, Khar RK, Talegaonkar S (2009) Nanocarrier for the transdermal delivery of an antiparkinsonian drug. AAPS PharmSciTech 10: 1093–1103. pmid:19757079
  29. 29. Bangham AD, Standish MM, Watkins JC (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol 13: 238–252. pmid:5859039
  30. 30. Paolino D, Cosco D, Cilurzo F, Trapasso E, Morittu VM, Celia C, et al. (2012) Improved in vitro and in vivo collagen biosynthesis by asiaticoside-loaded ultradeformable vesicles. J Control Release 162: 143–151. pmid:22698941
  31. 31. Cevc G, Blume G (1992) Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force. Biochim Biophys Acta 1104: 226–232. pmid:1550849
  32. 32. Malakar J, Sen SO, Nayak AK, Sen KK (2012) Formulation, optimization and evaluation of transferosomal gel for transdermal insulin delivery. Saudi Pharm J 20: 355–363. pmid:23960810
  33. 33. Gillet A, Grammenos A, Compere P, Evrard B, Piel G (2009) Development of a new topical system: drug-in-cyclodextrin-in-deformable liposome. Int J Pharm 380: 174–180. pmid:19576972
  34. 34. Song CK, Balakrishnan P, Shim CK, Chung SJ, Chong S, Kim DD (2012) A novel vesicular carrier, transethosome, for enhanced skin delivery of voriconazole: characterization and in vitro/in vivo evaluation. Colloids Surf B Biointerfaces 92: 299–304. pmid:22205066
  35. 35. Elsayed MM, Abdallah OY, Naggar VF, Khalafallah NM (2006) Deformable liposomes and ethosomes: mechanism of enhanced skin delivery. Int J Pharm 322: 60–66. pmid:16806755
  36. 36. Ghanbarzadeh S, Arami S (2013) Enhanced transdermal delivery of diclofenac sodium via conventional liposomes, ethosomes, and transfersomes. Biomed Res Int 2013: 616810. pmid:23936825
  37. 37. Lewis GA, Mathieu D, Phan-Tan-Luu R (1999) Pharmaceutical Experimental Design. Dekker, New York.
  38. 38. Huang YB, Lee KF, Huang CT, Tsai YH, Wu PC (2010) The effect of component of cream for topical delivery of hesperetin. Chem Pharm Bull (Tokyo) 58: 611–614.
  39. 39. Lademann J, Jacobi U, Surber C, Weigmann HJ, Fluhr JW (2009) The tape stripping procedure—evaluation of some critical parameters. Eur J Pharm Biopharm 72: 317–323. pmid:18775778
  40. 40. Vicentini FT, Simi TR, Del Ciampo JO, Wolga NO, Pitol DL, Iyomasa MM, et al. (2008) Quercetin in w/o microemulsion: in vitro and in vivo skin penetration and efficacy against UVB-induced skin damages evaluated in vivo. Eur J Pharm Biopharm 69: 948–957. pmid:18304790
  41. 41. De Paula D, Martins CA, Bentley MV (2008) Development and validation of HPLC method for imiquimod determination in skin penetration studies. Biomed Chromatogr 22: 1416–1423. pmid:18655215
  42. 42. Mutalik S, Udupa N (2004) Glibenclamide transdermal patches: physicochemical, pharmacodynamic, and pharmacokinetic evaluations. J Pharm Sci 93: 1577–1594. pmid:15124215
  43. 43. Yeagle PL (1985) Cholesterol and the cell membrane. Biochim Biophys Acta 822: 267–287. pmid:3904832
  44. 44. Lasic DD (1997) Liposomes in Gene Delivery. CRC Press, Boca Raton, New York.
  45. 45. Krause MR, Regen SL (2014) The Structural Role of Cholesterol in Cell Membranes: From Condensed Bilayers to Lipid Rafts. Acc Chem Res.
  46. 46. Semple SC, Chonn A, Cullis PR (1996) Influence of cholesterol on the association of plasma proteins with liposomes. Biochemistry 35: 2521–2525. pmid:8611555
  47. 47. Coderch L, Fonollosa J, De Pera M, Estelrich J, De La Maza A, Parra JL (2000) Influence of cholesterol on liposome fluidity by EPR. Relationship with percutaneous absorption. J Control Release 68: 85–95. pmid:10884582
  48. 48. Vrhovnik K, Kristl J, Sentjurc M, Smid-Korbar J (1998) Influence of liposome bilayer fluidity on the transport of encapsulated substance into the skin as evaluated by EPR. Pharm Res 15: 525–530. pmid:9587946
  49. 49. Li L, Hoffman RM (1997) Topical liposome delivery of molecules to hair follicles in mice. J Dermatol Sci 14: 101–108. pmid:9039973
  50. 50. Manosroi A, Jantrawut P, Manosroi J (2008) Anti-inflammatory activity of gel containing novel elastic niosomes entrapped with diclofenac diethylammonium. Int J Pharm 360: 156–163. pmid:18539416
  51. 51. Verma DD, Verma S, Blume G, Fahr A (2003) Particle size of liposomes influences dermal delivery of substances into skin. Int J Pharm 258: 141–151. pmid:12753761
  52. 52. Xia S, Tan C, Zhang Y, Abbas S, Feng B, Zhang X, et al. (2015) Modulating effect of lipid bilayer-carotenoid interactions on the property of liposome encapsulation. Colloids Surf B Biointerfaces 128: 172–180. pmid:25747311
  53. 53. Ge Y, Ge M (2015) Development of tea tree oil-loaded liposomal formulation using response surface methodology. J Liposome Res: 1–10.
  54. 54. Magarkar A, Dhawan V, Kallinteri P, Viitala T, Elmowafy M, Rog T, et al. (2014) Cholesterol level affects surface charge of lipid membranes in saline solution. Sci Rep 4: 5005. pmid:24845659
  55. 55. Li C, Zhang X, Huang X, Wang X, Liao G, Chen Z (2013) Preparation and characterization of flexible nanoliposomes loaded with daptomycin, a novel antibiotic, for topical skin therapy. Int J Nanomedicine 8: 1285–1292. pmid:23569376
  56. 56. Al-Suwayeh SA, Taha EI, Al-Qahtani FM, Ahmed MO, Badran MM (2014) Evaluation of skin permeation and analgesic activity effects of carbopol lornoxicam topical gels containing penetration enhancer. ScientificWorldJournal 2014: 127495. pmid:25045724
  57. 57. Nawaz A, Jan SU, Khan NR, Hussain A, Khan GM (2013) Formulation and in vitro evaluation of clotrimazole gel containing almond oil and tween 80 as penetration enhancer for topical application. Pak J Pharm Sci 26: 617–622. pmid:23625439
  58. 58. Elnaggar YS, El-Refaie WM, El-Massik MA, Abdallah OY (2014) Lecithin-based nanostructured gels for skin delivery: an update on state of art and recent applications. J Control Release 180: 10–24. pmid:24531009
  59. 59. Verma DD, Verma S, Blume G, Fahr A (2003) Liposomes increase skin penetration of entrapped and non-entrapped hydrophilic substances into human skin: a skin penetration and confocal laser scanning microscopy study. Eur J Pharm Biopharm 55: 271–277. pmid:12754000
  60. 60. Cevc G, Blume G (2001) New, highly efficient formulation of diclofenac for the topical, transdermal administration in ultradeformable drug carriers, Transfersomes. Biochim Biophys Acta 1514: 191–205. pmid:11557020