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Influence of chemical penetration enhancers on skin permeability of ellagic acid-loaded niosomes


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Original Research Paper

Influence of chemical penetration enhancers on skin

permeability of ellagic acid-loaded niosomes

Varaporn Buraphacheep Junyaprasert


, Pratyawadee Singhsa, Anchalee Jintapattanakit

Excellent Center of Innovative Drug Delivery and Nanomedicine, Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Rajathavee, Bangkok 10400, Thailand

a r t i c l e i n f o

Article history:

Received 28 November 2012 Received in revised form 24 March 2013

Accepted 1 April 2013 Keywords:

Ellagic acid Niosomes

Chemical penetration enhancer Skin permeation

Dermal delivery

a b s t r a c t

This study aimed to develop niosomes of ellagic acid (EA), a potent antioxidant phyto-chemical substance, for dermal delivery and to investigate the influence of phyto-chemical penetration enhancers on the physicochemical properties of EA-loaded niosomes. The EA niosomes were prepared by reverse phase evaporation method using Span 60, Tween 60 and cholesterol as vesicle forming agents and Solulan C24 as a steric stabilizer. Poly-ethylene glycol 400 (PEG) was used as a solubilizer while dimethylsulfoxide (DMSO) orN -methyl-2-pyrrolidone (NMP) was used as a skin penetration enhancer. It was found that the mean particle sizes of EA-loaded niosomes were in the range of 312e402 nm with PI values of lower than 0.4. The niosomes were determined to be spherical multilamellar vesicles as observed by transmission electron microscope and optical microscopy. All niosomes were stable after 4 months storage at 4C.In vitroskin permeation through human epidermis revealed that the skin enhancers affected the penetration of EA from the niosomes at 24 h. The DMSO niosomes showed the highest EA amount in epidermis; whereas the NMP niosomes had the highest EA amount in the acceptor medium. Concomitantly, the skin distribution by confocal laser scanning microscopy showed the high fluorescence intensity of the DMSO niosomes and NMP niosomes at a penetration depth of between 30e90mm (the epidermis layer) and 90e120mm (the dermis layer) under the skin, respectively. From the results, it can be concluded that the DMSO niosomes are suitable for epidermis delivery of EA while the NMP niosomes can be used for dermis delivery of EA.

ª2013 Shenyang Pharmaceutical University. Production and hosting by Elsevier B.V. All rights reserved.

*Corresponding author. Excellent Center of Innovative Drug Delivery and Nanomedicine, Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Rajathavee, Bangkok 10400, Thailand. Tel.:þ662 644 8677 91; fax:þ662 644 8694.

E-mail address:varaporn.jun@mahidol.ac.th(V.B. Junyaprasert). Peer review under responsibility of Shenyang Pharmaceutical University

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The self-assembly of non-ionic surfactants into vesicles rep-resents an interesting opportunity to achieve vesicular colloidal drug carriers, so-called niosomes, which resemble liposomes in their architecture and can be used as an effective alternative to liposomal drug carriers[1]. Niosomes have been proposed for a number of potential therapeutic applications. In particular, they have been intensively used as effective dermal and transdermal drug delivery systems[2e5]. Ellagic acid (EA) is a potent antioxidant phytochemical substance which has skin-whitening property with photoprotective ef-fect[6-7]. However, it is poorly soluble in water (w9.7mg/ml)[8] and not soluble in many organic solvents, leading to diffi-culties in the design of pharmaceutical formulations. In our previous study[9], we demonstrated that the 2:1 Span 60 and Tween 60 niosomes were able to encapsulate and deliver EA through human epidermis and dermis.

One generalized approach to overcome the barrier prop-erties of the skin for drugs and biomolecules is an incorpora-tion of suitable vehicles or other chemical compounds into transdermal delivery systems. Substances that promote the penetration of topically applied drugs through stratum cor-neum and epidermis are commonly referred to as skin permeation enhancers, accelerants, adjuvants, or sorption promoters [10]. Chemical skin permeation enhancers have been generally used to promote the drug permeation through the skin [11,12]. Dimethylsulfoxide (DMSO) is one of the earliest and most widely studied penetration enhancers and often used in many areas of pharmaceutical sciences as a “universal solvent”. It has been postulated that DMSO de-natures the intercellular structural protein of the stratum corneum[13], or promote lipid fluidity by disruption of the ordered structure of the lipid chains[14]. Although DMSO at high concentration of>60% was reported to induce erythema and wheals of the stratum corneum, it has been used as nonirritant skin penetration enhancer at 10% in many skin formulations [15]. N-methyl-2-pyrrolidone (NMP) is a polar aprotic solvent and miscible with most common solvents including water and alcohols. NMP can partition well into human stratum corneum. Within the tissue, it may act by altering the solvent nature of membrane and has been used to generate “reservoirs” within skin membranes[16]. It was also used as skin penetration enhancer in many topical for-mulations at the concentration up to 40% without any skin sensitization[17e19]. However, it has been reported that NMP metabolite, 5-hydroxy-N-methyl pyrrolidone was observed in urine after skin exposure with 5% solution of NMP[20]. To our knowledge, there are no reports on use of NMP as a penetra-tion enhancer in formulapenetra-tion of liposomes and niosomes. Hence, it is of our interest to investigate the potential of DMSO and NMP to enhance skin permeation of EA when added into niosomal formulations. In this study, the EA-loaded niosomes were prepared by using mixture of two non-ionic surfactants, 2:1 Span 60 and Tween 60, and cholesterol as the main com-ponents of the vesicular bilayer. In addition, Solulan C24 and two chemical penetration enhancers (10% v/v DMSO and 1% v/ v NMP) were used as membrane additives. The vesicular sys-tems were characterized in order to evaluate the capability of

the permeation enhancers to destabilize the lipid bilayers and to improve dermal delivery of EA.


Materials and methods

2.1. Materials

Sorbitan monostearate (Span 60) and polyethylene glycol sorbitan monostearate (Tween 60) were obtained from Uni-qema (Chicago, USA). Ellagic acid (EA) and polyethylene glycol 400 (PEG) were purchased from Fluka (West St. Paul, USA). Cholesteryl poly-24-oxyethylene ether (Solulan C24) was received as a gift sample from Amerchol (New Jersey, USA). Cholesterol and N-methyl-2-pyrrolidone (NMP) were pur-chased from Sigma-Aldrich (St. Louis, MO, USA). Dime-thylsulfoxide (DMSO) was obtained from Amresco (Ohio, USA). All other solvents were of HPLC grade and triple distilled water was used.

2.2. Preparation of EA-loaded niosomes

The vesicles were prepared by reverse phase evaporation (REV) method [21] using 1:1 mole ratio of surfactants and cholesterol as vesicle forming agents. The surfactants (2:1 Span 60 and Tween 60), cholesterol, 5 mol% and Solulan C24 and were dissolved in diethyl ether (organic phase) with and without addition of 10% v/v DMSO or 1% v/v NMP. To solubilize EA, 15% v/v PEG was added into an aqueous phase containing 1 mol% EA. At 1.5:1 the organic phase: the aqueous phase, the aqueous phase was added to the lipid mixture and mixed until a homogeneous w/o emulsion was obtained. The w/o emul-sion was sonicated at 7 C for 10 min. Afterwards, diethyl ether was slowly removed under reduced pressure using a rotary evaporator (60 rpm) until niosomal suspension was completely formed. The dispersion was evaporated about 15 min and then nitrogen gas was purged into dispersion for substitution of diethyl ether. In order to obtain niosomes with homogeneous size, REV vesicles were sonicated for 15 min and submitted to extrusion process for 3 times by an extruder device (Lipex, Northern Lipids, BC, Canada), equipped with a 400 nm pore size polyester membrane (Cyclopore, Whatman, NJ, USA). The maximum pressure was set at 800 psi as well as the temperature was controlled at 45C during the extrusion process.

2.3. Characterization of niosomes

Niosomes were characterized by optical microscopy and transmission electron microscopy (TEM) for vesicle formation and morphology. Optical micrographs were obtained with a Nikon TE-2000 inverted light microscope (magnification 900). For TEM, a drop of vesicle dispersion was applied to a 200 mesh formvar copper grid and was stained with a 1% phosphotungstic acid. Then samples were observed under a transmission electron microscope (Hitachi Model H-7000, Tokyo, Japan).

Size and size distribution of niosomes were determined by photon correlation spectroscopy (PCS) with a Malvern


Zetasizer ZS (Malvern Instruments, UK). The mean particle size (z-ave) and polydispersity index (PI) values were obtained by averaging of 10 measurements at an angle of 90in 10 mm diameter cells at 25 C. The real refractive index and the imaginary refractive index were set at 1.456 and 0.01, respectively.

2.4. Determination of entrapment efficiency

The percentage of EA-loaded niosomes (% E.E.) was deter-mined by ultrafiltration using centrifugal filter tubes with a molecular weight cut-off of 30 kDa which were centrifuged (Optima Model LE-80K, Beckman, USA) at 10,000 rpm for 15 min. Niosomal pellets were dissolved in methanol and then diluted 10 folds with methanol and 0.1% v/v phosphoric acid in water (1:1 v/v). The concentrations of EA in EA-loaded niosomes and ultrafiltrate (free drug) were analyzed using HPLC method. Each formulation was performed in duplicates and each replicate sample was diluted and analyzed for three times. The percentage of EA entrapment efficiency was calculated using the following equation:

% E:E:¼Amount of loaded EA in niosomes

Total amount of added EA 100

2.5. HPLC analysis

EA concentrations were measured by HPLC (Agilent 1200 series, Waldbronn, Germany). The stationary phase was HypersilODS C18 reversed-phase column (250 mm

4.6 mm5 mm) used in isocratic mode at ambient temper-ature. The mobile phase was a mixture of 0.1% phosphoric acid, methanol and acetronitrile (42:48:10, v/v). The UV detection was performed at a wavelength of 254 nm, the flow rate was 0.8 ml/min and the injection volume was 20ml. Prior to the analysis, the HPLC validation parameters including precision, accuracy and linearity were determined according to USP guideline[22]. Linearity was observed in concentration range of 0.05e10mg/ml with a correlation coefficient value of 0.9999. The valves of coefficients of variation for intraday and interday were less than 2% and the percent recovery was 100.15%.

2.6. Stability of EA-loaded niosomes

The niosomal dispersions were stored at 4, 25 and 40C. The % remaining of EA in the niosomes was evaluated by HPLC spectrophotometry over 4-month periods.

2.7. Skin permeation study

2.7.1. Skin preparation

Human skin samples were obtained by abdominoplasty sur-geries from female patient ranging in age from 25 to 60 years supported by Yanhee Hospital, Bangkok, Thailand. The study was carried out with the approval of the Committee on Human Rights Related to Human Experimentation, Mahidol University, Bangkok, Thailand. The excess adipose layer was sectioned off from the received tissues by dissecting with the surgical scissors. Then, the skin composed of epidermis and

underlying dermis was further used forin vitroskin distribu-tion study. Forin vitropermeation study, the epidermis was separated from the dermis using the heat separation tech-nique. The skin was immersed into hot water controlled temperature at 60C for 1 min. Then, the epidermal layer was carefully separated from the dermis using blunt forceps to produce intact sheets ready for mounting on diffusion cells. The obtained epidermis was wrapped with aluminium foil and stored at 20C until used. The stored epidermis was allowed to thaw, cut into 4.54.5 cm2pieces and hydrated by

placing in isotonic phosphate buffer in a refrigerator (at about 4C) overnight before used.

2.7.2. In vitropermeation study

In vitropermeation studies through human epidermis were investigated using Franz diffusion cells. The diffusion cells were thermo-regulated with a water jacket at 37 C. The epidermis was excised from human skins and mounted on Franz diffusion cells. A mixture of isotonic phosphate buffer pH 5.5 and isopropyl alcohol at the ratios of 90 and 10 (%, v/v) was used as an acceptor medium to provide the sink condition during the experiment and because of an appropriate solubi-lity of EA in isopropyl alcohol. After 24 h, the amounts of EA in acceptor fluids and in the epidermis were analyzed by HPLC spectroscopy.

To determine EA in the epidermis, the epidermis was removed and rinsed with water and isopropanol and subse-quently dried with a cotton swab. This procedure was done in triplicate. Briefly, the skins were cut into small pieces and soaked in 1 ml of methanol for 12 h in a closed tube. Subse-quently, it was subjected to an ultrasonic for three cycles of 15 min to avoid the raise in the temperature during extraction process. To ensure sufficient extraction of EA from skin, the residual extracted skin was performed in the same manner as mentioned above. It was found that no HPLC peak signal of EA was detected (LOD¼10 ng/ml) indicating a complete extrac-tion at the first extracextrac-tion.

2.8. Skin distribution study

To investigate the distribution of EA from niosomal formula-tions after being applied on the human skin, confocal laser scanning microscopy (CLSM) was performed. Fortunately, EA molecule can be detected by fluorescence detector. Therefore, EA distribution in the different skin layers was investigated using CLSM (FluoView FV1000 with IX2 inverted microscope: Multi-line Argon laser exciting wavelengths of 457 and 488 nm, and HeliumeNeon green laser exciting wavelength of 543 nm). The emission wavelengths were set at 488 and 547 nm. The images were recorded by setting the camera integration time to 10 ms. Sample speed was 4.0mm/pixel. The objective lens was in the magnification of 20(Olympus, mi-croscope, Japan).

In this study, the mixture of phosphate buffer pH 5.5 and isopropyl alcohol at the ratio of 90 and 10 (%, v/v) was used as an acceptor medium. After applying EA-loaded niosomes and EA solution on the skin surface, at 24 h, the skin was rinsed with distilled water. Then, the treated skins were removed from the Franz diffusion cells. The skin was sectioned into the pieces of 1 cm2size and evaluated for depth of EA penetration.


The full skin thickness (epidermis and underlying dermis) was optically scanned at different increments through thez-axis of a CLS microscope. The skin slices were investigated under both normal light and fluorescence microscopes. The images from the two techniques were overlaid to obtain the infor-mation of the EA distribution in the different skin layers.

2.9. Statistical analysis

Statistical analysis of difference in the physicochemical properties among predetermined intervals in the some formulation and between formulations was performed by using paired-ttest and one-way analysis of variance (one-way ANOVA), respectively. The level of significance was taken atp value of 0.05.


Results and discussion

3.1. Morphology, vesicle size and % entrapment


Vesicle formation in the presence of the enhancers was confirmed by optical microscopy and TEM. Fig. 1 (A1, A2) shows the obtained niosomes were spherical shape and multilamellar vesicles under optical microscope which the

mean vesicle sizes in submicron range. In addition, the similar results were displayed by TEM images as shown inFig. 1(A2, B2). In general, an addition of chemical penetration enhancers (10% v/v DMSO or 1% v/v NMP) into the niosomal formulations did not alter vesicle formation. The vesicles could normally

Fig. 2ePercentage of EA remaining after 4 months of storage at 4, 25, 40C for EA-loaded niosomes containing 15% v/v PEG without chemical enhancers (E-PEG) and with 10% v/v DMSO (E-DPEG) or 1% v/v NMP (E-NPEG).

Fig. 1ePhotomicrographs of EA-loaded niosomes containing 15% v/v PEG and (A1, A2) 10% v/v DMSO or (B1, B2) 1% v/v NMP from; (A1, B1) optical microscope (magnification 9003) and (A2, B2) transmission electron microscopy (TEM).


form with the presence of enhancers, and homogeneous white dispersion of niosomes without large dispersed parti-cles or curd was obtained.

The vesicle sizes of all EA-free niosomes were in the range of 396e511 nm, as seen inTable 1. After EA was entrapped into the niosomes, the vesicle size of the niosomes significantly increased (p>0.05). In addition, after submitted to extrusion process, the niosomes of small and homogeneous size were obtained with the mean vesicle size of 312e402 nm and PI values lower than 0.4 (Table 1). Interestingly, after extrusion the vesicle sizes of the DMSO niosomes were larger than the NMP niosomes which may be due to the fact that NMP could have more effect on increasing fluidity of vesicular membrane resulting in ease of size reduction when subjected to extrusion process. The change of niosomes size is an important parameter which can influence both vesicular properties such as entrapment efficiency and stability, as well as skin-vesicle interactions.

The % E.E. of all niosomes was in between 25.63% and 38.73% (Table 1). In particular, the addition of DMSO and NMP increased % E.E of the niosomes. This may be resulted from the high solubility of EA in the enhancers (6.15 mg/ml in DMSO and 55.42 mg/ml in NMP). The DMSO niosomes had the lower entrapment efficiency than the NMP niosomes which may be described as the lower solubility of EA in DMSO. The higher % E.E. of NMP niosomes could be explained from the high solu-bility of EA in both PEG and NMP, and PEG molecules can localize between double vesicular layers which may also carry associated NMP with them. In addition, it was reported that NMP acts as penetration enhancer by fluidizing lipid domain (alkyl chains) of the stratum corneum[23,24]. The presence of NMP in the niosome bilayer could produce the same effect, fluidizing lipid domain of vesicle membrane, leading to an increased space in the bilayer for the EA incorporation.

3.2. Stability of EA-loaded niosomes

The results of the stability study of EA entrapped in various niosomes stored at 4, 25 and 40C for 4 months are shown in Fig. 2. At 4C, the EA in all niosomes exhibited a good stability, which the percentages of EA remaining were almost 100. At higher temperature (25C), the percentages of EA remaining decreased to 40%e60%. Likewise, at 40C, they dramatically decreased to below 30%. A direct relationship between a decrease in % E.E. and the storage time as increasing the

temperature indicated the high degree of leaching. The higher EA leakage at higher temperature may be due to the higher fluidity of lipid bilayers at high temperature[25].

It is well known that the gel to liquid phase transition temperature of the vesicular bilayers played an important role in niosome formation and stability which was influenced by types of non-ionic surfactants, mole ratio of surfactant and cholesterol[1]. The stability of niosomes would be improved by increasing the phase transition temperature of the system by changing surfactant type and the surfactant/lipid level. 3.3. In vitropermeation of EA-loaded niosomes

Fig. 3 shows the amount of EA in the epidermis and the acceptor medium after applying EA-loaded niosomes or EA solution for 24 h. The amount of EA in epidermis after applying all niosomes was much higher than the EA solution. In addition, the DMSO niosomes gave significantly higher amount of EA in epidermis than the NMP niosomes. Conversely, the NMP niosomes gave significantly higher amount of EA in acceptor medium than the DMSO niosomes. This indicated that the NMP niosomes could penetrate through epidermis into dermis higher than the DMSO nio-somes which may be resulted from the higher penetration enhancement efficiency of NMP [26] which could promote Table 1eMean vesicle size (z-ave), polydispersity index (PI) and % entrapment efficiency of EA-loaded niosomes.

Formulation Before extrusion After extrusion Entrapment


EA-free niosomes EA-loaded niosomes EA-loaded niosomes

z-ave (nm) PI z-ave (nm) PI z-ave (nm) PI

E-PEGa 4085 0.3870.021 4274 0.4250.011 3903 0.3600.016 25.631.04

E-DPEGb 5113 0.2730.006 51517 0.4240.041 4024 0.2350.014 31.042.01

E-NPEGc 3964 0.4230.023 56028 0.5040.066 3122 0.2260.037 38.731.58

a The 2:1 Span 60:Tween 60 niosomes contained 15% v/v PEG without skin enhancers. bThe 2:1 Span 60:Tween 60 niosomes contained 15% v/v PEG with 10% v/v DMSO. c The 2:1 Span 60:Tween 60 niosomes contained 15% v/v PEG with 1% v/v NMP. d The values are expressed as the meanSD from at least three batches.

Fig. 3eIn vitroskin permeation study of EA solution, EA-loaded niosomes containing 15% v/v PEG without chemical enhancers (E-PEG) and with 10% v/v DMSO (E-DPEG) or 1% v/v NMP (E-NPEG) after applying for 24 h (n[3).


lipid fluidity by disruption of the ordered structure of the lipid chains[27]. Therefore, the DMSO niosomes gave an advantage on the delivery to the epidermis layer, while the NMP nio-somes were better for delivering EA to the dermis layer.

3.4. Skin distribution of EA-loaded niosomes

CLSM studies were conducted to evaluate the extent of penetration and transdermal potential of the permeated sys-tem as depicted an increase in both the depth of penetration and in fluorescence intensity. The CLSM images were ob-tained from overlaying the bright field images and fluores-cence images of the same area. Each image was one of the z-stack at 15mm depth.Fig. 4shows the fluorescence images of the epidermis and dermis layers of human skin after being treated with the EA-loaded niosomes. The CLSM images of the DMSO niosomes (Fig. 4A) showed effectiveness in permeating EA up to 120mm which showed high fluorescence intensity at the penetration depth between 30e90 mm (epidermis layer depth) and lower fluorescence intensity at the depth up to 120mm (underlying dermis depth). Thus, the DMSO niosomes

had high ability to deliver EA to the viable epidermis layer which small amount of EA penetrated into dermis layer. Meanwhile, the penetration of the NMP niosomes to dermis layer was better than the DMSO niosomes as revealed by the higher fluorescence intensity of EA at the deeper skin layer (90e120mm depth), as shown inFig. 4B. According to the ob-tained results, it confirmed that the skin enhancers affected the permeation of EA to the epidermis or dermis in accordance with the results fromin vitropermeation study.



The niosomes of 2:1 Span 60 and Tween 60 using PEG as the solubilizer could be formed in the presence of chemical en-hancers (DMSO and NMP). The vesicle shape was spherical multilamellar vesicles as assessed by optical microscopy and TEM. The mean particle sizes were in range of 312e402 nm and PI values were lower than 0.4. An addition of enhancers increased the entrapment efficiency of the EA-loaded nio-somes. The results of the skin distribution of EA-loaded

Fig. 4eCLSM images revealing the penetration and distribution of EA within the human skin after treated with (A) the DMSO niosomes and (B) the NMP niosomes for 24 h.


niosomes by CLSM were in agreement with the results of in vitropermeation which showed that the DMSO niosomes had the higher fluorescence intensity in the epidermis layer whereas the NMP niosomes had the higher fluorescence in-tensity in the dermis layer. Therefore, the chemical enhancers influenced the permeation of EA from the EA-loaded niosomes through the human skin.


This project is supported by the Office of the High Education Commission and Mahidol University under the National Research Universities Initiative.

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