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Niosomes containing hydroxyl additives as percutaneous penetration enhancers: Effect on the transdermal delivery of sulfadiazine sodium salt Rita Muzzalupo a,∗ , Lorena Tavano a,b , Francesco Lai c , Nevio Picci a a Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria, Edificio Polifunzionale, 87036 Arcavacata di Rende, Cosenza, Italy b Dipartimento di Ingegneria Informatica, Modellistica, Elettronica e Sistemistica, Università della Calabria, Via P. Bucci Cubo 39/C, 87036 Arcavacata di Rende, Cosenza, Italy c Dipartimento Scienze della Vita e dell’Ambiente, Università degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
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Article history: Received 5 June 2014 Received in revised form 1 September 2014 Accepted 8 September 2014 Available online xxx Keywords: Niosomes Ethanol Propylene glycol Glycerol Sulfadiazine sodium Skin permeation
a b s t r a c t The aim of this study was to improve the transdermal permeation of sulfadiazine sodium, employing synergistic combination of surfactants (in the form of niosomes) and additives with different number of hydroxylic groups, (following referred to as “alcohol”), as component of the bilayer. In particular the effect of different concentration of each alcohol (ethanol, propylene glycol or glycerol, from 5%, to 40% v/v) on niosomes size and distribution, drug entrapment efficiencies and ex vivo drug percutaneous permeation were evaluated, identifying formulations giving the best performances. The findings revealed that the presence of alcohol critically affect the physico-chemical properties of niosomes, with regards to dimensions, drug encapsulation and permeation. Vesicular size increased with the amount of alcohol and at the same alcohol concentration, follow the sequence ethanol > propylene glycol > glycerol. Loaded niosomes were larger than empty ones. Low E% values were found for ethanol, even less in propylene glycol and glycerol based samples, confirming that the chemical structure of the alcohol and its physico-chemical properties, affected the sulfadiazine entrapment efficiency. The comparative evaluation of percutaneous permeation profiles showed that the cumulative amount of permeated drug increases with alcohol concentration up to 20% v/v. Higher concentration (40% v/v) resulted in a strong decrease of the potential skin permeation. Best performances were obtained with glycerol. In all cases ex vivo sulfadiazine percutaneous permeations are controlled and improved respect to the corresponding free drug solutions and traditional niosomes used as controls. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Transdermal administration of many drugs is generally a problem because of the stratum corneum barrier. To reduce this diffusional barrier, penetration enhancers, pharmacologically inert and having an immediate but reversible effect on the sc, can be added to formulations. In general, permeation enhancers may contain a wide variety of different chemical functional groups and act by a multiplicity of mechanisms to enhance transport of small molecules as well as large drugs across the skin. In addition they also show an effect on the solubility of the drug in the vehicle [1]. Permeation enhancers can directly exert their effect on
∗ Corresponding author. Tel.: +(0039)0984493173. E-mail address: [email protected] (R. Muzzalupo).
skin structure by acting on intercellular lipids or corneocytes. In particular, they can extract lipids from the skin, thereby creating diffusion pathways for the drug to permeate through or they can partition themselves into the lipid bilayers thus disrupting the highly ordered lipid lamellae and causing their fluidization [1]. Alcohols including alkanols, alkenols, glycols, polyglycols and glycerols are frequently used as vehicles, solvents, or penetration enhancers to improve transdermal delivery of drugs. In particular, alcohols can enhance skin permeation by extraction of lipids and proteins, swelling of the stratum corneum, improvement of drug partitioning into the skin or drug solubility in the formulation [2,3]. Obviously, the alkyl chain length of the alcohol is an important parameter in permeation enhancement [4]: lower molecular weight molecules act as solvents, enhancing the solubility of drugs in the matrix of the sc, while disruption of its integrity through extraction of biochemicals, occurs with the more hydrophobic
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Please cite this article in press as: R. Muzzalupo, et al., Niosomes containing hydroxyl additives as percutaneous penetration enhancers: Effect on the transdermal delivery of sulfadiazine sodium salt, Colloids Surf. B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.09.017
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alcohols [5]. Often, the use of alcohol in pharmaceutical formulations is limited by the accompanying skin irritation, and for these reasons the possibility to use niosomes (vesicular structures composed of surfactant molecules assembled into bilayers, acting themselves as percutaneous permeation enhancers) [6], containing a certain amount of alcohol in their formulations, have been proposed to reduce side effects and promote the drug percutaneous permeation by synergistic combination [7]. In these lights ethosomes are relatively new types of vesicle systems, primarily composed of water, ethanol and phospholipids, that have been reported to be effective at delivering molecules to and through the skin; ethanol, in fact, may provide vesicles with soft flexible characteristics, which allow them to penetrate more easily into the deeper layers of the skin [8]. In ethosomes, due to the presence of ethanol, the solubility of many drugs increases and high encapsulation efficiency values for a wide range of molecules are obtained [9]. Furthermore, transfersomes consist of phospholipids and an edge activator, that increases the deformability of the bilayers, conferring vesicles ultradeformable properties suitable for drug transport across the skin [10]. Recently Barichello et al. studied the combined effect of vesiculation and addition of glycerol on the transdermal delivery of isosorbide 5-nitrate, confirming that glycerol action on sc is useful to facilitate skin permeation and accumulation of drugs formulated in vesicles [11]. To our knowledge, there are no comparative studies concerning the influence of alcohols with different chemical structure on vesicles physico-chemical properties and drug permeation. In this report we investigated the effect of ethanol, propylene glycol and glycerol as component of niosomes, on the transdermal delivery of sulfadiazine sodium, an antibiotic usually used for the topical cure of infected burns. In particular, the effects of different amounts of alcohol in the formulations were evaluated. All formulations were compared in terms of dimensions, morphology and polydispersity index (PI), while ex vivo percutaneous permeation profiles were investigated by using the Franz diffusion cells. 2. Materials and methods 2.1. Chemicals 2.1.1. Preparation of niosomes Multilamellar niosomal vesicles (MLVs) were prepared by the film hydration method [12]. Accurately weighed amounts of Tween 60 were dissolved in chloroform in a round-bottom flask. After mixing, the solvent was evaporated under reduced pressure and constant rotation to form a thin lipid film. The lipid film was then hydrated with 10 mL of sodium sulfadiazine aqueous solution (1.46 × 10−3 M) or sodium sulfadiazine hydroalcoholic solutions (additive/water 5%, 10%, 20% and 40% v/v) at 60 ◦ C for 30 min to form large multilamellar vesicles (MLV), at 1 × 10−2 M of total lipid concentration (Table 1). After preparation, the dispersion was left to equilibrate at 25 ◦ C overnight, to allow complete annealing and partitioning of the drug between the lipid bilayer and the aqueous phase. Small unilamellar vesicles (SUV) were prepared starting from MLV by sonication in an ultrasonic bath for 30 min at 60 ◦ C. The purification of niosomes was carried out by exhaustive dialysis for 4 h, using Visking tubing (Spectra/Por® , cut-off 12–14 kD), manipulated before use in according to Fenton’s method [13]. After purification, niosomes were stored at 4 ◦ C and in the dark until used in subsequent experiments. 2.2. Zeta-potential and size distribution analysis The Z-potential of the formulations was measured with the laser Doppler electrophoretic mobility measurements using the
Zetasizer ZS (Malvern Instruments Ltd., Malvern, U.K.), at 25.0 ± 0.1 ◦ C. All analyses were done in triplicate. Z-potential values and standard deviations were elaborated directly from the instrument. The niosomes size and distribution were determined by Dynamic Light Scattering (DLS) analyses using Zetasizer ZS (Malvern Instruments Ltd., Malvern, U.K.), at 25.0 ± 0.1 ◦ C by measuring the autocorrelation function at 90◦ . The laser was operating at 658 nm. The distribution size was directly obtained from the instrument fitting data by the inverse “Laplace transformation” method [14]. The PI was used as a measure of the width of size distribution. Polydispersity index less than 0.3 indicates a homogenous population for colloidal systems [15]. Each sample was measured three times and the results are expressed as mean ± standard deviation. 2.3. Transmission electron microscopy (TEM) The morphological analysis of vesicles was carried out by transmission electron microscopy (TEM), using a ZEISS EM 900 unit working at an accelerating voltage of 80 kV. A drop of the vesicular formulation was placed on a carbon-coated copper grid, and the sample in excess was removed using a piece of filter paper. A drop of 2% (w/v) PTA (phosphotungstic acid solution) was then stratified on the carbon grid and left to stay for 2 min. Once the excess of staining agent was removed by filter paper, the samples were air-dried and the thin film of stained niosomes was observed. Each experiment was carried out in triplicate. 2.4. Drug entrapment efficiency Drug encapsulation efficiency was determined using the dialysis technique for separating the non-entrapped drug from niosomes [16]. According to this method, 3 mL of drug-loaded niosomal dispersion were dropped into a dialysis bag immersed in 100 mL of distilled water and magnetically stirred (Visking tubing, Spectra/Por® , cut-off 12–14 kD). Free drug was dialyzed for 30 min each time and the dialysis was complete when no drug was detectable in the recipient solution. The percentage of encapsulation efficiency (E%) was expressed as the percentage of the drug entrapped into niosomes referred to the total amount of drug present in non-dialyzed samples. It was determined by diluting 1 mL of dialyzed and 1 mL of non-dialyzed niosomes in 25 mL of methanol, and by measuring the absorbance of these solutions at 270 nm. This procedure was necessary to break the niosomal membrane. Absorption spectra were recorded with a UV ± vis JASCO V-530 spectrometer using 1 cm quartz cells. Each experiment was carried out in triplicate and the results are expressed as mean ± standard deviation. 2.5. Ex vivo permeation studies The experiments were carried out in the vertical Franz diffusion cells for 24 h at 37 ◦ C, through rabbit ear skin obtained from a local slaughterhouse. The skin, previously frozen at −18 ◦ C, was pre-equilibrated in physiological solution at room temperature for 2 h before the experiments. A circular piece of this skin was sandwiched securely between the receptor and donor compartments with the dermal side in contact with the receiver medium and the epidermis side in contact with the donor chamber (contact area = 0.416 cm2 ). The donor compartment was charged with an appropriate volume of sample to keep constant the drug moles and the receptor compartment was filled with 5.5 mL of distilled water. At regular intervals up to 24 h, the medium in the receiver compartment was removed and replaced with an equal volume of pre-thermostated (37 ± 0.5 ◦ C) fresh medium [17]. The content of
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Table 1 Composition, hydrodynamic diameter and polydispersity index of empty vesicular systems at 25 ◦ C. Values represent mean ± SD (n = 3). Formulation
Surfactant (mg)
Alcohol
% Alcohol Hydrating solution
Diameter (nm)
PI
T60 T60E5 T60E10 T60E20 T60E40 T60P5 T60P10 T60P20 T60P40 T60G5 T60G10 T60G20 T60G40
131.42 ± 0.01 131.76 ± 0.01 130.49 ± 0.01 131.23 ± 0.01 130.55 ± 0.01 130.21 ± 0.01 131.22 ± 0.01 131.61 ± 0.01 130.45 ± 0.01 130.88 ± 0.01 131.70 ± 0.01 130.58 ± 0.01 130.44 ± 0.01
– Ethanol Ethanol Ethanol Ethanol Propylene glycol Propylene glycol Propylene glycol Propylene glycol Glycerol Glycerol Glycerol Glycerol
– 5 10 20 40 5 10 20 40 5 10 20 40
476 ± 11 342 ± 14 484 ± 10 528 ± 10 544 ± 10 400 ± 13 430 ± 12 449 ± 13 470 ± 11 326 ± 15 346 ± 14 407 ± 13 434 ± 12
0.235 0.239 0.225 0.217 0.298 0.280 0.247 0.209 0.237 0.271 0.258 0.223 0.239
sulfadiazine sodium in the samples was analyzed by UV–vis spectrometry. Each experiment was carried out in triplicate, and the results were in agreement within ± standard deviation. 3. Discussion Vesicles embodying alcohol in relatively high concentration are interesting and innovative vesicular systems that have appeared in the field of pharmaceutical technology and drug delivery in the last years. These carriers present interesting features correlated with their ability to permeate intact through the skin due to high deformability and to transport active substances more efficaciously through the sc than conventional vesicles [18]. Niosomal formulations were prepared using Tween 60 and different concentration of ethanol, propylene glycole or glycerol, as reported in Table 1. DLS measurements were performed to determine both the hydrodynamic size and the PI of the vesicles, while Zeta-potential measurements were achieved to characterize the electrostatic properties of niosomes, since Zeta-potential is a measure of the electrical charge close to the surface of the vesicles [19]. As shown, traditional vesicles generally have larger size than those containing alcohol, except in the case of ethanol. Whereas the alcohol itself, vesicular size increased with the amount of additive present in the formulation: in the case of ethanol, the diameter ranged from 342 nm (5% v/v) to 544 nm (40% v/v), while in the case of propylene glycol and glycerol diameter were found to increase from 400 nm to 470 nm (5% v/v) and 326–434 nm (40% v/v), respectively. On the other hand, at the same alcohol concentration bigger vesicles were obtained with ethanol, followed by propylene glycol and glycerol. PI values indicated suitable measurements and good quality of colloidal systems [6]. Both traditional and elastic niosomes gave similar Zeta-potential values (about −22.50 ± 5.28 mV), showing that this parameter was not influenced either by the type of alcohol or its concentration. All niosomal dispersions were translucent with no sedimentation or layer separation. The morphology of the niosomes is spherical in shape when observed under transmission electron microscope (Fig. 1a–c) and their size correlated well with laser diffraction results. The chemical structure of the encapsulated drug and its affinity for niosomal bilayers strongly affect its partition therein, and influence vesicles size too [20]. Table 2 reported hydrodynamic diameter, PI and sulfadiazine sodium entrapment efficiency for drug loaded vesicles. Loaded niosomes were larger than empty ones: generally T60 vesicles resulted bigger with respect ones containing alcohol up to 10 v/v%; on the other hand, increasing amount
of ethanol, propylene glycol or glycerol (20% and 40 v/v%) resulted in bigger diameters. As reported for empty vesicles, vesicular size increased with the amount of alcohol present in the formulation. At the same alcohol concentration no linear relationship between dimension and alcohol chemical structure occurred. Frequently alcohols are considered as cosurfactants and cosolvents. One reason is that it is very difficult to directly measure the composition of the surfactant layer. Entrapment efficiency of the vesicular dispersions containing different concentrations of alcohol is reported in Table 2. Lowest E% value was achieved by T60 sample, while the highest one was obtained in the case of T60G40. As shown, ethanol entrapment efficiency ranged from 38% to 40%, independently on the concentration of the percutaneous permeation enhancer up to 20% v/v. Similar behavior was found in the case of propylene glycol and glycerolbased vesicles. The increase of alcohol concentration up to 40% v/v led to a significant increase of E% that were found to be about 47%, 47% and 76% for T60E40, T60P40 and T60G40, respectively. It is important to note that lower E% values were found for T60E samples, followed by T60P and finally for T60G ones: this means that the chemical structure of the alcohol and its physico-chemical properties affected the sulfadiazine sodium entrapment efficiency. In fact ethanol, propylene glycol and glycerol differs from the number of hydroxyl groups: one in the case of ethanol, two in the case of propylene glycol an three in the case of glycerol. Since sulfadiazine sodium is an hydrophilic drug, much more interactions (H bonds) may occur between drug and polyol or diol respect to ethanol: probably this results in higher solubility of sulfadiazine sodium in presence of glycerol that led to a higher accumulation of the drug in the vesicular systems. No relationships between diameters and entrapment efficiency values were detected. 3.1. Ex vivo permeation studies The exact mechanism for better permeation into deeper skin layers from vesicles containing alcohol is still not clear: probably synergistic effects of combination of surfactant and high concentration of alcohol in vesicular formulations are responsible for deeper distribution and penetration in the lipid bilayer [21]. Presently no scientific reports concerning the influence of alcohols with different chemical structure (and their concentration) on the transdermal delivery of sulfadiazine sodium from niosomal vesicles were reported. For these reasons, equal amounts of sulfadiazine sodium from different formulations, including solution, were applied on the skin surface in donor compartment, to make a comparison among their drug penetration ability through rabbit ear skin. Amount of drug permeated through skin over 24 h was plotted versus time (Fig. 2).
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Fig. 1. Typical TEM photomicrograph for: a) T60E5, b) T60P5 and c) T60G5 samples.
Table 2 Composition, hydrodynamic diameter, polydispersity index and entrapment efficiency of loaded vesicular systems at 25 ◦ C. Values represent mean ± SD (n = 3). Formulation
Alcohol
% alcohol Hydrating solution v/v
Diameter (nm)
PI
SulfadiazineE%
Sulfadiazine encapsulated moles (10−6 )
T60 T60E5 T60E10 T60E20 T60E40 T60P5 T60P10 T60P20 T60P40 T60G5 T60G10 T60G20 T60G40
– Ethanol Ethanol Ethanol Ethanol Propylene glycol Propylene glycol Propylene glycol Propylene glycol Glycerol Glycerol Glycerol Glycerol
– 5 10 20 40 5 10 20 40 5 10 20 40
526 ± 13 488 ± 15 506 ± 14 685 ± 10 766 ± 10 508 ± 14 545 ± 11 559 ± 12 641 ± 11 485 ± 15 507 ± 14 552 ± 12 560 ± 12
0.190 0.245 0.229 0.200 0.266 0.180 0.199 0.228 0.269 0.210 0.193 0.258 0.246
35.94 ± 6.4 38.70 ± 5.5 40.21 ± 5.0 38.29 ± 6.3 47.04 ± 4.8 44.00 ± 5.3 37.87 ± 6.1 41.69 ± 6.4 47.12 ± 4.3 50.05 ± 3.8 51.45 ± 3.5 49.76 ± 3.9 76.55 ± 2.9
5.24 ± 0.93 5.65 ± 0.80 5.84 ± 0.73 5.57 ± 0.92 6.91 ± 0.70 6.42 ± 0.73 5.51 ± 0.89 6.07 ± 0.93 6.92 ± 0.63 7.30 ± 0.54 7.50 ± 0.51 7.25 ± 0.57 11.2 ± 0.42
Fig. 2. Ex vivo sulfadiazine sodium permeation from the different formulations through rabbit skin at 37 ◦ C: a) T60 (), drug solution (X); b) T60E5 (•), T60E10 (), T60E20 (), T60E40 (X); c) T60P5 (•), T60P10 (), T60P20 (), T60P40 (X); d) T60G5 (•), T60G10 (), T60G20 (), T60G40 (X); (mean ± SE; n = 3).
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As shown, the cumulative amount of drug permeated up to 24 h for both sulfadiazine sodium solution and T60 niosomes (32% and 48%, Fig. 2a) was significantly lower compared alcohol based formulations, regardless of the type of alcohol and its concentration. Results indicated that vesicular systems can improve skin delivery of hydrophilic drugs and that alcohols affected drug permeation across the skin in a relevant manner, confirming the synergistic effects of combining surfactant and alcohol. Polysorbates based vesicles have been reported to possess some deformability owing to the influence of the surfactant compositions: the high curvature of the membrane surface gave high elastic bending energy, thereby a more deformability of the vesicular membrane was obtained [22,23]. The presence of alcohol has been reported to reduced the phase transition temperature of system and to enhance the penetration to deep tissues because it interacts with lipid molecules in the polar head group region, resulting in an increased fluidity which leads to improved membrane permeability. Moreover alcohols provide the vesicles with softness and flexibility that allows to squeeze more easily and penetrate across the skin [24]. As reported in Fig. 2, percutaneous permeation profiles obtained for ethanol, propylene glycol and glycerol based formulations followed the same trend: cumulative amount of permeated sulfadiazine sodium increased with alcohol concentration: in the case of ethanol (Fig. 3b), permeated drug amount increase from 52%, to 58% and finally to 81%, for T60E5, T60E10 and T60E20, respectively. Similar behaviors were found both for propylene glycol and glycerol based formulations (Fig. 3c and d). In addition, it is important to note that the percentage of permeated drug increased with the number of hydroxyl groups of the alcohol: intermediate values (55%, 60% and 83%) were obtained for T60P5, T60P10 and T60P20 samples, respectively, while highest values were obtained with glycerol based ones (59%, 65% and 85% for T60G5, T60G10 and T60G20, respectively). This means that also the chemical structure and its physico-chemical properties affected the sulfadiazine sodium permeation across the skin. Conversely, all samples containing 40% v/v of alcohol showed a completely different trend respect to the corresponding formulations prepared with lower amount of alcohol: the cumulative percentage of permeated drug decreased to 50%, 51% and 46% for T60E40, T60P40 and T60G40, respectively. Suddenly, in all cases, these values were lower than ones obtained with the 5% v/v of alcohol: the higher values of entrapment efficiency, the lower release profiles. Water is the most natural penetration enhancer. Its hydration is one of the primary causes to increase the skin penetration of both hydrophilic and lipophilic molecules: usually, increased hydration of the sc enhances transdermal flux of a variety of drugs [25]. Glycerol possess three hydroxyl groups through which hydrogen bonds can be formed with water molecules, bringing about considerable water uptake, with respect to ethanol and propylene glycol. Glycerol is a major determinant of sc water retention [26] and this may explain the higher values of percutaneous permeation obtained in the case of T60G formulations at concentrations of alcohol up to 20% v/v. As reported before, alcohols are able to intercalate into the lipid bilayers allowing a temporary appropriate environment for drug diffusion through the deeper layers of the skin and posterior blood circulation. Obviously this behavior is more pronounced with increasing concentration (up to 20% v/v). It seems that an excessive amount of alcohol may lead to a worsening of performance in terms of percutaneous permeation across skin and may represents a limitation of the drug carriers potential. As reported elsewhere, in some cases more is not always better [27]. These results may be explained as reported by Barrichello et al. in 2008: the authors demonstrated that addition of glycerol to liposome formulations resulted in an increase of drug permeated through rat skin up to 10% of alcohol, while further addition of glycerol resulted in a decrease of the cumulative permeation [11]. In our
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case, it can be assumed that concentration up to 20% v/v of alcohol might facilitate the release of surfactant from niosomes, giving them the possibility to eventually penetrate the sc and occasionally be incorporated into the lipid layer. This would imply enough plasticity and hydration to skin to become even more attractive for hydrophilic drug permeation. This effect might thus facilitate an uptake of water by the sc, enhancing the permeation of a drug as reported in literature [28]. Probably at higher percentage of alcohol (40%), the physico-chemical properties of the skin may be compromise by alteration of the flux of surfactant and drug through the skin, causing less permeation of drug. 4. Conclusion The objective of this study was to evaluate the synergistic combination of surfactant (in the form of niosomes) and different concentration of cosurfactants on the transdermal permeation of sulfadiazine sodium. In particular, ethanol, propylene glycol or glycerol were used as additives and their effect was studied in terms of niosome size and distribution, drug entrapment efficiencies and ex vivo drug percutaneous permeation. Results indicated that the presence of alcohol critically affected the physico-chemical properties of niosomes: vesicular size increased with the amount of alcohol and at the same alcohol concentration, bigger vesicles were obtained with ethanol, followed by propylene glycol and glycerol. Also the introduction of sulfadiazine sodium influenced niosomes size: loaded niosomes were larger than empty ones. Lower E% values were found for ethanol based samples, followed by propylene glycol and glycerol based ones. Higher values of entrapment efficiency were obtained increasing the percentages of alcohol up to 40% v/v, regardless the alcohol type. The comparative evaluation of the drug percutaneous permeation profiles showed that cumulative amount of permeated drug increased with alcohol concentration, up to 20% v/v; higher concentration (40% v/v) resulted in a strong decrease of the potential skin permeation. Best performances were obtained with glycerol based formulation. In all cases ex vivo sulfadiazine sodium percutaneous permeations appeared to be controlled and improved respect to the corresponding free drug solution and traditional niosomes used as controls. Acknowledgments MIUR, the Italian Ministry for University, is acknowledged for financial support (EX-60%, PRIN 2010-2011, Prot. N. 2010H834LS004). Moreover, the project has been co-funded with support from the Commission European Social Fund and Region of Calabria (Italy). References [1] R.B. Walker, E.W. Smith, Adv. Drug Del. Rev. 18 (1996) 295. [2] A.C. Williams, B.W. Barry, Crit. Rev. Ther. Drug Carrier Syst. 9 (1992) 305. [3] B. Mollgaard, Synergistic effects in percutaneous enhancement, in: K. Walters, J. Hadgraft (Eds.), Pharmaceutical Skin Penetration Enhancement, Marcell Dekker, New York–Hong Kong–Basel, 1993. [4] Y.W. Chien, H. Xu, C.C. Chiang, Y.C. Hung, Pharm. Res. 5 (1988) 103. [5] D. Friend, P. Catz, J. Heller, J. Reid, R. Baker, J. Control. Release 7 (1988) 243. [6] R. Muzzalupo, L. Tavano, R. Cassano, S. Trombino, T. Ferrarelli, N. Picci, Eur. J. Pharm. Biopharm. 79 (2011) 28. [7] P. Karande, S. Mitragotri, Biochim. Biophys. Acta 1788 (2009) 2362. [8] E. Touitou, N. Dayan, L. Bergelson, B. Godin, M. Eliaz, J. Control. Release 65 (2000) 403. [9] Y.F. Rao, F. Zheng, X. Zhang, J. Gao, W. Liang, AAPS Pharmscitech. 9 (2008) 860. [10] G. Cevc, Crit. Rev. Ther. Drug Carrier Syst. 13 (1996) 257. [11] J.M. Barichello, N. Yamakawa, M. Kisyuku, H. Handa, T. Shibata, T. Ishida, H. Kiwada, Int. J. Pharm. 357 (2008) 199. [12] A.D. Bangham, M.M. Standish, J.C. Watkins, J. Mol. Biol. 13 (1965) 238. [13] R.R. Fenton, W.J. Easdable, H. Meng, E.S.M. Omara, M.J. Mckeage, P.J. Russel, T.W. Hambley, J. Med. Chem. 40 (1997) 1090. [14] S.W. Provencher, Comput. Phys. Commun. 27 (1982) 229.
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Niosomes containing hydroxyl additives as percutaneous penetration enhancers: Effect on the transdermal delivery of sulfadiazine sodium salt Rita Muzzalupo a,∗ , Lorena Tavano a,b , Francesco Lai c , Nevio Picci a a Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria, Edificio Polifunzionale, 87036 Arcavacata di Rende, Cosenza, Italy b Dipartimento di Ingegneria Informatica, Modellistica, Elettronica e Sistemistica, Università della Calabria, Via P. Bucci Cubo 39/C, 87036 Arcavacata di Rende, Cosenza, Italy c Dipartimento Scienze della Vita e dell’Ambiente, Università degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
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Article history: Received 5 June 2014 Received in revised form 1 September 2014 Accepted 8 September 2014 Available online xxx Keywords: Niosomes Ethanol Propylene glycol Glycerol Sulfadiazine sodium Skin permeation
a b s t r a c t The aim of this study was to improve the transdermal permeation of sulfadiazine sodium, employing synergistic combination of surfactants (in the form of niosomes) and additives with different number of hydroxylic groups, (following referred to as “alcohol”), as component of the bilayer. In particular the effect of different concentration of each alcohol (ethanol, propylene glycol or glycerol, from 5%, to 40% v/v) on niosomes size and distribution, drug entrapment efficiencies and ex vivo drug percutaneous permeation were evaluated, identifying formulations giving the best performances. The findings revealed that the presence of alcohol critically affect the physico-chemical properties of niosomes, with regards to dimensions, drug encapsulation and permeation. Vesicular size increased with the amount of alcohol and at the same alcohol concentration, follow the sequence ethanol > propylene glycol > glycerol. Loaded niosomes were larger than empty ones. Low E% values were found for ethanol, even less in propylene glycol and glycerol based samples, confirming that the chemical structure of the alcohol and its physico-chemical properties, affected the sulfadiazine entrapment efficiency. The comparative evaluation of percutaneous permeation profiles showed that the cumulative amount of permeated drug increases with alcohol concentration up to 20% v/v. Higher concentration (40% v/v) resulted in a strong decrease of the potential skin permeation. Best performances were obtained with glycerol. In all cases ex vivo sulfadiazine percutaneous permeations are controlled and improved respect to the corresponding free drug solutions and traditional niosomes used as controls. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Transdermal administration of many drugs is generally a problem because of the stratum corneum barrier. To reduce this diffusional barrier, penetration enhancers, pharmacologically inert and having an immediate but reversible effect on the sc, can be added to formulations. In general, permeation enhancers may contain a wide variety of different chemical functional groups and act by a multiplicity of mechanisms to enhance transport of small molecules as well as large drugs across the skin. In addition they also show an effect on the solubility of the drug in the vehicle [1]. Permeation enhancers can directly exert their effect on
∗ Corresponding author. Tel.: +(0039)0984493173. E-mail address: [email protected] (R. Muzzalupo).
skin structure by acting on intercellular lipids or corneocytes. In particular, they can extract lipids from the skin, thereby creating diffusion pathways for the drug to permeate through or they can partition themselves into the lipid bilayers thus disrupting the highly ordered lipid lamellae and causing their fluidization [1]. Alcohols including alkanols, alkenols, glycols, polyglycols and glycerols are frequently used as vehicles, solvents, or penetration enhancers to improve transdermal delivery of drugs. In particular, alcohols can enhance skin permeation by extraction of lipids and proteins, swelling of the stratum corneum, improvement of drug partitioning into the skin or drug solubility in the formulation [2,3]. Obviously, the alkyl chain length of the alcohol is an important parameter in permeation enhancement [4]: lower molecular weight molecules act as solvents, enhancing the solubility of drugs in the matrix of the sc, while disruption of its integrity through extraction of biochemicals, occurs with the more hydrophobic
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alcohols [5]. Often, the use of alcohol in pharmaceutical formulations is limited by the accompanying skin irritation, and for these reasons the possibility to use niosomes (vesicular structures composed of surfactant molecules assembled into bilayers, acting themselves as percutaneous permeation enhancers) [6], containing a certain amount of alcohol in their formulations, have been proposed to reduce side effects and promote the drug percutaneous permeation by synergistic combination [7]. In these lights ethosomes are relatively new types of vesicle systems, primarily composed of water, ethanol and phospholipids, that have been reported to be effective at delivering molecules to and through the skin; ethanol, in fact, may provide vesicles with soft flexible characteristics, which allow them to penetrate more easily into the deeper layers of the skin [8]. In ethosomes, due to the presence of ethanol, the solubility of many drugs increases and high encapsulation efficiency values for a wide range of molecules are obtained [9]. Furthermore, transfersomes consist of phospholipids and an edge activator, that increases the deformability of the bilayers, conferring vesicles ultradeformable properties suitable for drug transport across the skin [10]. Recently Barichello et al. studied the combined effect of vesiculation and addition of glycerol on the transdermal delivery of isosorbide 5-nitrate, confirming that glycerol action on sc is useful to facilitate skin permeation and accumulation of drugs formulated in vesicles [11]. To our knowledge, there are no comparative studies concerning the influence of alcohols with different chemical structure on vesicles physico-chemical properties and drug permeation. In this report we investigated the effect of ethanol, propylene glycol and glycerol as component of niosomes, on the transdermal delivery of sulfadiazine sodium, an antibiotic usually used for the topical cure of infected burns. In particular, the effects of different amounts of alcohol in the formulations were evaluated. All formulations were compared in terms of dimensions, morphology and polydispersity index (PI), while ex vivo percutaneous permeation profiles were investigated by using the Franz diffusion cells. 2. Materials and methods 2.1. Chemicals 2.1.1. Preparation of niosomes Multilamellar niosomal vesicles (MLVs) were prepared by the film hydration method [12]. Accurately weighed amounts of Tween 60 were dissolved in chloroform in a round-bottom flask. After mixing, the solvent was evaporated under reduced pressure and constant rotation to form a thin lipid film. The lipid film was then hydrated with 10 mL of sodium sulfadiazine aqueous solution (1.46 × 10−3 M) or sodium sulfadiazine hydroalcoholic solutions (additive/water 5%, 10%, 20% and 40% v/v) at 60 ◦ C for 30 min to form large multilamellar vesicles (MLV), at 1 × 10−2 M of total lipid concentration (Table 1). After preparation, the dispersion was left to equilibrate at 25 ◦ C overnight, to allow complete annealing and partitioning of the drug between the lipid bilayer and the aqueous phase. Small unilamellar vesicles (SUV) were prepared starting from MLV by sonication in an ultrasonic bath for 30 min at 60 ◦ C. The purification of niosomes was carried out by exhaustive dialysis for 4 h, using Visking tubing (Spectra/Por® , cut-off 12–14 kD), manipulated before use in according to Fenton’s method [13]. After purification, niosomes were stored at 4 ◦ C and in the dark until used in subsequent experiments. 2.2. Zeta-potential and size distribution analysis The Z-potential of the formulations was measured with the laser Doppler electrophoretic mobility measurements using the
Zetasizer ZS (Malvern Instruments Ltd., Malvern, U.K.), at 25.0 ± 0.1 ◦ C. All analyses were done in triplicate. Z-potential values and standard deviations were elaborated directly from the instrument. The niosomes size and distribution were determined by Dynamic Light Scattering (DLS) analyses using Zetasizer ZS (Malvern Instruments Ltd., Malvern, U.K.), at 25.0 ± 0.1 ◦ C by measuring the autocorrelation function at 90◦ . The laser was operating at 658 nm. The distribution size was directly obtained from the instrument fitting data by the inverse “Laplace transformation” method [14]. The PI was used as a measure of the width of size distribution. Polydispersity index less than 0.3 indicates a homogenous population for colloidal systems [15]. Each sample was measured three times and the results are expressed as mean ± standard deviation. 2.3. Transmission electron microscopy (TEM) The morphological analysis of vesicles was carried out by transmission electron microscopy (TEM), using a ZEISS EM 900 unit working at an accelerating voltage of 80 kV. A drop of the vesicular formulation was placed on a carbon-coated copper grid, and the sample in excess was removed using a piece of filter paper. A drop of 2% (w/v) PTA (phosphotungstic acid solution) was then stratified on the carbon grid and left to stay for 2 min. Once the excess of staining agent was removed by filter paper, the samples were air-dried and the thin film of stained niosomes was observed. Each experiment was carried out in triplicate. 2.4. Drug entrapment efficiency Drug encapsulation efficiency was determined using the dialysis technique for separating the non-entrapped drug from niosomes [16]. According to this method, 3 mL of drug-loaded niosomal dispersion were dropped into a dialysis bag immersed in 100 mL of distilled water and magnetically stirred (Visking tubing, Spectra/Por® , cut-off 12–14 kD). Free drug was dialyzed for 30 min each time and the dialysis was complete when no drug was detectable in the recipient solution. The percentage of encapsulation efficiency (E%) was expressed as the percentage of the drug entrapped into niosomes referred to the total amount of drug present in non-dialyzed samples. It was determined by diluting 1 mL of dialyzed and 1 mL of non-dialyzed niosomes in 25 mL of methanol, and by measuring the absorbance of these solutions at 270 nm. This procedure was necessary to break the niosomal membrane. Absorption spectra were recorded with a UV ± vis JASCO V-530 spectrometer using 1 cm quartz cells. Each experiment was carried out in triplicate and the results are expressed as mean ± standard deviation. 2.5. Ex vivo permeation studies The experiments were carried out in the vertical Franz diffusion cells for 24 h at 37 ◦ C, through rabbit ear skin obtained from a local slaughterhouse. The skin, previously frozen at −18 ◦ C, was pre-equilibrated in physiological solution at room temperature for 2 h before the experiments. A circular piece of this skin was sandwiched securely between the receptor and donor compartments with the dermal side in contact with the receiver medium and the epidermis side in contact with the donor chamber (contact area = 0.416 cm2 ). The donor compartment was charged with an appropriate volume of sample to keep constant the drug moles and the receptor compartment was filled with 5.5 mL of distilled water. At regular intervals up to 24 h, the medium in the receiver compartment was removed and replaced with an equal volume of pre-thermostated (37 ± 0.5 ◦ C) fresh medium [17]. The content of
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Table 1 Composition, hydrodynamic diameter and polydispersity index of empty vesicular systems at 25 ◦ C. Values represent mean ± SD (n = 3). Formulation
Surfactant (mg)
Alcohol
% Alcohol Hydrating solution
Diameter (nm)
PI
T60 T60E5 T60E10 T60E20 T60E40 T60P5 T60P10 T60P20 T60P40 T60G5 T60G10 T60G20 T60G40
131.42 ± 0.01 131.76 ± 0.01 130.49 ± 0.01 131.23 ± 0.01 130.55 ± 0.01 130.21 ± 0.01 131.22 ± 0.01 131.61 ± 0.01 130.45 ± 0.01 130.88 ± 0.01 131.70 ± 0.01 130.58 ± 0.01 130.44 ± 0.01
– Ethanol Ethanol Ethanol Ethanol Propylene glycol Propylene glycol Propylene glycol Propylene glycol Glycerol Glycerol Glycerol Glycerol
– 5 10 20 40 5 10 20 40 5 10 20 40
476 ± 11 342 ± 14 484 ± 10 528 ± 10 544 ± 10 400 ± 13 430 ± 12 449 ± 13 470 ± 11 326 ± 15 346 ± 14 407 ± 13 434 ± 12
0.235 0.239 0.225 0.217 0.298 0.280 0.247 0.209 0.237 0.271 0.258 0.223 0.239
sulfadiazine sodium in the samples was analyzed by UV–vis spectrometry. Each experiment was carried out in triplicate, and the results were in agreement within ± standard deviation. 3. Discussion Vesicles embodying alcohol in relatively high concentration are interesting and innovative vesicular systems that have appeared in the field of pharmaceutical technology and drug delivery in the last years. These carriers present interesting features correlated with their ability to permeate intact through the skin due to high deformability and to transport active substances more efficaciously through the sc than conventional vesicles [18]. Niosomal formulations were prepared using Tween 60 and different concentration of ethanol, propylene glycole or glycerol, as reported in Table 1. DLS measurements were performed to determine both the hydrodynamic size and the PI of the vesicles, while Zeta-potential measurements were achieved to characterize the electrostatic properties of niosomes, since Zeta-potential is a measure of the electrical charge close to the surface of the vesicles [19]. As shown, traditional vesicles generally have larger size than those containing alcohol, except in the case of ethanol. Whereas the alcohol itself, vesicular size increased with the amount of additive present in the formulation: in the case of ethanol, the diameter ranged from 342 nm (5% v/v) to 544 nm (40% v/v), while in the case of propylene glycol and glycerol diameter were found to increase from 400 nm to 470 nm (5% v/v) and 326–434 nm (40% v/v), respectively. On the other hand, at the same alcohol concentration bigger vesicles were obtained with ethanol, followed by propylene glycol and glycerol. PI values indicated suitable measurements and good quality of colloidal systems [6]. Both traditional and elastic niosomes gave similar Zeta-potential values (about −22.50 ± 5.28 mV), showing that this parameter was not influenced either by the type of alcohol or its concentration. All niosomal dispersions were translucent with no sedimentation or layer separation. The morphology of the niosomes is spherical in shape when observed under transmission electron microscope (Fig. 1a–c) and their size correlated well with laser diffraction results. The chemical structure of the encapsulated drug and its affinity for niosomal bilayers strongly affect its partition therein, and influence vesicles size too [20]. Table 2 reported hydrodynamic diameter, PI and sulfadiazine sodium entrapment efficiency for drug loaded vesicles. Loaded niosomes were larger than empty ones: generally T60 vesicles resulted bigger with respect ones containing alcohol up to 10 v/v%; on the other hand, increasing amount
of ethanol, propylene glycol or glycerol (20% and 40 v/v%) resulted in bigger diameters. As reported for empty vesicles, vesicular size increased with the amount of alcohol present in the formulation. At the same alcohol concentration no linear relationship between dimension and alcohol chemical structure occurred. Frequently alcohols are considered as cosurfactants and cosolvents. One reason is that it is very difficult to directly measure the composition of the surfactant layer. Entrapment efficiency of the vesicular dispersions containing different concentrations of alcohol is reported in Table 2. Lowest E% value was achieved by T60 sample, while the highest one was obtained in the case of T60G40. As shown, ethanol entrapment efficiency ranged from 38% to 40%, independently on the concentration of the percutaneous permeation enhancer up to 20% v/v. Similar behavior was found in the case of propylene glycol and glycerolbased vesicles. The increase of alcohol concentration up to 40% v/v led to a significant increase of E% that were found to be about 47%, 47% and 76% for T60E40, T60P40 and T60G40, respectively. It is important to note that lower E% values were found for T60E samples, followed by T60P and finally for T60G ones: this means that the chemical structure of the alcohol and its physico-chemical properties affected the sulfadiazine sodium entrapment efficiency. In fact ethanol, propylene glycol and glycerol differs from the number of hydroxyl groups: one in the case of ethanol, two in the case of propylene glycol an three in the case of glycerol. Since sulfadiazine sodium is an hydrophilic drug, much more interactions (H bonds) may occur between drug and polyol or diol respect to ethanol: probably this results in higher solubility of sulfadiazine sodium in presence of glycerol that led to a higher accumulation of the drug in the vesicular systems. No relationships between diameters and entrapment efficiency values were detected. 3.1. Ex vivo permeation studies The exact mechanism for better permeation into deeper skin layers from vesicles containing alcohol is still not clear: probably synergistic effects of combination of surfactant and high concentration of alcohol in vesicular formulations are responsible for deeper distribution and penetration in the lipid bilayer [21]. Presently no scientific reports concerning the influence of alcohols with different chemical structure (and their concentration) on the transdermal delivery of sulfadiazine sodium from niosomal vesicles were reported. For these reasons, equal amounts of sulfadiazine sodium from different formulations, including solution, were applied on the skin surface in donor compartment, to make a comparison among their drug penetration ability through rabbit ear skin. Amount of drug permeated through skin over 24 h was plotted versus time (Fig. 2).
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Fig. 1. Typical TEM photomicrograph for: a) T60E5, b) T60P5 and c) T60G5 samples.
Table 2 Composition, hydrodynamic diameter, polydispersity index and entrapment efficiency of loaded vesicular systems at 25 ◦ C. Values represent mean ± SD (n = 3). Formulation
Alcohol
% alcohol Hydrating solution v/v
Diameter (nm)
PI
SulfadiazineE%
Sulfadiazine encapsulated moles (10−6 )
T60 T60E5 T60E10 T60E20 T60E40 T60P5 T60P10 T60P20 T60P40 T60G5 T60G10 T60G20 T60G40
– Ethanol Ethanol Ethanol Ethanol Propylene glycol Propylene glycol Propylene glycol Propylene glycol Glycerol Glycerol Glycerol Glycerol
– 5 10 20 40 5 10 20 40 5 10 20 40
526 ± 13 488 ± 15 506 ± 14 685 ± 10 766 ± 10 508 ± 14 545 ± 11 559 ± 12 641 ± 11 485 ± 15 507 ± 14 552 ± 12 560 ± 12
0.190 0.245 0.229 0.200 0.266 0.180 0.199 0.228 0.269 0.210 0.193 0.258 0.246
35.94 ± 6.4 38.70 ± 5.5 40.21 ± 5.0 38.29 ± 6.3 47.04 ± 4.8 44.00 ± 5.3 37.87 ± 6.1 41.69 ± 6.4 47.12 ± 4.3 50.05 ± 3.8 51.45 ± 3.5 49.76 ± 3.9 76.55 ± 2.9
5.24 ± 0.93 5.65 ± 0.80 5.84 ± 0.73 5.57 ± 0.92 6.91 ± 0.70 6.42 ± 0.73 5.51 ± 0.89 6.07 ± 0.93 6.92 ± 0.63 7.30 ± 0.54 7.50 ± 0.51 7.25 ± 0.57 11.2 ± 0.42
Fig. 2. Ex vivo sulfadiazine sodium permeation from the different formulations through rabbit skin at 37 ◦ C: a) T60 (), drug solution (X); b) T60E5 (•), T60E10 (), T60E20 (), T60E40 (X); c) T60P5 (•), T60P10 (), T60P20 (), T60P40 (X); d) T60G5 (•), T60G10 (), T60G20 (), T60G40 (X); (mean ± SE; n = 3).
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As shown, the cumulative amount of drug permeated up to 24 h for both sulfadiazine sodium solution and T60 niosomes (32% and 48%, Fig. 2a) was significantly lower compared alcohol based formulations, regardless of the type of alcohol and its concentration. Results indicated that vesicular systems can improve skin delivery of hydrophilic drugs and that alcohols affected drug permeation across the skin in a relevant manner, confirming the synergistic effects of combining surfactant and alcohol. Polysorbates based vesicles have been reported to possess some deformability owing to the influence of the surfactant compositions: the high curvature of the membrane surface gave high elastic bending energy, thereby a more deformability of the vesicular membrane was obtained [22,23]. The presence of alcohol has been reported to reduced the phase transition temperature of system and to enhance the penetration to deep tissues because it interacts with lipid molecules in the polar head group region, resulting in an increased fluidity which leads to improved membrane permeability. Moreover alcohols provide the vesicles with softness and flexibility that allows to squeeze more easily and penetrate across the skin [24]. As reported in Fig. 2, percutaneous permeation profiles obtained for ethanol, propylene glycol and glycerol based formulations followed the same trend: cumulative amount of permeated sulfadiazine sodium increased with alcohol concentration: in the case of ethanol (Fig. 3b), permeated drug amount increase from 52%, to 58% and finally to 81%, for T60E5, T60E10 and T60E20, respectively. Similar behaviors were found both for propylene glycol and glycerol based formulations (Fig. 3c and d). In addition, it is important to note that the percentage of permeated drug increased with the number of hydroxyl groups of the alcohol: intermediate values (55%, 60% and 83%) were obtained for T60P5, T60P10 and T60P20 samples, respectively, while highest values were obtained with glycerol based ones (59%, 65% and 85% for T60G5, T60G10 and T60G20, respectively). This means that also the chemical structure and its physico-chemical properties affected the sulfadiazine sodium permeation across the skin. Conversely, all samples containing 40% v/v of alcohol showed a completely different trend respect to the corresponding formulations prepared with lower amount of alcohol: the cumulative percentage of permeated drug decreased to 50%, 51% and 46% for T60E40, T60P40 and T60G40, respectively. Suddenly, in all cases, these values were lower than ones obtained with the 5% v/v of alcohol: the higher values of entrapment efficiency, the lower release profiles. Water is the most natural penetration enhancer. Its hydration is one of the primary causes to increase the skin penetration of both hydrophilic and lipophilic molecules: usually, increased hydration of the sc enhances transdermal flux of a variety of drugs [25]. Glycerol possess three hydroxyl groups through which hydrogen bonds can be formed with water molecules, bringing about considerable water uptake, with respect to ethanol and propylene glycol. Glycerol is a major determinant of sc water retention [26] and this may explain the higher values of percutaneous permeation obtained in the case of T60G formulations at concentrations of alcohol up to 20% v/v. As reported before, alcohols are able to intercalate into the lipid bilayers allowing a temporary appropriate environment for drug diffusion through the deeper layers of the skin and posterior blood circulation. Obviously this behavior is more pronounced with increasing concentration (up to 20% v/v). It seems that an excessive amount of alcohol may lead to a worsening of performance in terms of percutaneous permeation across skin and may represents a limitation of the drug carriers potential. As reported elsewhere, in some cases more is not always better [27]. These results may be explained as reported by Barrichello et al. in 2008: the authors demonstrated that addition of glycerol to liposome formulations resulted in an increase of drug permeated through rat skin up to 10% of alcohol, while further addition of glycerol resulted in a decrease of the cumulative permeation [11]. In our
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case, it can be assumed that concentration up to 20% v/v of alcohol might facilitate the release of surfactant from niosomes, giving them the possibility to eventually penetrate the sc and occasionally be incorporated into the lipid layer. This would imply enough plasticity and hydration to skin to become even more attractive for hydrophilic drug permeation. This effect might thus facilitate an uptake of water by the sc, enhancing the permeation of a drug as reported in literature [28]. Probably at higher percentage of alcohol (40%), the physico-chemical properties of the skin may be compromise by alteration of the flux of surfactant and drug through the skin, causing less permeation of drug. 4. Conclusion The objective of this study was to evaluate the synergistic combination of surfactant (in the form of niosomes) and different concentration of cosurfactants on the transdermal permeation of sulfadiazine sodium. In particular, ethanol, propylene glycol or glycerol were used as additives and their effect was studied in terms of niosome size and distribution, drug entrapment efficiencies and ex vivo drug percutaneous permeation. Results indicated that the presence of alcohol critically affected the physico-chemical properties of niosomes: vesicular size increased with the amount of alcohol and at the same alcohol concentration, bigger vesicles were obtained with ethanol, followed by propylene glycol and glycerol. Also the introduction of sulfadiazine sodium influenced niosomes size: loaded niosomes were larger than empty ones. Lower E% values were found for ethanol based samples, followed by propylene glycol and glycerol based ones. Higher values of entrapment efficiency were obtained increasing the percentages of alcohol up to 40% v/v, regardless the alcohol type. The comparative evaluation of the drug percutaneous permeation profiles showed that cumulative amount of permeated drug increased with alcohol concentration, up to 20% v/v; higher concentration (40% v/v) resulted in a strong decrease of the potential skin permeation. Best performances were obtained with glycerol based formulation. In all cases ex vivo sulfadiazine sodium percutaneous permeations appeared to be controlled and improved respect to the corresponding free drug solution and traditional niosomes used as controls. Acknowledgments MIUR, the Italian Ministry for University, is acknowledged for financial support (EX-60%, PRIN 2010-2011, Prot. N. 2010H834LS004). Moreover, the project has been co-funded with support from the Commission European Social Fund and Region of Calabria (Italy). References [1] R.B. Walker, E.W. Smith, Adv. Drug Del. Rev. 18 (1996) 295. [2] A.C. Williams, B.W. Barry, Crit. Rev. Ther. Drug Carrier Syst. 9 (1992) 305. [3] B. Mollgaard, Synergistic effects in percutaneous enhancement, in: K. Walters, J. Hadgraft (Eds.), Pharmaceutical Skin Penetration Enhancement, Marcell Dekker, New York–Hong Kong–Basel, 1993. [4] Y.W. Chien, H. Xu, C.C. Chiang, Y.C. Hung, Pharm. Res. 5 (1988) 103. [5] D. Friend, P. Catz, J. Heller, J. Reid, R. Baker, J. Control. Release 7 (1988) 243. [6] R. Muzzalupo, L. Tavano, R. Cassano, S. Trombino, T. Ferrarelli, N. Picci, Eur. J. Pharm. Biopharm. 79 (2011) 28. [7] P. Karande, S. Mitragotri, Biochim. Biophys. Acta 1788 (2009) 2362. [8] E. Touitou, N. Dayan, L. Bergelson, B. Godin, M. Eliaz, J. Control. Release 65 (2000) 403. [9] Y.F. Rao, F. Zheng, X. Zhang, J. Gao, W. Liang, AAPS Pharmscitech. 9 (2008) 860. [10] G. Cevc, Crit. Rev. Ther. Drug Carrier Syst. 13 (1996) 257. [11] J.M. Barichello, N. Yamakawa, M. Kisyuku, H. Handa, T. Shibata, T. Ishida, H. Kiwada, Int. J. Pharm. 357 (2008) 199. [12] A.D. Bangham, M.M. Standish, J.C. Watkins, J. Mol. Biol. 13 (1965) 238. [13] R.R. Fenton, W.J. Easdable, H. Meng, E.S.M. Omara, M.J. Mckeage, P.J. Russel, T.W. Hambley, J. Med. Chem. 40 (1997) 1090. [14] S.W. Provencher, Comput. Phys. Commun. 27 (1982) 229.
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