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Novel gel-niosomes formulations as multicomponent systems for transdermal drug delivery
Colloids and Surfaces B: Biointerfaces 110 (2013) 281–288
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Novel gel-niosomes formulations as multicomponent systems for transdermal drug delivery Lorena Tavano a,b , Luigi Gentile c , Cesare Oliviero Rossi c , Rita Muzzalupo a,∗ a
Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria, Ed. Polifunzionale, 87036 Arcavacata di Rende (CS), Italy Dipartimento di Ingegneria Informatica, Modellistica, Elettronica e Sistemistica, Università della Calabria, Via P. Bucci Cubo 39/C, 87036 Arcavacata di Rende (CS), Italy c Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, Ponte P. Bucci, Cubo 14/D, 87036 Arcavacata di Rende (CS), Italy b
a r t i c l e
i n f o
Article history: Received 12 February 2013 Received in revised form 27 March 2013 Accepted 18 April 2013 Available online 6 May 2013 Keywords: Pluronic AOT Liquid crystals Niosomes Transdermal delivery
a b s t r a c t The percutaneous permeation profiles of sulfadiazine sodium salt, propranolol hydrochloride and tyrosol from novel liquid crystal-niosomes formulations as multicomponent systems, were investigated. The new carriers were prepared from mixture of water/surfactant, AOT or Pluronic L64 as anionic and nonionic surfactants, respectively, in order to obtain lamellar LLC phases. The same surfactants were used to prepare also the vesicular systems (niosomes) that were added to the corresponding gel. The obtained multicomponent drug carrier was characterized by deuterium nuclear magnetic resonance spectroscopy, in order to understand if the introduction of the drug or drug-loaded niosomal suspension, as third component in the formulations, could influence the microstructure of the system and then the drug delivery across the skin. Simple AOT and L64-based niosomal formulations and LLCs phases were then prepared and used as control. Different drugs percutaneous availability was achieved, and the results revealed that the obtained gel-niosomes carriers were affected by the chemical structure of the drugs and by their affinity for the components. As a consequence these systems could be proposed as novel transdermal drug delivery systems, since they were found able to control the percutaneous permeation of small drugs across the skin. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In recent years, the transdermal route of administration has found wide applications and gained considerable commercial success, winning the competition with oral, intravenous or intramuscular routes [1]. A problem that has been faced is the low penetration of most compounds through the outermost layer of the skin, the stratum corneum (SC). The SC consists of terminally differentiated keratinocytes, referred to as corneocytes, embedded in a lipid-rich intercellular matrix. The main constituents of this matrix are ceramides (CER), cholesterol (CHOL) and long-chain free fatty acids. These lipids are organized in crystalline lamellae at room temperature and play a key role in the barrier function of human skin. Absorption of substances into and across skin takes place by passive diffusion through the lipid domains of SC, since the driving force is the difference in the drug concentration between drug carriers and blood. In order to reduce temporary the SC barrier and improve drug vehiculation, penetration enhancers are developed to alter the SC lipid structural organization, avoiding side effects and
∗ Corresponding author. E-mail address: [email protected] (R. Muzzalupo). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.04.017
allowing a better control than other conventional delivery methods [2]. Among the several approaches, one possibility to increase the amount of drug vehiculated across the SC, is the use of lyotropic liquid crystals (LLCs), due to their stability, low interfacial tension arising at the oil/water interface and capability to increase the solubility of drugs, which are either insoluble or slightly soluble in water [3]. The LLC are excellent examples of self-assembling nanomaterials. They are usually formed from water and one or more surfactants at very definite proportions. Their phase sequence (cubic, hexagonal, lamellar) depends on both the different components concentration and temperature. Among the different mesophases, the lamellar ones represent the best approaches to be used as transdermal drug delivery systems, due to their special skin similarly structure. In fact, the structural units for the lamellar phase are double layers formed by surfactant molecules disposed in a bidimensional stacking of infinite layers, delimited by water. The polar heads of the molecules are in contact with the aqueous medium, while the hydrocarbon chains are interdigitating in order to avoid water. This phase is rather fluid and the bilayers can slip easily one on the other. This structure is very similar to that occurring in living organisms
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and for this reason lamellar LLC represents optimal candidate for the transdermal vehiculation of drugs [4]. An improvement of the pharmaceutical properties of these drug delivery systems can be obtained by the addition of vesicular systems in the LLCs lamellae. Several works, in fact, reported that the LLC networks can serve as an encapsulating matrix, escorting and protecting the embedded vesicles entrapping active entities and can enhance their stability prolonging the circulation time without losing drug [5]. In addition these multicomponent systems have attracted particular interest in the design of dermal and transdermal delivery systems due to the possibility to achieve prolonged and programmed drug percutaneous permeation through the skin [6]. In a previous work, our research group has carried out investigation on the complex systems based on Pluronic F127 and Tween 60 vesicles, to study the rheological interaction between polymer and vesicles and to evaluate the potential use of the systems as efficient drug delivery formulation [7]. Results demonstrated that the binary systems can act as transdermal controlled and prolonged delivery systems of diclofenac sodium salt. In this light, we decide to evaluate the effect of the chemical structure of different surfactants and vehiculated drug on the physico-chemical properties and drug percutaneous permeation profiles of novel multicomponent formulations. For these reasons we used Pluronic L64 or Aerosol OT (AOT) as surfactants and sulfadiazine sodium salt (Sul), propranolol hydrochloride (Pro) and tyrosol (Tyr) as model drugs. LLCs samples were prepared at fixed ratio between Pluronic L64 or AOT and water, in order to obtain lamellar LLC phases and the corresponding surfactant was used to prepare also the vesicular systems (niosomes) that were added to the gel. Pluronic L64 is a non-ionic copolymer of ethylene oxide (EO) and propylene oxide (PO) blocks arranged in a triblock structure, in which the hydrophobic polypropylene oxide (PPO) group links two hydrophilic polyethylene oxide (PEO). The amphiphilic nature of Pluronic makes it extremely useful in various fields as emulsifier, stabilizer and pharmaceutical additive. The presence of ethylene oxide moieties may reduce opsonisation and clearance by the reticuloendothelial system, leading to improved pharmacokinetic properties of the carriers. Several works reported the use of L64 as surfactant to obtain niosomes useful as parenteral and transdermal delivery systems vehiculation of different drugs [8]. AOT is a versatile double-tailed anionic surfactant whose phase equilibria with water and organic solvent has been extensively investigated, because its non toxicity, spontaneously formed and thermodynamically stable long lived micellar forms and it is known as a transdermal drug delivery vehicle [9]. Drugs with anionic, cationic and nonionic chemical structures and different properties such as Sul, Pro and Tyr, respectively, were incorporated into L64 and AOT lamellar LLC phases as free drug solutions or as drugloaded niosomal suspension. Deuterium resonance spectroscopy (2 H NMR) were used to evaluate the occurred structural modifications caused by the incorporation of drugs as a third component in the surfactant–water systems. Finally the percutaneous permeation profile of all the drugs from the lamellar LLC phases obtained from L64 and AOT were performed, in order to understand how the different formulations could give rise to a different transdermal delivery of drugs. 2. Materials and methods 2.1. Materials Pluronic L64, poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) copolymer, was provided from BASF (Mount Olive, NJ, USA). AOT (sodium bis(2-ethylhexyl) sulfosuccinate), sulfadiazine sodium salt, propranolol hydrochloride and tyrosol were supplied by Sigma–Aldrich, Milan, Italy. The drug
Table 1 Details on the preparation of drug-loaded gels. Samples
Surfactant name
Surfactant (g)
D2 O (g)
Drug
Drug (g)
G-AOT-Sul
AOT
0.90
2.07
0.03
G-AOT-Pro
AOT
0.90
2.07
G-AOT-Tyr G-L64-Sul
AOT L64
0.90 2.25
2.07 0.72
G-L64-Pro
L64
2.25
0.72
G-L64-Tyr
L64
2.25
0.72
Sulfadiazine sodium salt Propranolol hydrochloride Tyrosol Sulfadiazine sodium salt Propranolol hydrochloride Tyrosol
0.03 0.03 0.03 0.03 0.03
content in the permeation studies was analyzed by UV-VIS JASCO V-530 spectrometer using 1 cm quartz cells at the pertinent wavelengths. Ultrapure water from a Millipore Synergy® purification unit was used. Deuterium oxide (Sigma–Aldrich, Milan, Italy) was used in order to perform 2 H NMR measurements. 2.2. Preparation of drug-loaded LLC gels LLC gel samples were prepared using a fixed ratio between surfactants and water, in order to obtain lamellar LLC phases. The percentage of drug added to each formulation was 1% in weight. Details on the preparation were reported in Table 1. Briefly, the preparation is as follows: 0.03 g of hydrophilic drug was dissolved in the appropriate amount of water and mixed with each surfactant. Samples were mixed several times and heated at 40 ◦ C, until a homogeneous mixture was obtained and stored at room temperature. The samples were analyzed only after one week. Details on the samples preparation are reported in Table 1. 2.3. Preparation of niosomes Multilamellar niosomal vesicles (MLVs) were prepared vortexing and subsequent sonication. Accurately weighed amounts of L64 or AOT were putted in a round-bottom flask in the presence of 10 mL of mixture of distilled and deuterated water (empty niosomes) or 10 mL of mixture of distilled and deuterated water containing 0.03 g of drug (drug-loaded niosomes) at 25 and 60 ◦ C for AOT and L64, respectively, for 30 min, to form large multilamellar vesicles. 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 25 and 60 ◦ C for AOT and L64, respectively. The niosomes purification was also carried out by exhaustive dialysis for 6 h, using Visking tubing (20/30), manipulated before use in according to Fenton’s method [10]. Final formulations were stored at 4 ◦ C until used in subsequent experiments (Table 2). 2.3.1. Characterization of niosomes 2.3.1.1. Morphology. The morphology of hydrated niosome dispersions was examined by transmission electron microscopy (TEM). A drop of dispersion was stratified onto a carbon-coated copper grid and left to adhere on the carbon substrate for about 1 min. The dispersion in excess was removed by a piece of filter paper. A drop of 2% phosphotungstic acid solution was stratified and, again, the solution in excess was removed by a tip of filter paper. The sample was air-dried and observed under a ZEISS EM 10 electron microscope at an accelerating voltage of 80 kV. 2.3.1.2. Size and distribution. The niosomes size and standard deviation were determined by dynamic light scattering (DLS) analysis using 90 Plus Particle Size Analyzer (Brookhaven Instruments
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Table 2 Details on the preparation and physico-chemical properties of drug-loaded niosomes. Samples
Surfactant name
Surfactant (g)
Drug
Drug (g)
D2 O (mL)
H2 O (mL)
Diameter (nm)
P.I.
E%
N-AOT-Sul N-AOT-Pro N-AOT-Tyr N-L64-Sul N-L64-Pro N-L64-Tyr
AOT AOT AOT L64 L64 L64
0.04 0.04 0.04 0.29 0.29 0.29
Sulfadiazine Propranolol Tyrosol Sulfadiazine Propranolol Tyrosol
0.03 0.03 0.03 0.03 0.03 0.03
5 5 5 5 5 5
5 5 5 5 5 5
408 274 384 366 361 391
0.29 0.19 0.27 0.24 0.21 0.27
30 71 24 12 10 13
Corporation, New York, USA) at 25.0 ± 0.1 ◦ C by measuring the autocorrelation function at 90◦ . The laser was operating at 658 nm. Data were fitted by the inverse “Laplace transformation” using Contin program [11]. The polydispersity index (P.I.) was used as a measure of the width of size distribution. P.I. less than 0.4 indicates a homogenous and monodisperse population. All the samples were analyzed 24 h after their preparation. They were diluted with distilled water before the measurements. In particular, 50 l of each vesicle dispersion was diluted to 10 ml with distilled water. Each sample was measured three times and results are expressed as mean ± standard deviation. 2.3.1.3. Entrapment efficiency. Drug encapsulation efficiency (E%) was determined by exhaustive dialysis for separating the nonentrapped drug from niosomes [12]. According to this method, 3 mL of drug-loaded niosomal dispersion were dropped into a dialysis bag (Spectra/Por, MW cut-off 12,000, Spectrum, Canada) immersed in 100 mL of distilled water and magnetically stirred. Free drug was dialyzed for 30 min each time and the dialysis was complete when no drug was detectable in the recipient solution. The E% was expressed as the percentage of the drug entrapped into niosomes referred to the total amount of drug that is present in the non-dialyzed sample. It was determined by diluting 1 mL of dialyzed and 1 mL of non-dialyzed niosomes in 25 mL of methanol, followed by the measurement of absorbance of these solutions at the respective wavelengths. This procedure is 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. 2.4. Preparation of gel-niosomes systems The gel-niosomes systems were prepared by dissolving proper amount of surfactant in a given volume of vesicles stock dispersions. All samples were stored at room temperature for 1 week before performing the experiments. All the prepared mixtures are listed and labeled in Table 3.
aggregates dispersed in deuterium oxide, 2 H NMR spectroscopy can be used to investigate both the local order of molecules and the structure of the aggregates. In our study, 2 H NMR experiments were performed at a resonance frequency of 46.53 MHz on a Bruker AVANCE 300 pulsed superconducting spectrometer working in Fourier Transform mode. The sample temperature was controlled during NMR measurements by passing air at the desired temperature (3.0 ± 0.1 ◦ C) through the sample holder. A quadrupole echo sequence with a /2 pulse width of 3.5 s was used for acquiring 2 H NMR spectra. The delay between the two /2 pulses was 40 s and repetition time was 1 s. To allow samples to reach thermal equilibrium, spectra were recorded 30 min after each temperature setting. 3.2. In vitro 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 [12]. 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 amount of sample, so as to keep constant the drug amount (5.0 and 2.7 mg for AOT and L64 based formulations, respectively) and the receptor compartment was filled with 5.5 mL of distilled water. During the study, the donor chamber was covered by parafilm. At regular intervals up to 8 h, the medium in the receiver compartment was removed and replaced with an equal volume of prethermostated (37 ± 0.5 ◦ C) freshly distilled water. The complete substitution of the medium was needed to ensure sink conditions and quantitative determination of the small amounts of drug permeated. The content of drug in the samples was analyzed by UV–Vis spectrometry. Each experiment was carried out in triplicate, and the results were in agreement within ±4% standard error.
3. Methods 4. Results and discussion 3.1.
2H
NMR theory and experiments
Phases and phase transitions in LLCs can be identified by Deuterium Nuclear Magnetic Resonance spectroscopy (2 H NMR) and polarized optical microscopy (POM) observations. In the case of
The design of novel formulations increasing the effectiveness of existing drugs is one of new trends observed in pharmaceutical technology in recent years [13]. In this context, LLCs have aroused great interest as novel dosage forms, due to their stability, low
Table 3 Details on the preparation of drug-loaded gel-niosomes systems. Samples
Surfactant name
Surfactant moles (×10−3 )
Niosomal solution (mL)
Drug
Final drug amount (g × 10−3 )
NG-AOT-Sul NG-AOT-Pro NG-AOT-Tyr NG-L64-Sul NG-L64-Pro NG-L64-Tyr
AOT AOT AOT L64 L64 L64
0.90 0.90 0.90 2.25 2.25 2.25
1.85 1.85 1.85 0.64 0.64 0.64
Sulfadiazine sodium salt Propranolol hydrochloride Tyrosol Sulfadiazine sodium salt Propranolol hydrochloride Tyrosol
5.5 5.5 5.5 1.9 1.9 1.9
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interfacial tension, and capability to increase the solubility of both oil and water soluble compounds. In addition, they exhibit good penetration across the skin, and show extensive similarity to living organisms biological membranes and chromosomes [14]. The LLCs samples were prepared using an accurate ratio between surfactant and water, in order to obtain lamellar LLC phases, for both L64 and AOT. In the case of the Pluronic L64, X-ray experiments reported in literature reveal a large lamellar LLC phase in the surfactant-rich region of the phase diagram [15] and for this reason we decided to prepare the sample at 75 wt% of the surfactant. For the AOT, since the published AOT–water diagram showed a lamellar phase extends from ca. 5 to ca. 70 wt%, the systems were prepared starting from 30 wt% of AOT, in order to minimize the final amount of surfactant present in the formulations [16]. The loaded-drugs formulations were prepared by adding drug solution or drug-loaded niosomal suspension in a fixed wt percentage, so as to keep constant the amount of drug in all the formulations. The obtained mesophases, were characterized by 2 H NMR to evaluate the structural modifications. In fact, it is known that the incorporation of drugs as a third component could induce structural modifications and these influence the in vitro penetration of the therapeutic agents in the skin. In the present study, drugs with anionic, cationic and nonionic chemical structures such as Sul, Pro and Tyr, respectively, were used as model drugs. Sul is an topical antibiotic used to protect infected burns; it prevents the growth of a wide array of bacteria, as well as yeast, on the damaged skin. Propranolol, one of the most widely prescribed -blockers in the long-term treatment of hypertension and in psychotherapy is usually taken orally. Following oral administration, it is rapidly and completely absorbed from gastrointestinal tract, still the oral bioavailability is low because of significant first pass hepatic metabolism. For this reason topical application is ideal for propranolol, but the transdermal absorption is poor, whereby different approaches to increase the permeation are needed. Tyr is a phenolic compound present in two of the traditional components of the Mediterranean diet: wine and virgin olive oil. It was shown to be able to carry out antioxidant activity in vitro studies and for these reasons it could be used to prevent or reduce erythema, photoaging, photocarcinogenesis, edema, and skin hypersensitivity associated with exposure to ultraviolet B radiation [17]. 4.1. AOT and L64-based niosomes The AOT/water binary system is dominated by a large lamellar LLC phase and at high surfactant content, a discontinuous cubic phase and a reversed hexagonal phase are formed, as well reported in literature. The lamellar microstructure of the system AOT/water has been subjected to different analysis in the past years: unusual results were observed, like the modification of the optical signal of the birefringence in the range between 30 and 40 wt% of AOT, the discontinuity of the electrical conductivity and the anomalies from the X-ray data that divided the lamellar mesophase in three different ranges. Two of them show an ideal swelling behavior and are separated by a no-swelling one [18]. Many hypothesis were studied and was observed that the systems were strongly influenced by mechanical stresses which force the lamellae to assume defective configurations: Coppola et al. showed that the lamellar phase of the AOT/water system is constituted by small non-interconnected domains [19]. AOT is well known to form monodispersed, unilamellar and stable vesicles in aqueous solution. The efficacy of these vesicles to host both charged and hydrophobic drugs has also been demonstrated in several works and makes them useful as drug delivery systems [20]. In our case the vesicles were prepared by vortex followed by sonication method, in order to obtain a control on the physico-chemical properties of the niosomes. Dynamic
light scattering measurements of the obtained empty vesicles were carried out in dilute aqueous solution at room temperature and indicated a diameter of 372 nm. The distribution profile was found to be unimodal with relatively low P.I., suggesting the existence of vesicles of uniform sizes. The introduction of equal amount of drugs was found to affect the vesicles size, depending on the molecular structure of the encapsulated molecules (Table 2). Hydrodynamic diameter of 274, 408 and 384 nm were found for the cationic, anionic and non-ionic drugs, respectively. Since AOT is an anionic surfactant, it is clear that these results are strongly connected with the charge of the drugs: in fact, ionic repulsions among anionic groups of AOT and sulfadiazine result in the higher increase of vesicles dimensions; a decrease of the hydrodynamic diameter was achieved in the presence of propranolol, since ionic attraction occurred, while in the case of tyrosol as non-ionic molecule, an intermediate value of diameter was found, probably due to the absence of net charge. The P.I. was between 0.19 and 0.29: these values were considered as evidence of homogeneous distribution of colloidal vesicles. Drug vesicle encapsulation efficiency is a product of different factors, the most important are: the ionization state of the drugs and the charges of the niosomal matrix. As reported before concerning the niosomal size, the chemical structure of the encapsulated drug and the affinity for the niosomal bilayer strongly affect the partitioning and location of drug molecules into niosomes. Sul, Pro and Tyr are hydrophilic drugs and they are located in the aqueous core of the vesicles. As reported in Table 2, E% were found to be 30, 71 and 24% for Sul, Pro and Tyr, respectively, and are dependent on the ionic and electrostatic interactions between the ionic groups of the carrier and the drugs. Pluronic L64 is a water-soluble triblock copolymer forming vesicles. These drug carriers were demonstrated to have a spherical structure, with the hydrophobic block forming the inner part of the bilayer, while the hydrophilic PEO blocks are withdrawing to the inside aqueous core of the vesicles or outside. These block copolymer vesicles showed properties as drug carriers for the transdermal and parenteral vehiculations of small molecules of pharmacological interest. As reported in Table 2, empty vesicles of L64 were prepared following the thin layer evaporation method, and the diameter was found to be 396 nm. This value was found to be similar to that reported in our previous work [8] and it was also similar to that obtained from AOT, even if, in this case no repulsions between similar charged groups resulting in an increase of vesicles diameter occurred. The introduction of drugs determined a decrease of vesicles size: this is very common when hydrophilic drugs were introduced into the niosomes. The chemical structure of the drugs did not affect in a relevant manner the hydrodynamic diameter of the carriers: in the case of Sul, this value was found to be 356 nm, while in the case of Pro and Tyr the dimensions increased up to 361 and 391 nm, respectively. The P.I. was between 0.21 and 0.27. The E% values of 12, 10 and 13% were found in the case of Sul, Pro and Tyr, respectively. These values are comparable and reflect the trend found in the case of vesicles size, in which the chemical structure of the drugs did not influence the dimensions. In fact, it seems that, regardless of the charge of the drug, the L64 based niosomes were able to encapsulate only a small amount of hydrophilic molecules. In the transmission electron micrograph, all niosomal formulations were spherical and homogeneous in shape and the size was well correlated with the results of the laser diffraction particle size (DLS). No sedimentation or flocculation were observed and the samples were found to be stable for 9 months. 4.2. AOT based liquid crystal and liquid crystal-niosomes systems Generally the 2 H NMR spectra obtained by the binary system AOT/water, at the concentration used in this study, did not
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show a classical lamellar structure, because of the presence of microdomains that gives rise to biaxial lineshapes. Previous investigations demonstrated that the structural organization of the systems can be attributed to a “defective” lamellar phase [19]. In our case, the addition of drugs to the liquid crystals samples, both as drug solution or niosomal suspension, resulted in a viscous, transparent and birefringent gel. Collected 2 H NMR spectra showed the presence of lamellar mesophases similar to those obtained without the drug: the third component did not change the phase diagram of the sample, at this surfactant concentration (Fig. 1). In fact, the drugs induced a weak size modifications of microdomains consequently, the systems keep the typical biaxial shape. Only when the sample was prepared in the presence of Pro loaded vesicular suspension, the recorded 2 H NMR spectrum showed significant modifications because of lamellar macrodomains presence (Fig. 1f). All systems were investigated after 6 months, the spectra were not reported because they showed similar lineshape. Just the systems containing Pro gave a lamellar uniaxial phase, due to the interaction between drug and surfactant, giving the formation in situ of propranolol-bis(2ethylhexyl)sulfosuccinate salt (Fig. 1g and h). In literature, it was reported that the system sodium dodecyl sulfate-propranolol hydrochloride (as surfactant and drug, respectively) affected the drug release in tablet [21]. 4.3. L64 based liquid crystal and liquid crystal-niosomes systems The G-L64 and NG-L64-Pro samples appeared as transparent and birefrangent lamellar gels, but less viscous than the corresponding ones based on AOT. The 2 H NMR spectra showed a pure lamellar mesophase and this means that the introduction of Pro as third component did not affect its microstructures (Fig. 2a and b). Similar trends were found in the case of Sul and Tyr-based formulations (data not shown). 4.4. Percutaneous permeation studies The success of a transdermal drug delivery system depends on the drug ability to permeate the skin in a sufficient amount to maintain its therapeutic levels. Drug compounds can be classified chemically as ionic, zwitterionic or neutral. It is well established that small, neutral compounds will permeate the skin barrier more readily than charged species, while charged drug molecules are, in general, considered to be unsuitable for transdermal drug delivery. An ideal drug candidate would have sufficient lipophilicity to partition into the SC, but also sufficient hydrophilicity to enable the second partitioning step into the viable epidermis and, eventually, the systemic circulation. However, research continues into possible methods of enhancing the transdermal permeation of these particular compounds: among these, LLC represents a valid alternative [22]. A good drug nanocarrier should prerequisitely be able to release a drug in a controlled manner and for this reason the percutaneous permeation profiles of the three different model drugs from all obtained formulations were evaluated. Results showed particular trends because the formation of ion pair between AOT and cationic drugs and repulsions between charged drugs and skin. 4.5. G-AOT and G-L64 systems percutaneuos permeation profiles The cumulative quantities of Prop, Sul and Tyr permeated from AOT and L64 gel systems across the skin are plotted versus time in Fig. 3a and b, respectively. In the case of AOT-based formulations, the permeation profiles ranged from 0.300 to 0.025 mg/cm2 within 8 h: the higher amount of permeated drug was obtained in the case of Tyr, the non-ionic
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molecule, while lower values were obtained by G-AOT-Sul; intermediate values were collected with G-AOT-Pro (0.139 mg/cm2 ). When L64 was used as surfactant, some differences in the permeation profiles occurred: in fact, the higher cumulative amount of drug permeated across the skin was obtained for G-L64-Pro, followed by G-L64-Tyr and G-L64-Sul samples. This trend could be ascribed to the synergistic action between the positive charge of the propranolol and the cell membrane negative potential of the stratum corneum, increasing the drug permeation. In both cases, the cumulative sulfadiazine percutaneous permeations achieved from G-AOT-Sul and G-L64-Sul were found to be very low and these behaviors appeared to confirm the repulsions between the negative charged drug and the cell membrane negative potential of the stratum corneum. In addition, as showed in Fig. 3a and b, the cumulative drugs permeations from AOT-based gel systems were found to be higher than those obtained from L64-based ones. An additional mechanism for the skin penetration enhancement by AOT could involve the hydrophobic interaction of its alkyl chains with the skin structure which leaves the end sulfonate group of the surfactant exposed, creating additional sites in the membrane which leads to permit an increase in skin hydration [23]. 4.6. N-AOT and N-L64 systems percutaneuos permeation profiles The drugs permeation profiles from AOT and L64 niosomal samples were reported in Fig. 4a and b. As showed, the trends found for these formulations were similar to those obtained in the case of gel systems. In fact the higher cumulative amount of permeated drug was obtained by N-AOT-Tyr, followed by N-AOT-Pro and then by N-AOT-Sul samples, while in the case of L64, the order is as follows: N-L64-Pro, N-L64-Tyr and N-L64-Sul. These results confirmed the hypothesis that the percutaneous permeations of the model drugs are strongly dependent on the physico-chemical nature of the surfactants and not by the kind of carrier used in the experimental (gel or niosomes). In fact, the drugs permeation order from niosomal systems was found to be the same obtained by the gel systems. 4.7. NG-AOT and NG-L64 systems percutaneous permeation profiles Generally, in order to make them suitable for transdermal administration, niosomes were usually incorporated in a gel. Once incorporated they showed slightly lower drug permeation, ascribed to the slow diffusion of drug through the gel network. Percutaneous permeation profiles of Pro, Sul and Tyr from niosomes-gel systems are reported in Fig. 5a and b. The presence of intact niosomes into the multicomponent systems was confirmed by diluting the formulation in aqueous medium and performing light scattering experiments. In fact, in the case of sulfadiazine, vesicles diameter after dilution was found to be 415 nm for AOT based-niosomes and 350 nm for L64 ones. Similar trends have been obtained for the other drugs. In niosome-gel samples, the drug is distributed inside and outside the vesicles: however, unlike simple niosomal systems, the drug outside the vesicles is not quickly available for the permeation, because it is embedded into the matrix and must diffuse through it to reach the skin and to permeate it. In this light the capability of the drug to interact with the gel matrix could affect in a relevant manner its diffusion through the network. As shown in Fig. 4b, the higher cumulative permeated drug amount was achieved by NGAOT-Pro, followed by NG-AOT-Tyr and NG-AOT-Sul. This behavior differs from those obtained in the case of corresponding gel or niosomes systems, in which the samples containing tyrosol showed the best permeation performance. This could be due to the formation of the lipophilic propranolol-bis(2-ethylhexyl)sulfosuccinate
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Fig. 1. 2 H NMR spectra of AOT lamellar mesophases: (a) G-AOT-Sul; (b) NG-AOT-Sul; (c) G-AOT-Tyr; (d) NG-AOT-Tyr; (e) G-AOT-Pro; (f) NG-AOT-Pro; (g) G-AOT-Pro after 6 months; (h) NG-AOT-Pro after 6 months.
Fig. 2.
2
H NMR spectra of L64 lamellar mesophases: (a) G-L64-Pro and (b) NG-L64-Pro.
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Fig. 3. Permeated drug cumulative amounts as a function of time from: (a) G-AOT formulations: (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt. (b) G-L64 formulations: (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt (mean ± SD; n = 3).
Fig. 4. Permeated drug cumulative amounts as a function of time from: (a) N-AOT formulations (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt. (b) N-L64 formulations: (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt (mean ± SD; n = 3).
salt that possesses an higher ability to diffuse into the network respect to the Pro temporarily altering the membrane permeability and enhancing the drug skin permeation. The formation of a more lipophilic salt is the reason because the propranolol, despite attractive interactions, permeated faster than the sulfadiazine. In the case of L64-based multicomponent systems, the higher cumulative permeated drug amount was achieved by NG-L64-Tyr, followed by NG-L64-Pro and NG-L64-Sul. Also in this case the trend is partially different from those obtained by the simple gel or niosomal systems. As reported above, since drugs entrapment efficiencies were found to be similar, the amount of drugs embedded into the network and then immediately available were comparable, whereby the only explanation could be related to the different interactions and affinity between each drug and its diffusion through the matrix. 4.8. General remarks The results obtained in this work are in line with recent findings suggesting that factors such as vehicle composition, drug solubility in the matrix and its viscosity, play an important role in the skin
permeation. In all cases it is important to note that both AOT and L64-based formulations act as percutaneous permeation enhancers of Sul, Pro and Tyr. In fact, all cumulative permeated drug amounts were always higher than those obtained from the corresponding free solutions. As consequence, these systems could be proposed as novel transdermal drug delivery systems. The diffusion mechanisms involved in the drugs permeation are probably different for the kind of systems (gel, niosomes and gel-niosomes formulations) and also for the different structures of the drugs. Clearly AOT and L64 both in the form of niosomes and as LLCs, interact with the lipid structure of the SC and also change its hydration level in such a way that facilitates the permeation of drugs across the skin. Several works reported that the hydrophobic interactions of the alkyl chain of an anionic surfactant (AOT) with the keratinous proteins, leaves the negative end group of the surfactant exposed and induces additional anionic sites in the membrane cells. This results in a greater repulsive force that separates the protein matrix and results in the softening of the SC. On the other hand, L64, as pluronic copolymer surfactant of ethylene oxide (EO) and propylene oxide (PO) blocks arranged in a triblock structure, is claimed to attract
Fig. 5. Permeated drug cumulative amounts as a function of time from: (a) NG-AOT formulations (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt. (b) NG-L64 formulations: (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt (mean ± SD; n = 3).
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water molecules and this could also increase skin hydration and lipid fluidization. 5. Conclusions In this paper we have developed novel multicomponent formulations based on liquid crystal-niosomes systems, for the transdermal drugs delivery. AOT and L64 were used as surfactants and Sul, Pro and Tyr were used as model drugs. Clearly, the vehicle composition, drug solubility and its chemical structure play an important role in skin permeation. In fact, it is not possible to establish a definite, common trend, since several variables could be considered. All the obtained formulations, such as gel-niosomes systems, niosomal suspensions and gels, act as percutaneous permeation enhancer respect to the corresponding free drug solutions used as control. Finally these formulations could be considered as effective functional materials for controlled transdermal delivery of small hydrophilic molecules. Acknowledgements MIUR, the Italian Ministry for University, is acknowledged for financial support (Grants # EX-60%, PRIN 2010/11). Moreover, the project has been co-funded with support from the Commission European Social Fund and Region of Calabria (Italy). References [1] H.Y. Thong, H. Zhai, H.I. Maibach, Skin Pharmacol. Physiol. 20 (2007) 272. [2] B.W. Barry, Dermatological Formulation: Percutaneous Absorption, Marcel Dekker, New York, 1983.
[3] G.A. Kossena, W.N. Charman, B.J. Boyd, C.J. Porter, J. Control. Release 99 (2004) 217. [4] D. Chapman, Lyotropic mesophase in biological systems, in: B. Bahadur (Ed.), Liquid Crystals Applications and Uses, World Scientific, Singapore, 1991. [5] G. Grassi, A. Crevatin, R. Farra, G. Guarnieri, A. Pascotto, B. Rehimers, R. Lapasin, M. Grassi, J. Coll. Interf. Sci. 301 (2006) 282. [6] A. Manosroi, P. Jantrawuta, J. Manosroi, Int. J. Pharm. 360 (2008) 156. [7] F.E. Antunes, L. Gentile, C. Oliviero Rossi, L. Tavano, G.A. Ranieri, Coll. Surf. B: Biointerfaces 1 (2011) 42. [8] L. Tavano, R. Muzzalupo, S. Trombino, R. Cassano, A. Pingitore, N. Picci, Coll. Surf. B: Biointerfaces 79 (2010) 227. [9] R. Muzzalupo, L. Tavano, R. Cassano, S. Trombino, T. Ferrarelli, N. Picci, Eur. J. Pharm. Biopharm. 79 (2011) 28. [10] 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. [11] S.W. Provencher, Comput. Phys. Commun. 27 (1982) 213. [12] L. Tavano, R. Muzzalupo, R. Cassano, S. Trombino, T. Ferrarelli, N. Picci, Coll. Surf. B: Biointerfaces 75 (2010) 319. [13] L. Tavano, P. Alfano, R. Muzzalupo, B. De Cindio, Coll. Surf. B: Biointerfaces 87 (2011) 333. [14] R. Muzzalupo, L. Tavano, F.P. Nicoletta, S. Trombino, R. Cassano, N. Picci, J. Drug Targets 8 (2010) 404. [15] P. Alexandridis, D. Zhou, A. Khan, Langmuir 12 (1996) 2690. [16] O. Robles-Vàsqez, S. Corona-Galvàn, J.F.A. Soltero, J.E. Puig, J. Coll. Interf. Sci. 160 (1993) 65. [17] N. Vassallo, Polyphenols and Health: New and Recent Advances, Nova Publishers, New York, 2008. [18] Park, J. Rogers, R.W. Toft, P.A. Winsor, Coll. Surf. B: Bionterfaces 32 (1970) 81. [19] L. Coppola, R. Muzzalupo, G.A. Ranieri, M. Terenzi, Langmuir 11 (1995) 1116. [20] R. Saha, P.K. Verma, R.K. Mitra, S.K. Pal, Coll. Surf. B: Biointerfaces 88 (2011) 345. [21] J.F. Ford, K. Mitchell, D. Sawh, S. Ramdour, D.J. Armstrong, P.N.C. Elliott, C. Rostron, J.E. Hogan, Int. J. Pharm. 71 (1991) 213. [22] Y.B. Bannon, J. Corish, O.I. Corrigan, J.G. Devane, M. Kavanagh, S. Mulligan, Eur. J. Clin. Pharmacol. 37 (1989) 285. [23] L.D. Rhein, C.R. Robbins, K. Fernee, R. Cantore, J. Soc. Cosmet. Chem. 37 (1986) 125.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Novel gel-niosomes formulations as multicomponent systems for transdermal drug delivery Lorena Tavano a,b , Luigi Gentile c , Cesare Oliviero Rossi c , Rita Muzzalupo a,∗ a
Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria, Ed. Polifunzionale, 87036 Arcavacata di Rende (CS), Italy Dipartimento di Ingegneria Informatica, Modellistica, Elettronica e Sistemistica, Università della Calabria, Via P. Bucci Cubo 39/C, 87036 Arcavacata di Rende (CS), Italy c Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, Ponte P. Bucci, Cubo 14/D, 87036 Arcavacata di Rende (CS), Italy b
a r t i c l e
i n f o
Article history: Received 12 February 2013 Received in revised form 27 March 2013 Accepted 18 April 2013 Available online 6 May 2013 Keywords: Pluronic AOT Liquid crystals Niosomes Transdermal delivery
a b s t r a c t The percutaneous permeation profiles of sulfadiazine sodium salt, propranolol hydrochloride and tyrosol from novel liquid crystal-niosomes formulations as multicomponent systems, were investigated. The new carriers were prepared from mixture of water/surfactant, AOT or Pluronic L64 as anionic and nonionic surfactants, respectively, in order to obtain lamellar LLC phases. The same surfactants were used to prepare also the vesicular systems (niosomes) that were added to the corresponding gel. The obtained multicomponent drug carrier was characterized by deuterium nuclear magnetic resonance spectroscopy, in order to understand if the introduction of the drug or drug-loaded niosomal suspension, as third component in the formulations, could influence the microstructure of the system and then the drug delivery across the skin. Simple AOT and L64-based niosomal formulations and LLCs phases were then prepared and used as control. Different drugs percutaneous availability was achieved, and the results revealed that the obtained gel-niosomes carriers were affected by the chemical structure of the drugs and by their affinity for the components. As a consequence these systems could be proposed as novel transdermal drug delivery systems, since they were found able to control the percutaneous permeation of small drugs across the skin. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In recent years, the transdermal route of administration has found wide applications and gained considerable commercial success, winning the competition with oral, intravenous or intramuscular routes [1]. A problem that has been faced is the low penetration of most compounds through the outermost layer of the skin, the stratum corneum (SC). The SC consists of terminally differentiated keratinocytes, referred to as corneocytes, embedded in a lipid-rich intercellular matrix. The main constituents of this matrix are ceramides (CER), cholesterol (CHOL) and long-chain free fatty acids. These lipids are organized in crystalline lamellae at room temperature and play a key role in the barrier function of human skin. Absorption of substances into and across skin takes place by passive diffusion through the lipid domains of SC, since the driving force is the difference in the drug concentration between drug carriers and blood. In order to reduce temporary the SC barrier and improve drug vehiculation, penetration enhancers are developed to alter the SC lipid structural organization, avoiding side effects and
∗ Corresponding author. E-mail address: [email protected] (R. Muzzalupo). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.04.017
allowing a better control than other conventional delivery methods [2]. Among the several approaches, one possibility to increase the amount of drug vehiculated across the SC, is the use of lyotropic liquid crystals (LLCs), due to their stability, low interfacial tension arising at the oil/water interface and capability to increase the solubility of drugs, which are either insoluble or slightly soluble in water [3]. The LLC are excellent examples of self-assembling nanomaterials. They are usually formed from water and one or more surfactants at very definite proportions. Their phase sequence (cubic, hexagonal, lamellar) depends on both the different components concentration and temperature. Among the different mesophases, the lamellar ones represent the best approaches to be used as transdermal drug delivery systems, due to their special skin similarly structure. In fact, the structural units for the lamellar phase are double layers formed by surfactant molecules disposed in a bidimensional stacking of infinite layers, delimited by water. The polar heads of the molecules are in contact with the aqueous medium, while the hydrocarbon chains are interdigitating in order to avoid water. This phase is rather fluid and the bilayers can slip easily one on the other. This structure is very similar to that occurring in living organisms
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and for this reason lamellar LLC represents optimal candidate for the transdermal vehiculation of drugs [4]. An improvement of the pharmaceutical properties of these drug delivery systems can be obtained by the addition of vesicular systems in the LLCs lamellae. Several works, in fact, reported that the LLC networks can serve as an encapsulating matrix, escorting and protecting the embedded vesicles entrapping active entities and can enhance their stability prolonging the circulation time without losing drug [5]. In addition these multicomponent systems have attracted particular interest in the design of dermal and transdermal delivery systems due to the possibility to achieve prolonged and programmed drug percutaneous permeation through the skin [6]. In a previous work, our research group has carried out investigation on the complex systems based on Pluronic F127 and Tween 60 vesicles, to study the rheological interaction between polymer and vesicles and to evaluate the potential use of the systems as efficient drug delivery formulation [7]. Results demonstrated that the binary systems can act as transdermal controlled and prolonged delivery systems of diclofenac sodium salt. In this light, we decide to evaluate the effect of the chemical structure of different surfactants and vehiculated drug on the physico-chemical properties and drug percutaneous permeation profiles of novel multicomponent formulations. For these reasons we used Pluronic L64 or Aerosol OT (AOT) as surfactants and sulfadiazine sodium salt (Sul), propranolol hydrochloride (Pro) and tyrosol (Tyr) as model drugs. LLCs samples were prepared at fixed ratio between Pluronic L64 or AOT and water, in order to obtain lamellar LLC phases and the corresponding surfactant was used to prepare also the vesicular systems (niosomes) that were added to the gel. Pluronic L64 is a non-ionic copolymer of ethylene oxide (EO) and propylene oxide (PO) blocks arranged in a triblock structure, in which the hydrophobic polypropylene oxide (PPO) group links two hydrophilic polyethylene oxide (PEO). The amphiphilic nature of Pluronic makes it extremely useful in various fields as emulsifier, stabilizer and pharmaceutical additive. The presence of ethylene oxide moieties may reduce opsonisation and clearance by the reticuloendothelial system, leading to improved pharmacokinetic properties of the carriers. Several works reported the use of L64 as surfactant to obtain niosomes useful as parenteral and transdermal delivery systems vehiculation of different drugs [8]. AOT is a versatile double-tailed anionic surfactant whose phase equilibria with water and organic solvent has been extensively investigated, because its non toxicity, spontaneously formed and thermodynamically stable long lived micellar forms and it is known as a transdermal drug delivery vehicle [9]. Drugs with anionic, cationic and nonionic chemical structures and different properties such as Sul, Pro and Tyr, respectively, were incorporated into L64 and AOT lamellar LLC phases as free drug solutions or as drugloaded niosomal suspension. Deuterium resonance spectroscopy (2 H NMR) were used to evaluate the occurred structural modifications caused by the incorporation of drugs as a third component in the surfactant–water systems. Finally the percutaneous permeation profile of all the drugs from the lamellar LLC phases obtained from L64 and AOT were performed, in order to understand how the different formulations could give rise to a different transdermal delivery of drugs. 2. Materials and methods 2.1. Materials Pluronic L64, poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) copolymer, was provided from BASF (Mount Olive, NJ, USA). AOT (sodium bis(2-ethylhexyl) sulfosuccinate), sulfadiazine sodium salt, propranolol hydrochloride and tyrosol were supplied by Sigma–Aldrich, Milan, Italy. The drug
Table 1 Details on the preparation of drug-loaded gels. Samples
Surfactant name
Surfactant (g)
D2 O (g)
Drug
Drug (g)
G-AOT-Sul
AOT
0.90
2.07
0.03
G-AOT-Pro
AOT
0.90
2.07
G-AOT-Tyr G-L64-Sul
AOT L64
0.90 2.25
2.07 0.72
G-L64-Pro
L64
2.25
0.72
G-L64-Tyr
L64
2.25
0.72
Sulfadiazine sodium salt Propranolol hydrochloride Tyrosol Sulfadiazine sodium salt Propranolol hydrochloride Tyrosol
0.03 0.03 0.03 0.03 0.03
content in the permeation studies was analyzed by UV-VIS JASCO V-530 spectrometer using 1 cm quartz cells at the pertinent wavelengths. Ultrapure water from a Millipore Synergy® purification unit was used. Deuterium oxide (Sigma–Aldrich, Milan, Italy) was used in order to perform 2 H NMR measurements. 2.2. Preparation of drug-loaded LLC gels LLC gel samples were prepared using a fixed ratio between surfactants and water, in order to obtain lamellar LLC phases. The percentage of drug added to each formulation was 1% in weight. Details on the preparation were reported in Table 1. Briefly, the preparation is as follows: 0.03 g of hydrophilic drug was dissolved in the appropriate amount of water and mixed with each surfactant. Samples were mixed several times and heated at 40 ◦ C, until a homogeneous mixture was obtained and stored at room temperature. The samples were analyzed only after one week. Details on the samples preparation are reported in Table 1. 2.3. Preparation of niosomes Multilamellar niosomal vesicles (MLVs) were prepared vortexing and subsequent sonication. Accurately weighed amounts of L64 or AOT were putted in a round-bottom flask in the presence of 10 mL of mixture of distilled and deuterated water (empty niosomes) or 10 mL of mixture of distilled and deuterated water containing 0.03 g of drug (drug-loaded niosomes) at 25 and 60 ◦ C for AOT and L64, respectively, for 30 min, to form large multilamellar vesicles. 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 25 and 60 ◦ C for AOT and L64, respectively. The niosomes purification was also carried out by exhaustive dialysis for 6 h, using Visking tubing (20/30), manipulated before use in according to Fenton’s method [10]. Final formulations were stored at 4 ◦ C until used in subsequent experiments (Table 2). 2.3.1. Characterization of niosomes 2.3.1.1. Morphology. The morphology of hydrated niosome dispersions was examined by transmission electron microscopy (TEM). A drop of dispersion was stratified onto a carbon-coated copper grid and left to adhere on the carbon substrate for about 1 min. The dispersion in excess was removed by a piece of filter paper. A drop of 2% phosphotungstic acid solution was stratified and, again, the solution in excess was removed by a tip of filter paper. The sample was air-dried and observed under a ZEISS EM 10 electron microscope at an accelerating voltage of 80 kV. 2.3.1.2. Size and distribution. The niosomes size and standard deviation were determined by dynamic light scattering (DLS) analysis using 90 Plus Particle Size Analyzer (Brookhaven Instruments
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Table 2 Details on the preparation and physico-chemical properties of drug-loaded niosomes. Samples
Surfactant name
Surfactant (g)
Drug
Drug (g)
D2 O (mL)
H2 O (mL)
Diameter (nm)
P.I.
E%
N-AOT-Sul N-AOT-Pro N-AOT-Tyr N-L64-Sul N-L64-Pro N-L64-Tyr
AOT AOT AOT L64 L64 L64
0.04 0.04 0.04 0.29 0.29 0.29
Sulfadiazine Propranolol Tyrosol Sulfadiazine Propranolol Tyrosol
0.03 0.03 0.03 0.03 0.03 0.03
5 5 5 5 5 5
5 5 5 5 5 5
408 274 384 366 361 391
0.29 0.19 0.27 0.24 0.21 0.27
30 71 24 12 10 13
Corporation, New York, USA) at 25.0 ± 0.1 ◦ C by measuring the autocorrelation function at 90◦ . The laser was operating at 658 nm. Data were fitted by the inverse “Laplace transformation” using Contin program [11]. The polydispersity index (P.I.) was used as a measure of the width of size distribution. P.I. less than 0.4 indicates a homogenous and monodisperse population. All the samples were analyzed 24 h after their preparation. They were diluted with distilled water before the measurements. In particular, 50 l of each vesicle dispersion was diluted to 10 ml with distilled water. Each sample was measured three times and results are expressed as mean ± standard deviation. 2.3.1.3. Entrapment efficiency. Drug encapsulation efficiency (E%) was determined by exhaustive dialysis for separating the nonentrapped drug from niosomes [12]. According to this method, 3 mL of drug-loaded niosomal dispersion were dropped into a dialysis bag (Spectra/Por, MW cut-off 12,000, Spectrum, Canada) immersed in 100 mL of distilled water and magnetically stirred. Free drug was dialyzed for 30 min each time and the dialysis was complete when no drug was detectable in the recipient solution. The E% was expressed as the percentage of the drug entrapped into niosomes referred to the total amount of drug that is present in the non-dialyzed sample. It was determined by diluting 1 mL of dialyzed and 1 mL of non-dialyzed niosomes in 25 mL of methanol, followed by the measurement of absorbance of these solutions at the respective wavelengths. This procedure is 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. 2.4. Preparation of gel-niosomes systems The gel-niosomes systems were prepared by dissolving proper amount of surfactant in a given volume of vesicles stock dispersions. All samples were stored at room temperature for 1 week before performing the experiments. All the prepared mixtures are listed and labeled in Table 3.
aggregates dispersed in deuterium oxide, 2 H NMR spectroscopy can be used to investigate both the local order of molecules and the structure of the aggregates. In our study, 2 H NMR experiments were performed at a resonance frequency of 46.53 MHz on a Bruker AVANCE 300 pulsed superconducting spectrometer working in Fourier Transform mode. The sample temperature was controlled during NMR measurements by passing air at the desired temperature (3.0 ± 0.1 ◦ C) through the sample holder. A quadrupole echo sequence with a /2 pulse width of 3.5 s was used for acquiring 2 H NMR spectra. The delay between the two /2 pulses was 40 s and repetition time was 1 s. To allow samples to reach thermal equilibrium, spectra were recorded 30 min after each temperature setting. 3.2. In vitro 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 [12]. 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 amount of sample, so as to keep constant the drug amount (5.0 and 2.7 mg for AOT and L64 based formulations, respectively) and the receptor compartment was filled with 5.5 mL of distilled water. During the study, the donor chamber was covered by parafilm. At regular intervals up to 8 h, the medium in the receiver compartment was removed and replaced with an equal volume of prethermostated (37 ± 0.5 ◦ C) freshly distilled water. The complete substitution of the medium was needed to ensure sink conditions and quantitative determination of the small amounts of drug permeated. The content of drug in the samples was analyzed by UV–Vis spectrometry. Each experiment was carried out in triplicate, and the results were in agreement within ±4% standard error.
3. Methods 4. Results and discussion 3.1.
2H
NMR theory and experiments
Phases and phase transitions in LLCs can be identified by Deuterium Nuclear Magnetic Resonance spectroscopy (2 H NMR) and polarized optical microscopy (POM) observations. In the case of
The design of novel formulations increasing the effectiveness of existing drugs is one of new trends observed in pharmaceutical technology in recent years [13]. In this context, LLCs have aroused great interest as novel dosage forms, due to their stability, low
Table 3 Details on the preparation of drug-loaded gel-niosomes systems. Samples
Surfactant name
Surfactant moles (×10−3 )
Niosomal solution (mL)
Drug
Final drug amount (g × 10−3 )
NG-AOT-Sul NG-AOT-Pro NG-AOT-Tyr NG-L64-Sul NG-L64-Pro NG-L64-Tyr
AOT AOT AOT L64 L64 L64
0.90 0.90 0.90 2.25 2.25 2.25
1.85 1.85 1.85 0.64 0.64 0.64
Sulfadiazine sodium salt Propranolol hydrochloride Tyrosol Sulfadiazine sodium salt Propranolol hydrochloride Tyrosol
5.5 5.5 5.5 1.9 1.9 1.9
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interfacial tension, and capability to increase the solubility of both oil and water soluble compounds. In addition, they exhibit good penetration across the skin, and show extensive similarity to living organisms biological membranes and chromosomes [14]. The LLCs samples were prepared using an accurate ratio between surfactant and water, in order to obtain lamellar LLC phases, for both L64 and AOT. In the case of the Pluronic L64, X-ray experiments reported in literature reveal a large lamellar LLC phase in the surfactant-rich region of the phase diagram [15] and for this reason we decided to prepare the sample at 75 wt% of the surfactant. For the AOT, since the published AOT–water diagram showed a lamellar phase extends from ca. 5 to ca. 70 wt%, the systems were prepared starting from 30 wt% of AOT, in order to minimize the final amount of surfactant present in the formulations [16]. The loaded-drugs formulations were prepared by adding drug solution or drug-loaded niosomal suspension in a fixed wt percentage, so as to keep constant the amount of drug in all the formulations. The obtained mesophases, were characterized by 2 H NMR to evaluate the structural modifications. In fact, it is known that the incorporation of drugs as a third component could induce structural modifications and these influence the in vitro penetration of the therapeutic agents in the skin. In the present study, drugs with anionic, cationic and nonionic chemical structures such as Sul, Pro and Tyr, respectively, were used as model drugs. Sul is an topical antibiotic used to protect infected burns; it prevents the growth of a wide array of bacteria, as well as yeast, on the damaged skin. Propranolol, one of the most widely prescribed -blockers in the long-term treatment of hypertension and in psychotherapy is usually taken orally. Following oral administration, it is rapidly and completely absorbed from gastrointestinal tract, still the oral bioavailability is low because of significant first pass hepatic metabolism. For this reason topical application is ideal for propranolol, but the transdermal absorption is poor, whereby different approaches to increase the permeation are needed. Tyr is a phenolic compound present in two of the traditional components of the Mediterranean diet: wine and virgin olive oil. It was shown to be able to carry out antioxidant activity in vitro studies and for these reasons it could be used to prevent or reduce erythema, photoaging, photocarcinogenesis, edema, and skin hypersensitivity associated with exposure to ultraviolet B radiation [17]. 4.1. AOT and L64-based niosomes The AOT/water binary system is dominated by a large lamellar LLC phase and at high surfactant content, a discontinuous cubic phase and a reversed hexagonal phase are formed, as well reported in literature. The lamellar microstructure of the system AOT/water has been subjected to different analysis in the past years: unusual results were observed, like the modification of the optical signal of the birefringence in the range between 30 and 40 wt% of AOT, the discontinuity of the electrical conductivity and the anomalies from the X-ray data that divided the lamellar mesophase in three different ranges. Two of them show an ideal swelling behavior and are separated by a no-swelling one [18]. Many hypothesis were studied and was observed that the systems were strongly influenced by mechanical stresses which force the lamellae to assume defective configurations: Coppola et al. showed that the lamellar phase of the AOT/water system is constituted by small non-interconnected domains [19]. AOT is well known to form monodispersed, unilamellar and stable vesicles in aqueous solution. The efficacy of these vesicles to host both charged and hydrophobic drugs has also been demonstrated in several works and makes them useful as drug delivery systems [20]. In our case the vesicles were prepared by vortex followed by sonication method, in order to obtain a control on the physico-chemical properties of the niosomes. Dynamic
light scattering measurements of the obtained empty vesicles were carried out in dilute aqueous solution at room temperature and indicated a diameter of 372 nm. The distribution profile was found to be unimodal with relatively low P.I., suggesting the existence of vesicles of uniform sizes. The introduction of equal amount of drugs was found to affect the vesicles size, depending on the molecular structure of the encapsulated molecules (Table 2). Hydrodynamic diameter of 274, 408 and 384 nm were found for the cationic, anionic and non-ionic drugs, respectively. Since AOT is an anionic surfactant, it is clear that these results are strongly connected with the charge of the drugs: in fact, ionic repulsions among anionic groups of AOT and sulfadiazine result in the higher increase of vesicles dimensions; a decrease of the hydrodynamic diameter was achieved in the presence of propranolol, since ionic attraction occurred, while in the case of tyrosol as non-ionic molecule, an intermediate value of diameter was found, probably due to the absence of net charge. The P.I. was between 0.19 and 0.29: these values were considered as evidence of homogeneous distribution of colloidal vesicles. Drug vesicle encapsulation efficiency is a product of different factors, the most important are: the ionization state of the drugs and the charges of the niosomal matrix. As reported before concerning the niosomal size, the chemical structure of the encapsulated drug and the affinity for the niosomal bilayer strongly affect the partitioning and location of drug molecules into niosomes. Sul, Pro and Tyr are hydrophilic drugs and they are located in the aqueous core of the vesicles. As reported in Table 2, E% were found to be 30, 71 and 24% for Sul, Pro and Tyr, respectively, and are dependent on the ionic and electrostatic interactions between the ionic groups of the carrier and the drugs. Pluronic L64 is a water-soluble triblock copolymer forming vesicles. These drug carriers were demonstrated to have a spherical structure, with the hydrophobic block forming the inner part of the bilayer, while the hydrophilic PEO blocks are withdrawing to the inside aqueous core of the vesicles or outside. These block copolymer vesicles showed properties as drug carriers for the transdermal and parenteral vehiculations of small molecules of pharmacological interest. As reported in Table 2, empty vesicles of L64 were prepared following the thin layer evaporation method, and the diameter was found to be 396 nm. This value was found to be similar to that reported in our previous work [8] and it was also similar to that obtained from AOT, even if, in this case no repulsions between similar charged groups resulting in an increase of vesicles diameter occurred. The introduction of drugs determined a decrease of vesicles size: this is very common when hydrophilic drugs were introduced into the niosomes. The chemical structure of the drugs did not affect in a relevant manner the hydrodynamic diameter of the carriers: in the case of Sul, this value was found to be 356 nm, while in the case of Pro and Tyr the dimensions increased up to 361 and 391 nm, respectively. The P.I. was between 0.21 and 0.27. The E% values of 12, 10 and 13% were found in the case of Sul, Pro and Tyr, respectively. These values are comparable and reflect the trend found in the case of vesicles size, in which the chemical structure of the drugs did not influence the dimensions. In fact, it seems that, regardless of the charge of the drug, the L64 based niosomes were able to encapsulate only a small amount of hydrophilic molecules. In the transmission electron micrograph, all niosomal formulations were spherical and homogeneous in shape and the size was well correlated with the results of the laser diffraction particle size (DLS). No sedimentation or flocculation were observed and the samples were found to be stable for 9 months. 4.2. AOT based liquid crystal and liquid crystal-niosomes systems Generally the 2 H NMR spectra obtained by the binary system AOT/water, at the concentration used in this study, did not
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show a classical lamellar structure, because of the presence of microdomains that gives rise to biaxial lineshapes. Previous investigations demonstrated that the structural organization of the systems can be attributed to a “defective” lamellar phase [19]. In our case, the addition of drugs to the liquid crystals samples, both as drug solution or niosomal suspension, resulted in a viscous, transparent and birefringent gel. Collected 2 H NMR spectra showed the presence of lamellar mesophases similar to those obtained without the drug: the third component did not change the phase diagram of the sample, at this surfactant concentration (Fig. 1). In fact, the drugs induced a weak size modifications of microdomains consequently, the systems keep the typical biaxial shape. Only when the sample was prepared in the presence of Pro loaded vesicular suspension, the recorded 2 H NMR spectrum showed significant modifications because of lamellar macrodomains presence (Fig. 1f). All systems were investigated after 6 months, the spectra were not reported because they showed similar lineshape. Just the systems containing Pro gave a lamellar uniaxial phase, due to the interaction between drug and surfactant, giving the formation in situ of propranolol-bis(2ethylhexyl)sulfosuccinate salt (Fig. 1g and h). In literature, it was reported that the system sodium dodecyl sulfate-propranolol hydrochloride (as surfactant and drug, respectively) affected the drug release in tablet [21]. 4.3. L64 based liquid crystal and liquid crystal-niosomes systems The G-L64 and NG-L64-Pro samples appeared as transparent and birefrangent lamellar gels, but less viscous than the corresponding ones based on AOT. The 2 H NMR spectra showed a pure lamellar mesophase and this means that the introduction of Pro as third component did not affect its microstructures (Fig. 2a and b). Similar trends were found in the case of Sul and Tyr-based formulations (data not shown). 4.4. Percutaneous permeation studies The success of a transdermal drug delivery system depends on the drug ability to permeate the skin in a sufficient amount to maintain its therapeutic levels. Drug compounds can be classified chemically as ionic, zwitterionic or neutral. It is well established that small, neutral compounds will permeate the skin barrier more readily than charged species, while charged drug molecules are, in general, considered to be unsuitable for transdermal drug delivery. An ideal drug candidate would have sufficient lipophilicity to partition into the SC, but also sufficient hydrophilicity to enable the second partitioning step into the viable epidermis and, eventually, the systemic circulation. However, research continues into possible methods of enhancing the transdermal permeation of these particular compounds: among these, LLC represents a valid alternative [22]. A good drug nanocarrier should prerequisitely be able to release a drug in a controlled manner and for this reason the percutaneous permeation profiles of the three different model drugs from all obtained formulations were evaluated. Results showed particular trends because the formation of ion pair between AOT and cationic drugs and repulsions between charged drugs and skin. 4.5. G-AOT and G-L64 systems percutaneuos permeation profiles The cumulative quantities of Prop, Sul and Tyr permeated from AOT and L64 gel systems across the skin are plotted versus time in Fig. 3a and b, respectively. In the case of AOT-based formulations, the permeation profiles ranged from 0.300 to 0.025 mg/cm2 within 8 h: the higher amount of permeated drug was obtained in the case of Tyr, the non-ionic
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molecule, while lower values were obtained by G-AOT-Sul; intermediate values were collected with G-AOT-Pro (0.139 mg/cm2 ). When L64 was used as surfactant, some differences in the permeation profiles occurred: in fact, the higher cumulative amount of drug permeated across the skin was obtained for G-L64-Pro, followed by G-L64-Tyr and G-L64-Sul samples. This trend could be ascribed to the synergistic action between the positive charge of the propranolol and the cell membrane negative potential of the stratum corneum, increasing the drug permeation. In both cases, the cumulative sulfadiazine percutaneous permeations achieved from G-AOT-Sul and G-L64-Sul were found to be very low and these behaviors appeared to confirm the repulsions between the negative charged drug and the cell membrane negative potential of the stratum corneum. In addition, as showed in Fig. 3a and b, the cumulative drugs permeations from AOT-based gel systems were found to be higher than those obtained from L64-based ones. An additional mechanism for the skin penetration enhancement by AOT could involve the hydrophobic interaction of its alkyl chains with the skin structure which leaves the end sulfonate group of the surfactant exposed, creating additional sites in the membrane which leads to permit an increase in skin hydration [23]. 4.6. N-AOT and N-L64 systems percutaneuos permeation profiles The drugs permeation profiles from AOT and L64 niosomal samples were reported in Fig. 4a and b. As showed, the trends found for these formulations were similar to those obtained in the case of gel systems. In fact the higher cumulative amount of permeated drug was obtained by N-AOT-Tyr, followed by N-AOT-Pro and then by N-AOT-Sul samples, while in the case of L64, the order is as follows: N-L64-Pro, N-L64-Tyr and N-L64-Sul. These results confirmed the hypothesis that the percutaneous permeations of the model drugs are strongly dependent on the physico-chemical nature of the surfactants and not by the kind of carrier used in the experimental (gel or niosomes). In fact, the drugs permeation order from niosomal systems was found to be the same obtained by the gel systems. 4.7. NG-AOT and NG-L64 systems percutaneous permeation profiles Generally, in order to make them suitable for transdermal administration, niosomes were usually incorporated in a gel. Once incorporated they showed slightly lower drug permeation, ascribed to the slow diffusion of drug through the gel network. Percutaneous permeation profiles of Pro, Sul and Tyr from niosomes-gel systems are reported in Fig. 5a and b. The presence of intact niosomes into the multicomponent systems was confirmed by diluting the formulation in aqueous medium and performing light scattering experiments. In fact, in the case of sulfadiazine, vesicles diameter after dilution was found to be 415 nm for AOT based-niosomes and 350 nm for L64 ones. Similar trends have been obtained for the other drugs. In niosome-gel samples, the drug is distributed inside and outside the vesicles: however, unlike simple niosomal systems, the drug outside the vesicles is not quickly available for the permeation, because it is embedded into the matrix and must diffuse through it to reach the skin and to permeate it. In this light the capability of the drug to interact with the gel matrix could affect in a relevant manner its diffusion through the network. As shown in Fig. 4b, the higher cumulative permeated drug amount was achieved by NGAOT-Pro, followed by NG-AOT-Tyr and NG-AOT-Sul. This behavior differs from those obtained in the case of corresponding gel or niosomes systems, in which the samples containing tyrosol showed the best permeation performance. This could be due to the formation of the lipophilic propranolol-bis(2-ethylhexyl)sulfosuccinate
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Fig. 1. 2 H NMR spectra of AOT lamellar mesophases: (a) G-AOT-Sul; (b) NG-AOT-Sul; (c) G-AOT-Tyr; (d) NG-AOT-Tyr; (e) G-AOT-Pro; (f) NG-AOT-Pro; (g) G-AOT-Pro after 6 months; (h) NG-AOT-Pro after 6 months.
Fig. 2.
2
H NMR spectra of L64 lamellar mesophases: (a) G-L64-Pro and (b) NG-L64-Pro.
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Fig. 3. Permeated drug cumulative amounts as a function of time from: (a) G-AOT formulations: (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt. (b) G-L64 formulations: (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt (mean ± SD; n = 3).
Fig. 4. Permeated drug cumulative amounts as a function of time from: (a) N-AOT formulations (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt. (b) N-L64 formulations: (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt (mean ± SD; n = 3).
salt that possesses an higher ability to diffuse into the network respect to the Pro temporarily altering the membrane permeability and enhancing the drug skin permeation. The formation of a more lipophilic salt is the reason because the propranolol, despite attractive interactions, permeated faster than the sulfadiazine. In the case of L64-based multicomponent systems, the higher cumulative permeated drug amount was achieved by NG-L64-Tyr, followed by NG-L64-Pro and NG-L64-Sul. Also in this case the trend is partially different from those obtained by the simple gel or niosomal systems. As reported above, since drugs entrapment efficiencies were found to be similar, the amount of drugs embedded into the network and then immediately available were comparable, whereby the only explanation could be related to the different interactions and affinity between each drug and its diffusion through the matrix. 4.8. General remarks The results obtained in this work are in line with recent findings suggesting that factors such as vehicle composition, drug solubility in the matrix and its viscosity, play an important role in the skin
permeation. In all cases it is important to note that both AOT and L64-based formulations act as percutaneous permeation enhancers of Sul, Pro and Tyr. In fact, all cumulative permeated drug amounts were always higher than those obtained from the corresponding free solutions. As consequence, these systems could be proposed as novel transdermal drug delivery systems. The diffusion mechanisms involved in the drugs permeation are probably different for the kind of systems (gel, niosomes and gel-niosomes formulations) and also for the different structures of the drugs. Clearly AOT and L64 both in the form of niosomes and as LLCs, interact with the lipid structure of the SC and also change its hydration level in such a way that facilitates the permeation of drugs across the skin. Several works reported that the hydrophobic interactions of the alkyl chain of an anionic surfactant (AOT) with the keratinous proteins, leaves the negative end group of the surfactant exposed and induces additional anionic sites in the membrane cells. This results in a greater repulsive force that separates the protein matrix and results in the softening of the SC. On the other hand, L64, as pluronic copolymer surfactant of ethylene oxide (EO) and propylene oxide (PO) blocks arranged in a triblock structure, is claimed to attract
Fig. 5. Permeated drug cumulative amounts as a function of time from: (a) NG-AOT formulations (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt. (b) NG-L64 formulations: (triangle) tyrosol; (square) propranolol hydrochloride; (diamond) sulfadiazine sodium salt (mean ± SD; n = 3).
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water molecules and this could also increase skin hydration and lipid fluidization. 5. Conclusions In this paper we have developed novel multicomponent formulations based on liquid crystal-niosomes systems, for the transdermal drugs delivery. AOT and L64 were used as surfactants and Sul, Pro and Tyr were used as model drugs. Clearly, the vehicle composition, drug solubility and its chemical structure play an important role in skin permeation. In fact, it is not possible to establish a definite, common trend, since several variables could be considered. All the obtained formulations, such as gel-niosomes systems, niosomal suspensions and gels, act as percutaneous permeation enhancer respect to the corresponding free drug solutions used as control. Finally these formulations could be considered as effective functional materials for controlled transdermal delivery of small hydrophilic molecules. Acknowledgements MIUR, the Italian Ministry for University, is acknowledged for financial support (Grants # EX-60%, PRIN 2010/11). Moreover, the project has been co-funded with support from the Commission European Social Fund and Region of Calabria (Italy). References [1] H.Y. Thong, H. Zhai, H.I. Maibach, Skin Pharmacol. Physiol. 20 (2007) 272. [2] B.W. Barry, Dermatological Formulation: Percutaneous Absorption, Marcel Dekker, New York, 1983.
[3] G.A. Kossena, W.N. Charman, B.J. Boyd, C.J. Porter, J. Control. Release 99 (2004) 217. [4] D. Chapman, Lyotropic mesophase in biological systems, in: B. Bahadur (Ed.), Liquid Crystals Applications and Uses, World Scientific, Singapore, 1991. [5] G. Grassi, A. Crevatin, R. Farra, G. Guarnieri, A. Pascotto, B. Rehimers, R. Lapasin, M. Grassi, J. Coll. Interf. Sci. 301 (2006) 282. [6] A. Manosroi, P. Jantrawuta, J. Manosroi, Int. J. Pharm. 360 (2008) 156. [7] F.E. Antunes, L. Gentile, C. Oliviero Rossi, L. Tavano, G.A. Ranieri, Coll. Surf. B: Biointerfaces 1 (2011) 42. [8] L. Tavano, R. Muzzalupo, S. Trombino, R. Cassano, A. Pingitore, N. Picci, Coll. Surf. B: Biointerfaces 79 (2010) 227. [9] R. Muzzalupo, L. Tavano, R. Cassano, S. Trombino, T. Ferrarelli, N. Picci, Eur. J. Pharm. Biopharm. 79 (2011) 28. [10] 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. [11] S.W. Provencher, Comput. Phys. Commun. 27 (1982) 213. [12] L. Tavano, R. Muzzalupo, R. Cassano, S. Trombino, T. Ferrarelli, N. Picci, Coll. Surf. B: Biointerfaces 75 (2010) 319. [13] L. Tavano, P. Alfano, R. Muzzalupo, B. De Cindio, Coll. Surf. B: Biointerfaces 87 (2011) 333. [14] R. Muzzalupo, L. Tavano, F.P. Nicoletta, S. Trombino, R. Cassano, N. Picci, J. Drug Targets 8 (2010) 404. [15] P. Alexandridis, D. Zhou, A. Khan, Langmuir 12 (1996) 2690. [16] O. Robles-Vàsqez, S. Corona-Galvàn, J.F.A. Soltero, J.E. Puig, J. Coll. Interf. Sci. 160 (1993) 65. [17] N. Vassallo, Polyphenols and Health: New and Recent Advances, Nova Publishers, New York, 2008. [18] Park, J. Rogers, R.W. Toft, P.A. Winsor, Coll. Surf. B: Bionterfaces 32 (1970) 81. [19] L. Coppola, R. Muzzalupo, G.A. Ranieri, M. Terenzi, Langmuir 11 (1995) 1116. [20] R. Saha, P.K. Verma, R.K. Mitra, S.K. Pal, Coll. Surf. B: Biointerfaces 88 (2011) 345. [21] J.F. Ford, K. Mitchell, D. Sawh, S. Ramdour, D.J. Armstrong, P.N.C. Elliott, C. Rostron, J.E. Hogan, Int. J. Pharm. 71 (1991) 213. [22] Y.B. Bannon, J. Corish, O.I. Corrigan, J.G. Devane, M. Kavanagh, S. Mulligan, Eur. J. Clin. Pharmacol. 37 (1989) 285. [23] L.D. Rhein, C.R. Robbins, K. Fernee, R. Cantore, J. Soc. Cosmet. Chem. 37 (1986) 125.