G Model
IJP 13084 1–10 ARTICLE IN PRESS
International Journal of Pharmaceutics xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
jo urnal homepage: www.elsevier.com/locate/ijpharm
1 Pharmaceutical nanotechnology
2 Bicellar systems as new delivery strategy for topical application of flufenamic acid
a,∗ a a a b a
3 Q1 L. Rubio ,C.Alonso , G. Rodríguez , M. Cócera , C. López-Iglesias , L. Coderch ,
a a a
4 A. De la Maza , J.L. Parra , O. López
a
5 Department of Chemical and Surfactants Technology (IQAC-CSIC), C/Jordi Girona 18-26, 08034 Barcelona, Spain
b
6 Scientific and Technological Centers-University of Barcelona (CCiT-UB), Barcelona Science Park, C/Baldiri Reixac, 10, 08028 Barcelona, Spain
7
8 a r t i c l e i n f o a b s t r a c t
9
10
Article history: In this work, bicellar systems, bilayered disc-shaped nanoaggregates formed in water by phospholipids,
11 Received 10 October 2012
are proposed as a novel strategy for delivery of the anti-inflammatory flufenamic acid (FFA) to the skin. A
12 Received in revised form 14 January 2013
comparative percutaneous penetration study of this drug in bicellar systems and other vehicles was
13 Accepted 17 January 2013
conducted. The effects induced on the skin by the application of FFA in the different vehicles were
Available online xxx
analyzed by attenuated total reflectance-fourier transform infrared (ATR-FTIR). Additionally, using the
14
microscopic technique freeze-substitution transmission electron microscopy (FSTEM) and X-ray scat-
15 Keywords:
tering technique using synchrotron radiation (SAXS-SR), we studied the possible microstructural and
16 Bicellar systems
organizational changes that were induced in the stratum corneum (SC) lipids and the collagen of the
18 DSC skin by the application of FFA bicellar systems. Bicellar systems exhibited a retarder effect on the per-
19 ATR-FTIR cutaneous absorption of FFA with respect to the other vehicles without promoting disruption in the SC
20 SAXS-SR barrier function of the skin. Given that skin disruption is one of the main effects caused by inflammation,
21
FS-TEM prevention of disruption and repair of the skin microstructure should be prioritized in anti-inflammatory
22 In vitro percutaneous absorption formulations.
23 Drug delivery © 2013 Published by Elsevier B.V.
24 1. Introduction (avoiding conventional surfactants) and nanodimensions of bicel- 36
lar systems, their use as delivery systems for topical applications 37
25 Bicellar systems are mixtures of two types of phospholipids has emerged as an interesting strategy. The use of bicellar systems 38
26 in aqueous medium, one with long alkyl hydrophobic chains and has been explored by our group for the transdermal delivery of the 39
27 another with short alkyl hydrophobic chains. These phospholipids amphiphilic drug diclofenac (Rubio et al., 2010). Additionally, we 40
28 are able to self-assemble, forming different nanoaggregates. The have demonstrated that these systems are able to work for skin 41
29 most common nanostructures formed are discoidal bicelles, which applications in two ways: as permeabilizing agents of the skin or 42
30 are intermediate forms between lipid vesicles and classical mixed as reinforcing agents of the lipid structures of the stratum corneum 43
31 micelles (Sanders and Prosser, 1998; Seddon et al., 2004). Long- (SC) (Barbosa-Barros et al., 2008b,c). The skin represents the largest 44
32 chain phospholipids constitute the bilayer structure in the central and most easily accessible organ of the body, and it is readily avail- 45
33 part of the bicelle, and the short-chain phospholipids complete able for drug application. Transdermal drug delivery offers many 46
34 the margins of the disc (Sanders and Schwonek, 1992; Vold and advantages compared to the conventional routes of application, 47
35 Prosser, 1996). Considering the structure, chemical composition including avoidance of the first-pass effect, stable blood levels, easy 48
application and higher compliance of the patient (Prausnitz et al., 49
2004; Thomas and Finnin, 2004) (Bal et al., 2010). To evaluate the 50
passage of substances through the skin and their distribution over 51
Abbreviations: SC, stratum corneum; FFA, flufenamic acid; DSC, differential scan- the different cutaneous compartments (Elias and Feingold, 2006; 52
ning calorimetry; ATR-FTIR, attenuated total reflectance-fourier transform infrared
OECD, 2004; Schaefer and Redermeier, 1996), in vitro percutaneous 53
spectroscopy; SAXS-SR, small angle X-ray scattering using synchrotron radiation;
absorption studies have been performed to determine the effec- 54
FSTEM, freeze-substitution transmission electron microscopy; DPPC, dipalmitoyl
tiveness of some vehicles as permeability enhancers of different 55
phosphatidylcholine; DHPC, dihexanoyl phosphatidylcholine; BSA, bovine serum
56
albumin; PBS, phosphate buffer saline; TEWL, transepidermal water loss; E, epider- compounds (Gwak and Chun, 2002).
mis; D, dermis; RF, receptor fluid; Perc. Abs., total percutanous absorption; HEX, Flufenamic acid (FFA) is a non-steroidal anti-inflammatory drug 57
hexagonal; OR, orthorhombic; C, corneocytes; L, lipids; CD, corneodesmosomes;
of the anthranilic acid group with potent anti-inflammatory and 58
DPPC, dimyristoyl phosphatidylcholine; Tm, gel-liquid phase transition tempera-
analgesic effects (Wagner et al., 2004). This drug, which is com- 59
ture; SPP, short periodicity phase.
∗ monly included in products for topical application, is insoluble 60
Corresponding author. Tel.: +34 93400 61 00; fax: +34 93204 59 04.
61
E-mail addresses: [email protected], [email protected] (L. Rubio). in water, and organic solvents that solubilize this drug could
0378-5173/$ – see front matter © 2013 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.ijpharm.2013.01.034
Please cite this article in press as: Rubio, L., et al., Bicellar systems as new delivery strategy for topical application of flufenamic acid. Int J
Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.01.034
G Model
IJP 13084 1–10 ARTICLE IN PRESS
2 L. Rubio et al. / International Journal of Pharmaceutics xxx (2013) xxx–xxx
62 impair the skin. Given that skin disruption is one of the major molar ratio 2:1. 10 mg of FFA was weighed and mixed with the 122
63 proinflammatory stimulators (Rahman et al., 2011), prevention of DPPC/DHPC chloroformic solution. The chloroform was removed by 123
64 disruption and reparation of the skin microstructure should be rota-evaporation, and the lipid film was dissolved in 1 ml of water 124
65 of primary importance in anti-inflammatory topical formulations. to reach a 20% (w/v) total lipid concentration and sonicated until a 125
66 Thus, in these formulations, not only should the drug be effec- transparent solution was obtained. pH measurements of FFA bicel- 126
67 tive, but the vehicle must also work in the same way to potentiate lar systems were performed with a Model 720 pH meter and a ROSS 127
68 the drug effect. Additionally, the deleterious effects of some anti- Model 8103 SC pH electrode (both from Orion Research, Cambridge, 128
69 inflammatory molecules themselves on skin structure (Schacke MA, USA). 129
70 et al., 2002) should be considered and avoided by using appropri-
71 ate skin-repairing vehicles. In this context, we think that the use
2.2.2. HPLC analysis 130
72 of bicellar systems as carriers of FFA may be a good alternative for
FFA was quantified by high-performance liquid chromatogra- 131
73 dermal applications.
phy (HPLC) using a Hitachi LaChrom Elite (Darmstadt, Germany). 132
74 Bicellar systems including FFA have been characterized (Rubio
The equipment consisted of an L-2130 pump, L-2200 autosam- 133
75 et al., 2011) using a combination of different techniques, such as
pler and L-400 UV detector. The system was operated using the 134
76 dynamic light scattering (DLS), freeze-fracture electron microscopy
Merck EZChrom Elite v3.1.3 software. The chromatographic sep- 135
77 (FFEM), cryo-transmission electron microscopy (Cryo-TEM), X-ray
aration was performed at room temperature using a Lichrocart 136
78 scattering and differential scanning calorimetry (DSC). The present
250-4/Lichrosorb RP-18 (5 m) column (Merck) with a flow rate 137
79 study investigates the percutaneous penetration of FFA in differ-
of 1.0 ml/min. 20 l of sample or calibration standard was injected 138
80 ent vehicles, as well as the effects induced on the skin by the
into the column and eluted with a mobile phase of methanol and 139
81 application of FFA bicellar systems. Attenuated total reflectance-
phosphate buffer (pH 2.5) (79:21, v/v). Detection was performed 140
82 fourier transform infrared (ATR-FTIR) spectroscopy, small-angle
by monitoring the absorbance signals at 284 nm. The calibration 141
83 X-ray scattering (SAXS) using synchrotron radiation (SR) and elec-
curve exhibited linear behavior (>0.99%) over the concentration 142
84 tron microscopy were used. ATR-FTIR is a suitable technique to
range of 0.078–97 g/ml. The intraday and interday variations of 143
85 determine the vibrational characteristic frequencies of the alkyl
the method were less than 2%. 144
86 chain lipids related to differently ordered phases (Boncheva et al.,
87 2008; Obata et al., 2010; Rodríguez et al., 2009). SAXS is an excellent
145
88 tool to study the structural organization of the collagen in the skin 2.2.3. In vitro percutaneous absorption studies
146
89 and the lipid lamellar organization of the SC (Cócera et al., 2011). The in vitro permeation experiments through porcine skin were
90 Freeze-substitution transmission electron microscopy (FSTEM) has performed using Franz-type diffusion cells with an exposure area 147
2
91 been commonly used to visualize the microstructure of the skin of 1.86 cm (Lara-Spiral, Courtenon, France). The porcine skin was 148
92 (Van den Bergh et al., 1997). In this work, the aforementioned excised from the dorsal region of female Landrace large white 149
93 techniques were used to evaluate the microstructure of skin after pigs weighing 30–40 kg. The pig skin was provided by the Clinical 150
94 treatment with FFA bicellar systems in vitro. Hospital of Barcelona, Spain. The bristles were removed carefully 151
with an animal clipper, and subsequently, the skin was washed 152
with water. The excised skin was dermatomed to a thickness of 153
95 2. Materials and methods
500 ± 50 m (Dermatome GA630, Aesculap, Tuttlingen, Germany). 154
Circular pieces of the dermatomed skin were obtained using an iron 155
96 2.1. Materials
punch (2.5 cm inner diameter) in such a way that they fit into the 156
◦
Franz diffusion cells. The skin discs were stored at −20 C. 157
97 Flufenamic acid (FFA) was purchased from Sigma–Aldrich (St.
Before the experiments, the skin discs were thawed and sand- 158
98 Louis, MO, USA). Dipalmitoyl phosphatidylcholine (DPPC) and
wiched securely between the two halves of the Franz cells (diffusion 159
99 dihexanoyl phosphatidylcholine (DHPC) were supplied by Avanti 2
area of 1.86 cm ) with the SC side facing the donor compartment. 160
100 Polar Lipids (Alabaster, AL, USA). Bovine serum albumin (BSA),
The receptor chamber was filled with a receptor fluid containing 161
101 phosphate-buffered saline (PBS) and gentamicin sulfate were
PBS (pH 7.4) in distilled water, 4% BSA, and 0.04% of gentamicin 162
102 obtained from Sigma–Aldrich. Methanol (HPLC grade), sodium
sulfate. 163
103 dihydrogen phosphate monohydrate and ortho-phosphoric acid ± ◦
The receptor phase was kept under constant stirring at 37 1 C 164
104 85% were acquired from Merck (Darmstadt, Germany). Purified
by means of a circulating water bath (Julabo Labortechnik GmbH, 165
105 water was obtained by an ultra-pure water system (Milli-Q plus
Germany) to ensure that the surface skin was maintained at 166
106 185, Millipore, Bedford, MA, USA). Ethanol was purchased from ◦
32 ± 1 C. The integrity of each skin sample was measured by deter- 167
107 Carlo Erba (Milano, Italy). Commercial topical gel containing FFA,
mining the transepidermal water loss (TEWL) using a Tewameter 168
108 salicylic acid and excipients such as isopropanol (Laboratorio Stada,
TM210 (Courage-Khazaka, Köln, Germany). The diffusion exper- 169
109 Sant Just Desvern, Spain) was purchased at a local pharmacy.
iment was initiated by applying each formulation to the entire 170
110 Trypsin (from porcine pancreas) was obtained from Sigma–Aldrich.
surface, delimited by the upper cell, 10 l of each of the ethanolic or 171
111 The chemicals for preparing microscopy samples were ruthe-
bicellar solution of FFA and approximately 4 mg of the commercial 172
112 nium tetroxide (RuO4), lowicryl HM20, glutaraldehyde, sodium
product of FFA. The doses applied were based on the OCDE rec- 173
113 cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA,
ommendations (OECD, 2004). The permeation experiments were 174
114 USA), methanol, potassium ferrocyanide (K4Fe(CN)6) (Merck), and
conducted in six replicates over 24 h. After 24 h, the content of 175
115 osmium tetroxide (OsO4) (Pelco International, Redding, CA, USA).
the donor compartment was washed with sodium lauryl ether sul- 176
fate and Milli-Q water following usual procedures, and the washing 177
116 2.2. Methods solution was collected. Then, the receptor fluid was removed from 178
the receptor compartment and brought up to 5 ml in a volumet- 179
117 2.2.1. Preparation of bicellar systems ric flask. Most of the SC of the treated skin area was removed by 180
®
181
118 Bicellar systems including FFA were formed with DPPC as the 8 successive tape-strippings using an adhesive tape (D-Squame ,
182
119 long-alkyl-chain phospholipid and DHPC as the short-alkyl-chain CuDerm Inc., Dallas, USA). After that, the viable epidermis (E) was
◦
183
120 phospholipid. An appropriate amount of DPPC was weighed and separated from the dermis (D) by heat treatment at 80 C for a few
184
121 mixed with a DHPC/chloroform solution to yield DPPC/DHPC in the seconds.
Please cite this article in press as: Rubio, L., et al., Bicellar systems as new delivery strategy for topical application of flufenamic acid. Int J
Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.01.034
G Model
IJP 13084 1–10 ARTICLE IN PRESS
L. Rubio et al. / International Journal of Pharmaceutics xxx (2013) xxx–xxx 3
185 The FFA in the washing solution and in the different skin layers 2.2.6. X-ray scattering measurements 248
186
(SC, E, and D) was extracted with methanol overnight. The different 2.2.6.1. Whole dermatomed skin. The SAXS profile of control skin 249
187
extracted samples and the receptor fluid were filtered through a and skin treated with FFA bicellar systems as described in the per- 250
188
0.45 m Acrodisc filter (Pall Gelman Sciences, Northampton, UK) cutaneous absorption section were analyzed to characterize the 251
189
and analyzed by HPLC as described above. organization of the collagen of the skin. SAXS measurements were 252
performed using the Spanish beamline (BM16) of the European 253
190
2.2.4. ATR-FTIR spectroscopy Synchrotron Radiation Facility (ESRF, Grenoble, France) at room 254
191
Four circular pieces of porcine skin were placed in Franz diffu- temperature. All of these skin samples were cut into sections 1 mm 255
192
sion cells as above. Three of them were treated with 10 l of FFA thick and 10 mm in diameter and were placed in the holder with the 256
193
ethanolic solution, FFA bicellar systems or approximately 10 mg of skin surface perpendicular to the beam. In the holder, skin pieces 257
194
FFA commercial product, respectively. The other sample was used were sandwiched between two aluminum plates with two holes, 258
195
as a control (untreated). The skin exposure lasted 24 h; after that, allowing the beam to pass directly through the samples. The energy 259
196