SLN

International Journal of Pharmaceutics 434 (2012) 169– 174 Contents lists available at SciVerse ScienceDirect International Journal of Pharmaceutics jo ur nal homep a ge: www.elsev ier .com Pharmaceutical Nanotechnology Ideben dr evaluat Lucia Mo ia C a Department o b Department o a r t i c l Article history: Received 2 Ap Received in re Accepted 21 M Available onlin Keywords: Idebenone Skin permeati Skin penetrati Solid lipid nan Skin delivery e of u ative licatio were diffe n par tratio e dif SLN u h-20 oun results suggest that the SLN tested could be an interesting carrier for IDE targeting to the upper skin layers. © 2012 Elsevier B.V. All rights reserved. 1. Introdu In recen antioxidant rier of the b of oxidative As respons (ROS) and o Maibach, 2 antioxidant min E, ubiq uric acid an is responsib idant (Thie to an increa defined as lipids, prote tains higher while the h 10 that, on tained in h regarded as ∗ Correspon E-mail add 0378-5173/$ – http://dx.doi.o ction t years, great interest has been focused on the use of s for topical administration. Being the outermost bar- ody, the skin is exposed to various exogenous sources stress, including ultraviolet radiation and pollutants. e to these oxidative attacks, reactive oxygen species ther free radicals are generated in the skin (Dreher and 001). To counteract the deleterious effects of ROS, an network consisting of a variety of lipophilic (e.g. vita- uinones, carotenoids) and hydrophilic (e.g. vitamin C, d glutathione) antioxidants is present in the skin and le for the balance between pro-oxidants and antiox- le et al., 2000). An impairment of this balance, due sed exposure to exogenous sources of ROS, has been “oxidative stress” and involves oxidative damages of ins and DNA (Sies, 1985). Generally, the epidermis con- concentrations of antioxidants compared to the dermis orny layer lacks of co-antioxidants such as ubiquinol the contrary, is the most abundant ubiquinone con- uman skin. Topical administration of antioxidants is an interesting strategy in reducing ROS induced skin ding author. Tel.: +39 095 738 40 10; fax: +39 095 738 42 11. ress: lmontene@unict.it (L. Montenegro). damages since it may improve skin antioxidant capacity (Dreher and Maibach, 2001). A topical supplementation with antioxidants could be particularly beneficial for the stratum corneum due to its high susceptibility for UV and ozone-induced depletion of antioxi- dants (Thiele et al., 1998). In the last decades, many colloidal carriers have been pro- posed for drug targeting to the skin, such as liposomes (Bernard et al., 1997; Mezei et al., 1994) and solid lipid nanoparticles (SLN) (Papakostas et al., 2011; Pardeike et al., 2009; Zhang and Smith, 2011). The latter show several advantages compared to other drug delivery systems: good local tolerability, improved drug stabil- ity, drug targeting, increased bioavailability, ability to incorporate drugs with different physico-chemical properties, high inclusion rate for lipophilic substances and small particle size allowing close contact to the stratum corneum (Müller et al., 2000; Mehnert and Mäeder, 2001). Recently, we have developed a novel technique to prepare SLN using low amounts of surfactants by means of the phase inver- sion temperature (PIT) method, that allowed us to obtain SLN with promising physico-chemical and technological properties such as good stability, small particle size, narrow size distribution and good loading capacity (Montenegro et al., 2011, 2012). Such SLN were loaded with idebenone (IDE, Fig. 1), a synthetic derivative of ubiquinone with a shorter carbon side chain and a subsequent increased solubility (Wieland et al., 1995). IDE anti-oxidant activ- ity is due to its structural analogy with coenzyme Q10, a natural see front matter © 2012 Elsevier B.V. All rights reserved. rg/10.1016/j.ijpharm.2012.05.046 one-loaded solid lipid nanoparticles for ion ntenegroa,∗, Chiara Sinicob, Ines Castangiab, Claud f Drug Sciences, University of Catania, V.le A. Doria, 6, 95125 Catania, Italy f Life and Environment Sciences, Via Ospedale 72, 09124 Cagliari, Italy e i n f o ril 2012 vised form 21 May 2012 ay 2012 e 29 May 2012 on on oparticles a b s t r a c t Idebenone (IDE), a synthetic derivativ beneficial in the treatment of skin oxid upper layers of the skin by topical app SLN loading different amounts of IDE cetyl palmitate as solid lipid and three 20. All IDE loaded SLN showed a mea distribution. In vitro permeation/pene diffusion cells. IDE penetration into th IDE permeation occurred from all the epidermis when SLN contained cetet upper skin layers depended on the am / locate / i jpharm ug delivery to the skin: In vitro arbonea, Giovanni Puglisi a biquinone, shows a potent antioxidant activity that could be damages. In this work, the feasibility of targeting IDE into the n of IDE-loaded solid lipid nanoparticles (SLN) was evaluated. prepared by the phase inversion temperature method using rent non-ionic surfactants: ceteth-20, isoceteth-20 and oleth- ticle size in the range of 30–49 nm and a single peak in size n experiments were performed on pig skin using Franz-type ferent skin layers depended on the type of SLN used while no nder investigation. The highest IDE content was found in the or isoceteth-20 as surfactant while IDE distribution into the t of IDE loaded when oleth-20 was used as surfactant. These 170 L. Montenegro et al. / International Journal of Pharmaceutics 434 (2012) 169– 174 antioxidant tronic trans IDE potent ability to in mitochondr 1989). IDE a in preventin ages due to et al., 1999) eases (Scho In recent to be effec (Junyaprase suggesting have signifi skin oxidati Therefor IDE into the mis) by top method. Wi were perfor ent amount various non paper had a for drug del palmitate w both after t Lukowski e of IDE-load together wi 2. Materia 2.1. Materi Polyoxy plied by F ether (Arlas (Milan, Ital 20, was bou (Tegin O®, G Cetyl Palmi Care Chem Wyeth Led methylisoth kindly supp F68) was a g lulose mem supplied by used in the H Merck (Dar grade and u 2.2. Prepara IDE-load prepared u Table 1 Composition (%, w/w) of IDE-loaded SLN. SLN Ceteth Isoceteth Oleth GO CP IDE Watera 8.7 – – 4.4 7.0 0.5 q b 100 8.7 8.7 – – – – – er con usly and ffere ; the mpe en c rring e tur ity m tric an O inyl lisoth deg ansm nega were torie he su s so al of ima l JEM oltag oton part ast t 90◦ las ter a ed fo iffere ana ed w feren imid one ≥99 o calibrate the calorimetric system in transition tempera- d enthalpy changes, following the procedure of the Mettler software. 100 �l of each SLN sample (unloaded SLN prepared he same procedures but without the addition of IDE) was Fig. 1. Chemical structure of IDE. of cell membranes involved in the mitochondrial elec- port chain (Crane, 2001; Dallner and Sindelar, 2000). antioxidant activity has been mainly attributed to its hibit lipid peroxidation (LPO), and to protect cell and ial membranes from oxidative damage (Imada et al., ntioxidant activity has been proposed to be beneficial g skin aging and to protect the skin from oxidative dam- its exposure to environmental oxidative agents (Hoppe , other than in the treatment of neurodegenerative dis- ls et al., 2004). years, nanostructured lipid carriers have been reported tive in increasing skin permeation of Coenzyme Q10 rt et al., 2009) and of idebenone (Li and Ge, 2012), thus that nanoparticles containing these antioxidants could cant potential use as topical formulations for reducing ve damages. e, in this work we assessed the feasibility of targeting upper layers of the skin (stratum corneum and epider- ical application of IDE-loaded SLN prepared by the PIT th this aim, in vitro permeation and penetration studies med on newborn pig skin using SLN loaded with differ- s of IDE, consisting of cetyl palmitate as lipid core and -ionic surfactants. IDE loaded SLN investigated in this composition similar to that of IDE loaded SLN described ivery to the brain (Montenegro et al., 2011, 2012). Cetyl as chosen as solid lipid because of its good tolerability opical and systemic administration (Wang et al., 2009; t al., 2000). After in vitro application on the skin surface ed SLN, IDE penetration into the different skin layers th its permeation through the skin were evaluated. ls and methods als ethylene-20-cetyl ether (Brij 58®, Ceteth-20) was sup- luka (Milan, Italy). Polyoxyethylene-20-isohexadecyl olve 200 L®, Isoceteth-20) was a kind gift of Bregaglio y). Polyoxyethylene-20-oleyl ether (Brij 98®, Oleth- ght from Sigma–Aldrich (Milan, Italy). Glyceryl oleate O) was obtained from Th. Goldschmidt Ag (Milan, Italy). tate (Cutina CP®, CP) was purchased from Cognis S.p.a. icals (Como, Italy). Idebenone (IDE) was a kind gift of erle (Catania, Italy). Methylchloroisothiazolinone and iazolinone (Kathon CG®), and imidazolidinyl urea were lied by Sinerga (Milan, Italy). Poloxamer 188 (Lutrol® ift of BASF (Ludwigshafen, Germany). Regenerated cel- branes (Spectra/Por CE; Mol. Wt. Cut off 3000) were Spectrum (Los Angeles, CA, USA). Methanol and water PLC procedures were of LC grade and were bought from mstadt, Germany). All other reagents were of analytical sed as supplied. C1 C2 C3 I1 I2 O1 O2 O3 a Wat previo phase and di ∼90 ◦C stant te was th ous sti mixtur ductiv an elec W/O to dazolid methy that no 2.3. Tr For sions Labora Then t aqueou remov before (mode ation v 2.4. Ph SLN a Zetam light a a 4 mW diame obtain 2.5. D DSC equipp The re (w/w) iazolin (purity used t ture an STARe using t tion of SLN ed SLN, whose composition is reported in Table 1, were sing the phase inversion temperature (PIT) method, as transferred submitted 65 ◦C, at the the rate of carried out – – 4.4 7.0 0.7 q b 100 – – 4.4 7.0 1.1 q b 100 10.6 – 3.5 7.0 0.5 q b 100 10.6 – 3.5 7.0 0.7 q b 100 – 7.5 3.7 7.0 0.5 q b 100 – 7.5 3.7 7.0 0.7 q b 100 – 7.5 3.7 7.0 1.1 q b 100 taining 0.35% (w/w) imidazolidinyl urea and 0.05% (w/w) Kathon CG. reported (Montenegro et al., 2011). Briefly, the aqueous the oil phase (cetyl palmitate, the selected emulsifiers nt percentages w/w of IDE) were separately heated at n the aqueous phase was added drop by drop, at con- rature and under agitation, to the oil phase. The mixture ooled to room temperature under slow and continu- . At the phase inversion temperature (PIT), the turbid ned into clear. PIT values were determined using a con- eter mod. 525 (Crison, Modena, Italy) which measured conductivity change when the phase inversion from a /W system occurred. Water contained 0.35% (w/w) imi- urea and 0.05% (w/w) methylchloroisothiazolinone and iazolinone as preservatives. A TLC analysis confirmed radation of IDE occurred under these conditions. ission electron microscopy (TEM) tive-staining electron microscopy, 5 �l of SLN disper- placed on a 200-mesh formvar copper grid (TAAB s Equipment, Berks, UK), and allowed to be adsorbed. rplus was removed by filter paper. A drop of 2% (w/v) lution of uranyl acetate was added over 2 min. After the the surplus, the sample was dried at room condition ging the SLN with a transmission electron microscope 2010, Jeol, Peabody, MA, USA) operating at an acceler- e of 200 kV. correlation spectroscopy (PCS) icle sizes were determined at room temperature using er S (Malvern Instruments, Malvern, UK), by scattering . The instrument performed particle sizing by means of er diode operating at 670 nm. The values of the mean nd polydispersity index were the averages of results r three replicates of two separate preparations. ntial scanning calorimetry (DSC) analyses lyses were performed using a Mettler TA STARe System ith a DSC 822e cell and a Mettler STARe V8.10 software. ce pan was filled with 100 �l of water containing 0.35% azolidinyl urea and 0.05% (w/w) methylchloroisoth- and methylisothiazolinone. Indium and palmitic acid .95% and ≥99.5%, respectively; Fluka, Switzerland) were into a 160 �l calorimetric pan, hermetically sealed and to DSC analysis as follows: (i) a heating scan from 5 to rate of 2 ◦C/min; (ii) a cooling scan from 65 to 5 ◦C, at 4 ◦C/min, for at least three times. Each experiment was in triplicate. L. Montenegro et al. / International Journal of Pharmaceutics 434 (2012) 169– 174 171 2.6. Stability tests Samples of SLN were stored in airtight jars, and then kept in the dark at room temperature and at 37 ◦C for two months, separately. Particle sured at fix weeks, one 2.7. Determ IDE wate excess of d at room tem photo-degr concentrati method des 2.8. In vitro IDE rele sured throu diffusion ce ature (Shah method for formulation The cell water for 1 type diffusi volume of receptor w pseudo-sink in the recei for in vitro r ticle integri receiving so to maintain lation was a conditions a toinstability from the li withdrawn tion pre-eq analyzed by content. At on the mem mine partic was perform 2.9. In vitro Experim order to ach of Franz dif 0.785 cm2 a cutaneous squares of pigs (∼1.2– slaughterho in physiolo experiment donor and r tum corneu compartme solution, w bar. A recep experiment water/ethanol (50/50, v/v) could damage the barrier integrity of animal skin in in vitro skin permeation experiments (Friend, 1992). Due to a slightly different design of Franz-cells used to perform in vitro skin permeation experiments, to reach the physiological mpe as s ted s n wa d wi alyze r 24 rem erm skin iderm pel. nol, s t by ults w e di on ( te th of p igh HPL chro 20 � CA, chr 4.6 c at ro nol/w ml/m anti ion t ting No in ed. T ults N ch -load revi rans inves egati n we ana re in melt at o lipids perfo out as ch t abo ell d wn) bout size and polydispersity index of the samples were mea- ed time intervals (24 h, one week, two weeks, three month, and two months) after their preparation. ination of IDE solubility r solubility was determined in triplicate by stirring an rug in 2 ml of solvent with a magnetic stirrer for 24 h perature and avoiding light exposure to prevent IDE adation. Thereafter, the mixture was filtered and IDE on in its saturated solution was determined by the HPLC cribed below. release experiments ase rates from the SLN under investigation were mea- gh regenerated cellulose membranes using Franz-type lls (LGA, Berkeley, CA, USA). As reported in the liter- et al., 1989), this technique is regarded as a suitable evaluating drug release from pharmaceutical topical s. ulose membranes were moistened by immersion in h at room temperature before being mounted in Franz- on cells. Diffusion surface area and receiving chamber the cells were, respectively, 0.75 cm2 and 4.5 ml. The as filled with water/ethanol (50/50, v/v) for ensuring conditions by increasing active compound solubility ving phase. This receiving phase has already been used elease studies of IDE from SLN and no sign of nanopar- ty change was observed (Montenegro et al., 2011). The lution was constantly stirred and thermostated at 35 ◦C the membrane surface at 32 ◦C. 200 �l of each formu- pplied on the membrane surface under non occlusion nd the experiments were run for 24 h. Due to IDE pho- , all the release experiments were carried out sheltered ght. At intervals, 200 �l of the receptor phase were and replaced with an equal volume of receiving solu- uilibrated to 35 ◦C. The receptor phase samples were the HPLC method described below to determine IDE the end of the experiments, samples of the SLN applied brane surface were withdrawn and analyzed to deter- le sizes and polydispersity indexes. Each experiment ed in triplicate. skin permeation/penetration experiments ents were performed in triplicate (at least five times in ieve statistical significance), non-occlusively by means fusion vertical cells with an effective diffusion area of nd skin fragments excised from new born pigs. The sub- fat was carefully removed and the skin was cut into 3 cm × 3 cm and randomized. Goland–Pietrain hybrid 1.5 kg), died by natural causes, were provided by a local use. The skin, stored at −80 ◦C, was pre-equilibrated gical solution (NaCl 0.9%, w/v) at 25 ◦C, 2 h before the s. Skin specimens were sandwiched securely between eceptor compartments of the Franz cells, with the stra- m (SC) side facing the donor compartment. The receptor nt was filled with 5.5 ml of a 5% Poloxamer 188 water hich was continuously stirred with a small magnetic tor fluid different from that reported for in vitro release s was used because a receiving phase consisting of skin te ature w the tes solutio replace and an Afte SC was burg, G on the The ep ile scal metha conten Res into th deviati evalua Values 2.10. H The liquid with a Cotati, Italy). The metry, Italy) metha rate 1 IDE. Qu in relat by rela areas. observ 3. Res 3.1. SL IDE those p Fig. 2, t under of aggr tigatio DSC lipid co as the than th of the ments carried bulk w peak a ited a w not sho ature a rature (i.e. 32 ± 1 ◦C) the thermostating bath temper- et at 37 ± 1 ◦C throughout the experiments. 200 �l of amples was placed onto the skin surface. The receiving s withdrawn after elapsed times of 1, 2, 4, 6, 8 and 24 h, th an equal volume of solution to ensure sink conditions d by HPLC for drug content. h, the skin surface of specimens was washed and the oved by stripping with adhesive tape Tesa® AG (Ham- any). Each piece of the adhesive tape was firmly pressed surface and rapidly pulled off with one fluent stroke. is was separated from the dermis with a surgical ster- Tape strips, epidermis, and dermis were placed each in onicated to extract the drug and then assayed for drug HPLC. ere expressed as cumulative amount of IDE penetrated fferent skin layers after 24 h. Mean values ± standard SD) were calculated and Student’s t-test was used to e significance of the difference between mean values. < 0.05 were considered statistically significant. performance liquid chromatography (HPLC) analysis C apparatus consisted of a Hewlett-Packard model 1050 matograph (Hewlett-Packard, Milan, Italy), equipped l Rheodyne model 7125 injection valve (Rheodyne, USA) and an UV-VIS detector (Hewlett-Packard, Milan, omatographic analyses were performed using a Sim- m × 15 cm reverse phase column (C18) (Waters, Milan, om temperature and a mobile phase consisting of a ater mixture (80:20, v/v). The column effluent (flow in) was monitored continuously at 280 nm to detect fying IDE was performed by measuring the peak areas o those of a standard calibration curve that was built up known concentrations of IDE with the respective peak terference of the other formulation components was he sensitivity of the HPLC method was 0.1 �g/ml. and discussion aracterization and stability ed SLN physico-chemical properties were similar to ously reported (Montenegro et al., 2011). As shown in mission electron microscopy (TEM) analyses of the SLN tigation showed spherical particles with no evident sign on. As all the images obtained from the SLN under inves- re similar, we reported only one picture as example. lysis can be used to determine the physical state of the SLN (Müller et al., 2000; Mehnert and Mäeder, 2001), ing peak of the lipid core occurs at a lower temperature f the bulk lipid, mainly due to the nanocrystalline size in the SLN (Westesen and Bunjees, 1995). The experi- rmed to assess the physical state of the lipid core were on unloaded SLN. While the calorimetric curve of CP aracterized by a broad peak at about 39 ◦C and a main ut 50.5 ◦C, the calorimetric curve of unloaded SLN exhib- efined peak at about 38 ◦C and a shoulder at 42 ◦C (data . The melting peak of these SLN, observed at a temper- 12 ◦C lo