Lipid Nanoparticles as Carrier for Octyl-Methoxycinnamate: In Vitro Percutaneous Absorption and Photostability Studies

Pharmaceutical Nanotechnology Lipid Nanoparticles as Carrier for Octyl-Methoxycinnamate: In Vitro Percutaneous Absorption and Photostability Studies CARMELO PUGLIA,1 FRANCESCO BONINA,1 LUISA RIZZA,1 PAOLO BLASI,2 AURELIE SCHOUBBEN,2 ROSARIO PERROTTA,3 MARIA STELLA TARICO,3 ELISABETTA DAMIANI4 1

Dipartimento di Scienze del Farmaco, Facolt`a di Farmacia, Universit`a di Catania, 95125 Catania, Italy

2

Dipartimento di Chimica e Tecnologia del Farmaco, Facolt`a di Farmacia, Universit`a di Perugia, 06123 Perugia, Italy

3

Dipartimento di Specialit`a Medico Chirurgiche, Sezione di Chirurgia Plastica, Azienda Ospedaliera per l’Emergenza “Cannizzaro,” 95126 Catania, Italy 4

Dipartimento Scienze della Vita e dell’Ambiente, Universit`a Politecnica delle Marche, 60131 Ancona, Italy

Received 14 June 2011; revised 5 August 2011; accepted 9 August 2011 Published online 8 September 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22741 ABSTRACT: The aim of the present study was the evaluation of lipid nanoparticles (solid lipid nanoparticles, SLN, and nanostructured lipid carriers, NLC) as potential carriers for octyl-methoxycinnamate (OMC). The release pattern of OMC from SLN and NLC was evaluated in vitro, determining its percutaneous absorption through excised human skin. Additional in vitro studies were performed in order to evaluate, after UVA radiation treatment, the spectral stability of OMC-loaded lipid nanoparticles. From the obtained results, ultrasonication method yielded both SLN and NLC in the nanometer range with a high active loading and a particle shape close to spherical. Differential scanning calorimetry data pointed out the key role of the inner oil phase of NLC in stabilizing the particle architecture and in increasing the solubility of OMC as compared with SLN. In vitro results showed that OMC, when incorporated in viscosized NLC dispersions (OMC–NLC), exhibited a lower flux with respect to viscosized SLN dispersions (OMC–SLN) and two reference formulations: a microemulsion (OMC-ME) and a hydroalcoholic gel (OMC-GEL). Photostability studies revealed that viscosized NLC dispersions were the most efficient at preserving OMC from ultraviolet-mediated photodegradation. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:301–311, 2012 Keywords: Nanoparticles; Percutaneous; Photodegradation; Calorimetry (DSC); In vitro models; Skin; Particle size; Ultrasound

INTRODUCTION On certain aspects, a minimal solar radiation level may be beneficial for all organisms,1 but excessive exposure is clearly detrimental, leading to the wellknown harmful effects of ultraviolet (UV) radiations. Health agencies worldwide have urged the public to take protective measures against UV damage, recommending the use of sunscreen products. Sunscreen products contain UV filters, that is, chemical filters that absorb UV radiation and/or physical filters that reflect and scatter UV radiation. The inorganic compounds titanium oxide and zinc oxide Correspondence to: Carmelo Puglia (Telephone: +390957384273; Fax: +39-095222239; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 101, 301–311 (2012) © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association

are commonly used as physical UV filters in sunscreen products, whereas a wider range of organic compounds are used as chemical UV filters. Octyl-p-methoxycinnamate, or 2-ethylhexyl-4methoxycinnamate (OMC), is a compound widely used in topical preparations as a UVB absorber in the wavelength region of 290–320 nm. Notwithstanding the beneficial effects of OMC in cosmetic products, irritant, allergic, and photoallergic contact dermatitis have been reported.2,3 These phenomena are often due to the choice of an unsuitable cosmetic vehicle or incorrect incorporation of the sunscreen into a cosmetic formulation. Under these conditions, OMC may degrade under sunlight, resulting in toxic degradation products4 and/or may penetrate the skin, exposing the organism to potential harmful effects.5 Owing to these problems, several studies have been

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012

301

302

PUGLIA ET AL.

aimed at developing efficient means for OMC topical administration to be able to reduce systemic absorption and consequently harmful side effects and, on the contrary, to increase filter photostability.6–9 Lipid nanoparticles (solid lipid nanoparticles, SLN, and nanostructured lipid carriers, NLC), based on nonirritating and nontoxic lipids, provide a carrier system for various active agents with controlled release characteristics.10,11 This becomes an important tool when it is necessary to supply an active substance over a prolonged period of time, to reduce systemic absorption, and when it produces irritation at high concentration.12 Furthermore, lipid nanoparticles exhibit high physical stability, protection of incorporated labile actives against degradation, and can also act as physical sunscreen on their own.13 The last feature, in particular, appears to be very important because the total concentration of incorporated sunscreen can be decreased while maintaining the protective efficacy against UV radiations, but lowering the risk associated with sunscreen side effects. Although both SLN and NLC are submicron-size particles (40–1000 nm) and are based on solid lipids, they can be distinguished by their inner structure. NLC are composed of a solid lipid and an oil phase that seem to be organized in nanocompartments inside a solid lipid matrix. This, more complex architecture guarantees higher stability and drug loading as compared with the first generation of lipid nanoparticles (i.e., SLN) composed only of solid lipids.14 In this study, we have evaluated the potential use of SLN and NLC as carriers for OMC. With this aim, both lipid nanoparticle types were formulated and characterized, and their influence on in vitro percutaneous absorption of OMC was evaluated too. Because the formulations tested are lipid based, and because it is well known that lipid peroxidation is promoted and propagated by UVA-induced reactive oxygen species (ROS), the extent of this autocatalytic and uncontrolled process was also measured in the formulations using the thiobarbituric acid assay, concomitant to the measurement of the spectral stability of OMC.

MATERIALS AND METHODS Materials R Compritol 888 ATO (glyceryl behenate, tribehenin), a mixture of mono, di, and triglycerides of behenic acid (C22), was a gift from Gattefoss´e (Milan, Italy). R 812 (caprylic/capric triglycerides) was Miglyol provided by Eigenmann & Veronelli S.p.A (Milan, R F68 was a gift from BASF ChemItaly). Lutrol Trade GmbH (Burgbernheim, Germany). OMC was purchased from Cognis S.p.A. (Milan, Italy).

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012

Xanthan gum was purchased from Sigma Chemicals R 934 P (CTFA: carbomer) (Milan, Italy). Carbopol was obtained from BFGoodrich (Cleveland, Ohio). High-performance liquid chromatography (HPLC)grade solvents and water were purchased from CarloErba Reagents (Milan, Italy). All the other chemicals and reagents were of the highest purity grade commercially available. SLN and NLC Preparation

Ultrasonication Method SLN and NLC were prepared by ultrasonication R 888 ATO (4 g) was method.15 Briefly, Compritol R ◦  melted at 80 C, and Miglyol 812 (1.52 g) and OMC (2 g) were added. The melted lipid phase was disR F68; persed in hot (80◦ C) surfactant solution (Lutrol 1.35%, w/v) by using a high-speed stirrer (UltraTurrax T25; IKA-Werke GmbH & Company KG, Staufen, Germany) at 8000 rpm for 10 min. The obtained preemulsion was ultrasonified for 10 min by using a UP 400 S Ultraschallprozessor (Dr. Hielscher GmbH, Teltow, Germany), maintaining the temperature at least 5◦ C above the lipid melting point. The hot dispersion was then cooled in an ice bath under highspeed homogenization (UltraTurrax T25; IKA-Werke GmbH & Company KG) at 8000 rpm for 5 min in order to solidify the lipid matrix and to form NLCs. The preparation of blank and active-loaded SLNs was very similar to the procedure previously deR oil scribed. In this case, the amount of Miglyol needed to prepare NLC formulation was substituted R 888 ATO. with the same amount of Compritol

Preparation of Formulations Lipid nanoparticles were formulated into hydrogel using glycerol and xanthan gum as excipients. Briefly, the hydrogel formulation was produced by adding 10% (w/w) glycerol and 1% (w/w) xanthan gum to 89% (w/w) OMC-loaded NLC suspension (OMC–NLC) or OMC-loaded SLN suspension (OMC–SLN) and stirring for about 5 min. Other two formulations containing the same amount of OMC were prepared and used as reference formulations: a microemulsion (OMC-ME), which was R 888 ATO with prepared by replacing Compritol R  Miglyol 812 in the lipid nanoparticle preparation and then viscosizing as previously described, and an hydroalcoholic gel (70:30 water–ethanol; OMC-GEL) containing Carbomer (1%, w/w) and trietanolamine (1%, w/w). All the formulations were stored at 4◦ C before use. Particle Size Distribution Mean particle size of the lipid dispersions was measured by photon correlation spectroscopy (PCS). Zetamaster (Malvern Instrument Ltd., Worcs, England), DOI 10.1002/jps

LIPID NANOPARTICLES AS CARRIERS FOR OMC

equipped with a solid-state laser having a nominal power of 4.5 mW and a maximum output of 5 mW at 670 nm, was employed. Analyses were performed at a 90◦ scattering angle, at 20 ± 0.2◦ C. Samples were prepared by diluting 10 :L of lipid nanoparticle suspension with 2 mL of deionized water previously filtered through a 0.2-:m Acrodisc LC 13 polyvinylidene fluoride filter (Pall Gelman Laboratory, Ann Arbor, Michigan). Dynamic light scattering has an upper size limit of 5 :m. In fact, for particles larger than 5 :m, molecular collisions that cause Brownian motion are integrated over such a large volume that insufficient motion is produced. Moreover, particles much larger than 4λ in size show significant diffraction. Anyhow, the presence of small amounts of large particles (>5 :m), generally, cause an increase in the Gaussian distribution width and, for this reason, analyses were performed on freshly prepared samples and filtered suspensions (pore size 2.5 :m), comparing mean size and the Gaussian distribution width. Analyses were performed in triplicate and results were expressed as mean hydrodynamic diameter ± Gaussian distribution width. Careful optical microscopy observation (Nikon Eclipse 80i; Nikon Corporation, Tokyo, Japan) was used to confirm the absence of very large particles. Determination of OMC Loading The percentage of OMC entrapped in the lipid matrix was determined as follows: A fixed amount of SLN or NLC dispersion was filtered using a Pellicon XLTM tangential ultrafiltration system (Millipore, Milan, Italy) equipped with a polyethersulfone Biomax 1000 membrane (Millipore, Milan, Italy) with a 1,000,000 daltons molecular weight cutoff. An amount of retained material was freeze dried, dissolved in chloroform, and analyzed by UV spectrophotometry at 310 nm (Lambda 52; PerkinElmer, Waltham, Massachusetts). Calibration curves for validated UV assays of OMC were performed on five solutions in the concentration range 10—100 mg/mL. The correlation coefficient was greater than 0.990. Each point represented the average of three measurements, and the error was calculated as standard deviation (± SD). OMC incorporation efficiency was expressed as active recovery and calculated using Eq. 1: Drug recovery (%) =

Mass of active in nanoparticles × 100 Mass of active fed to the system

(1) Possible lipid interferences during UV determination of OMC were also investigated by comparing the standard curve of the substance alone and in the presence of lipids. The differences observed between the standard curves were within the experimental DOI 10.1002/jps

303

error, thus inferring that no lipid interference occurred (data not shown). Differential Scanning Calorimetry Studies Differential scanning calorimetry (DSC) was used to characterize suspension thermal behavior. In particular, lipid melting temperature depression due to the colloidal size, the presence of surfactant, and OMC inclusion was investigated.16 Experiments were performed on a DSC821e calorimeter (Mettler Toledo, Greifensee, Switzerland) equipped with a liquid nitrogen cooling system. A nitrogen purge at a flow rate of 50 cm3 /min was used to provide an inert gas atmosphere in the measurement cell. The system was previously calibrated using indium, with a hermetically sealed empty pan as reference. Aluminum pans (40 :L) were loaded with 40 :L of lipid suspension and hermetically sealed. Hermetically sealed empty pans were used as reference. Samples were submitted to two heating runs from 10 to 85◦ C. In order to study the effect of formulation dehydration once applied on the skin, 40 :L of each lipid suspension was loaded into the pan and stored at 35◦ C for 1 h. Then, pans were hermetically sealed and submitted to a single heating ramp from 10◦ C to 85◦ C. Prior to the first heating ramp, all samples were equilibrated at 10◦ C for 3 min and, in all cases, heating or cooling rate of 5◦ C/min was used. Data were treated with STARe software (Mettler Toledo) and the results were expressed as the mean of two determinations. Morphology Studies Lipid nanoparticles were morphologically characterized by means of transmission electron microscopy (TEM) (Philips EM 400T microscope; Philips; Eindhoven, Netherlands). Samples were prepared by simply depositing a drop of the lipid suspension on the R -coated copper grid (200 mesh) surface of a Formvar (TAAB Laboratories Equipment Ltd., Aldermaston, Berks, England) and letting it dry overnight. Analyses were performed without staining.

In vitro Studies Skin Membrane Preparation Samples of adult human skin (mean age 36 ± 8 years) were obtained from breast reduction operations. Subcutaneous fat was carefully trimmed and the skin was immersed in distilled water at 60 ± 1◦ C for 2 min,17 after which SCE (stratum corneum and epidermis) was removed from the dermis using a dull scalpel blade. Epidermal membranes were dried in a desiccator at approximately 25% relative humidity. The dried samples were wrapped in aluminum foil and stored at 4 ± 1◦ C until use. Previous research work demonstrated the maintenance of SC barrier characteristics after storage in the reported JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012

304

PUGLIA ET AL.

conditions.18 Besides, preliminary experiments were carried out in order to assess the barrier integrity of SCE samples by measuring the in vitro permeability of [3 H]water through the membranes using the Franz cell method described below. The value of calculated permeability coefficient (Pm ) for [3 H]water agreed well with those previously reported.19

was always pre-run for 10 min to allow the output to stabilize. Samples were irradiated for 10 min corresponding to an incident dose of UVA of 270 kJ/m2 , that is, the dose approximately equivalent to about 90 min of sunshine at the French Riviera (Nice) in summer at noon.23 The nonirradiated control plates were kept in dark at room temperature for 30 min.

In vitro Skin Permeation Experiments Samples of dried SCE were rehydrated by immersion in distilled water at room temperature for 1 h before being mounted in Franz-type diffusion cells supplied by LGA (Berkeley, California). The exposed skin surface area was 0.75 cm2 and the receiver compartment volume was of 4.5 mL. The receptor compartment was filled with a water–ethanol solution (50:50) (to allow the establishment of sink conditions and to sustain permeant solubilization),20 stirred at 500 rpm, and thermostated at 32 ± 1◦ C during all experiments.21 Approximately 300 mg of each formulation (OMC–SLN, OMC–NLC, OMC-ME, and OMC-GEL) were placed on the skin surface in the donor compartment R (Pechiney and the latter was covered with Parafilm Plastic Packaging Company, Chicago, IL, USA). Each experiment was run in duplicate for 24 h using three different donors (n = 6). At predetermined intervals, samples (200 :L) of receiving solution were withdrawn and replaced with fresh solution. The samples were analyzed for active content by HPLC as described below. OMC fluxes through the skin were calculated by plotting the cumulative amounts of compound penetrating the skin against time and determining the slope of the linear portion of the curve and the χ-intercept values (lag time) by linear regression analysis. OMC fluxes [:g/(cm2 h)], at steady state, were calculated by dividing the slope of the linear portion of the curve by the area of the skin surface through which diffusion took place. Statistical analysis of data was performed using Student’s t-test.

In vitro Photostability Studies UVA Exposure. An in vitro study was performed using a method reported elsewhere22 to evaluate lipid nanoparticle photoprotective effect against UVA radiation. Briefly, each formulation was loaded onto a 5 × 5 cm2 glass (2 mg/cm2 ) and spread with a gloved finger. After leaving the plates to dry in the dark for 20 min, they were placed on a brass block embedded on ice at a distance of 20 cm from the light source consisting of a commercial UVA sun lamp, Philips Original Home Solarium (model HB 406/A; Philips, Groningen, Netherlands) equipped with a 400-W ozone-free Philips HPA lamp (Philips), UV type 3. The output of UVA was measured with a UV Power Puck radiometer (EIT, Inc., Sterling, Virginia, USA), and it corresponded to 0.045 W/cm2 . The lamp JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012

Optical Absorption Spectra. After UVA exposure, irradiated and control plates containing the formulations were immersed for 30 min in a beaker containing 10 mL ethyl acetate. From this organic solution, 50 :L were added to 2450 :L ethyl acetate in a quartz cuvette and its absorption spectra was measured on a Varian Cary 50 UV–visible spectrophotometer (Agilent Technologies Italia S.p.A., Milan, Italy) against a blank containing ethyl acetate. Evaluation of Lipid Peroxidation. The remaining, dissolved formulation from the above treatment was evaporated under vacuum by Rotavapor (BUCHI Italia S.r.l., Milan, Italy). To the residue, 3 mL of thiobarbituric acid (TBA)–trichloroacetic acid (TCA)–HCl (0.375% w/v TBA, 15% w/v TCA, and 0.2 M HCl) was added, followed by 0.1 mM butylated hydroxytoluene (BHT) to prevent possible lipid peroxidation during TBA assay. The samples were heated for 30 min at 95◦ C, followed by cooling and centrifugation. The absorbance of the pink chromophore formed in the supernatant by TBA and aldehydic breakdown products of lipid peroxidation (TBA reactive substances, TBARS) was measured at 532 nm and compared with the absorbance of malondialdehyde obtained from the calibration curve of 1,1,3,3-tetraethoxypropane reacted with TBA.24 Statistical analysis of data was performed using the Student’s t-test. High-Performance Liquid Chromatography The HPLC apparatus consisted of a Shimadzu LC10 AT VP equipped with a 20-:L loop injector and a SPD-M 10 A VP Shimadzu photodiode array UV detector (Shimadzu, Milan, Italy). Chromatography was performed using a Symmetry Shield Waters C18 RP column (particle size, 5 :m; 25 cm × 4.6 mm internal diameter; Waters, Italy). The mobile phase was composed of methanol and water (95:5). The flow rate was set at 1 mL/min, the detection was effected at 310 nm, and the retention time was 5.4 min.

RESULTS AND DISCUSSION SLN and NLC Preparation and Characterization As previously reported, ultrasonication method yielded lipid particles in the nanometer range with Gaussian distribution width lower than 150 nm and DOI 10.1002/jps

LIPID NANOPARTICLES AS CARRIERS FOR OMC

no particles larger than 1 :m (OMC–SLN, 385 ± 142 nm; OMC–NLC, 365 ± 147 nm). Owing to the dynamic light-scattering upper size limit (5 :m), particle size analyses were repeated after filtration (2.5 :m pore size membrane). Filtration did not appreciably reduce the Gaussian distribution width, showing the absence of large particles and/or aggregates. In fact, the presence of large objects generally increases the Gaussian distribution width. In addition, light microscopy observation ruled out the presence of particles and/or aggregates in the micrometer range. In both cases, a high OMC loading was achieved, whereas its encapsulation produced a moderate increase in the mean hydrodynamic diameter and Gaussian distribution width with respect to empty nanoparticles (SLN, 233 ± 97 nm; NLC, 297 ± 113 nm).6,15,25–27 The same trend was not observed for the reference microemulsion (OMC-ME, 313 ± 35 nm; ME not loaded with OMC, 308 ± 31 nm). R 812 in NLC suspension The presence of Miglyol was useful to increase the active recovery from 72% to 88% in comparison with SLN. This result is due to R 812 as compared higher OMC solubility in Miglyol R solid lipid with sunscreen solubility in Compritol 28 matrix. Mean hydrodynamic diameters were similar to previous reports, and the observed differences may be likely ascribed to formulation conditions (e.g., preparation method and sunscreen mixture). In fact, Xia et al.29 successfully reported the preparation of NLC containing a sunscreen mixture (including OMC) with target loadings from 20% to 70% (w/w) and particle size decreasing as a function of the sunscreen content. The formulation containing 20% (w/w) of OMC had a mean size of 472 nm and was not stable (gelation) at 30 days, whereas 4% of OMC (w/w) did not destabilize the colloidal suspension up to 3 months.29 Figure 1 shows TEM photographs of different lipid nanoparticles. According to TEM analysis, particle sizes are in agreement with PCS data, and the particle shape appears close to spherical. Figure 2 illustrates the DSC data obtained submitting the raw materials and different nanoparticle formulations to two consequent heating ramps, and Table 1 shows the values of onset, minimum, and width for the observed endothermic events as the mean of two independent measurements ± SD. As expected, DSC data showed neither endotherR 812 and OMC, mic nor exothermic events for Miglyol R , an endotheralthough, in the case of bulk Compritol mic event around 72◦ C (Table 1), previously ascribed to the melting of the lipid material, was observed.26 R When Compritol was formulated as colloidal dispersion (SLN), a slight depression in the melting point and a doubled peak width were observed (Fig. 2a). This is generally due to the colloidal size DOI 10.1002/jps

305

Figure 1. Transmission electron microscopy photographs of different lipid nanoparticles. (a) SLN dispersion, (b) OMC–SLN dispersion, (c) NLC dispersion, and (d) OMC–NLC dispersion. The bar equals 500 nm in panels a, b, and c, and 200 nm in panel d.

and the increased surface area, as expected for all nanodispersed materials, according to Gibbs–Thomson equation.30 Even though melting depression is always observed in the case of colloidal lipids, the effect is quite important in saturated monoacid triglycerides with a melting point reduction of about 20◦ C.31 A less pronounced effect is generally seen for complex R . The adsorpglyceride mixtures such as Compritol tion of an amphiphilic molecule (stabilizer) on particle surface is credited to be also responsible for this physicochemical change.16 In the case of NLC, a lower and wider melting event was recorded (Fig. 2a; Table 1). As well known, during the formulation of NLC, an oil is added to the solid lipid matrix to overcome some of the SLN limitations (i.e., fast crystallization, active squeezing, and low physical stability).32,33 Obviously, the oil, functioning as impurity for the main lipid, has an additional and greater effect with respect to colloidal size and surfactant on matrix crystallization.12 A similar consideration may be drawn when loading the carriers with OMC, a highly lipophilic liquid UV filter. In fact, in OMC-loaded nanocarriers, melting peaks were recorded at lower temperatures with respect to the relatively empty particles (Fig. 2a; Table 1). After complete melting, the lipid material was allowed to crystallize (–5◦ C/min) to be submitted to a R , as a bulk matesecond melting (Fig. 2b). Compritol rial, started to crystallize at about 60◦ C, generating a shoulder around 64◦ C and two superimposed peaks at approximately 69◦ C and approximately 70◦ C. Analogously, SLN showed three exothermic events at approximately 66◦ C, 63◦ C, and approximately 56◦ C. The former two have been previously ascribed to β and α-modifications, whereas the latter could be sub JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012

306

PUGLIA ET AL.

Figure 2. Differential scanning calorimetry data of raw materials, blank SLN and NLC, and OMC–SLN and NLC. (a) First heating ramp, (b) cooling ramp, and (c) second heating ramp.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012

DOI 10.1002/jps

LIPID NANOPARTICLES AS CARRIERS FOR OMC

Table 1.

DSC Data of Different Lipid Nanoparticle Preparations

Formulation First heating ramp R Compritol ATO 888 SLN OMC–SLN NLC OMC–NLC Second heating ramp R Compritol ATO 888a SLN OMC–SLN NLC OMC–NLC aA

307

Peak Onset (◦ C)

Peak Minimum (◦ C)

Peak Width (◦ C)

69.97 ± 0.13 68.27 ± 0.17 58.23 ± 0.16 58.19 ± 0.12 53.50 ± 0.95

71.94 ± 0.01 71.33 ± 0.13 66.03 ± 0.01 67.63 ± 1.07 64.39 ± 0.01

1.70 ± 0.18 3.44 ± 0.04 6.90 ± 0.03 10.21 ± 0.45 9.42 ± 0.08

69.44 ± 0.01 68.43 ± 0.58 54.29 ± 0.01 59.98 ± 0.42 49.84 ± 0.64

71.79 ± 0.01 71.20 ± 0.07 63.46 ± 0.01 66.47 ± 0.02 61.27 ± 0.16

1.96 ± 0.01 2.93 ± 0.03 8.59 ± 0.30 7.46 ± 0.31 10.23 ± 0.21

peak shoulder was present at 67.17 ± 0.06◦ C.

α-modification caused by non-negligible fraction of R 34 . Crystallizamonoglyceride present in Compritol tion temperature was further depressed in other samR 812, OMC, or both ples due to the presence of Miglyol (Fig. 2b). During the second heating scan (Fig. 2c), lower melting temperatures were recorded (Table 1) for all R DSC data indicated the samples. Bulk Compritol presence of a shoulder around 67◦ C, previously attributed to the melting of unstable α-modification.34 The same effect has been reported after bulk R aging for 1 week at 40◦ C.35 Compritol These data clearly confirm OMC embedding into R allowed the forthe lipid matrixes. In fact, Compritol mation of solid mixtures (melting–solidification) containing up to 80% (w/w) of sunscreen mixture, and NLC showed a similar behaviour for contents up to 14%. Owing to the dermal use of the presented formulations, DSC was performed on dried samples as well. In fact, after skin application, because of vehicle water

loss and contractive capillary forces of the nanometer pores between the particles, SLN tend to fuse, forming a dense film.36,37 This process may affect the lipid physicochemical properties during real use conditions. DSC data of lipid films, obtained by drying nanoparticle suspensions for 1 h at 35◦ C, showed a trend of melting point reduction similar to that observed in the case of lipid nanoparticle suspensions (Fig. 3).

In vitro Skin Permeation Experiments In Figure 4, the plots of the cumulative amounts of OMC permeated through human SCE membranes as a function of time are shown. The flux values of OMC from OMC–SLN, OMC–NLC, OMC-ME, and OMC-GEL formulations calculated from the linear segments at steady-state are reported in Figure 5. Statistical analysis revealed no significant differences between the steady-state flux values obtained with OMC–SLN and OMC–ME forms (p > 0.05). Significant differences of OMC fluxes between OMC–NLC

Figure 3. Differential scanning calorimetry data of the films formed by the surfactant solution and different lipid nanoparticles after exposure to 35◦ C for 1 h in an open pan. DOI 10.1002/jps

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012

308

PUGLIA ET AL.

Figure 4. Permeation profiles of OMC from different formulations through SCE membranes. (䉬) OMC–NLC formulation, (䊏) OMC–SLN formulation, (䉱) OMC-ME formulation, and (䊊) OMC-GEL formulation.

lipid matrix containing oil nanocompartments, which can influence NLC features. The importance of oils R 812 in the lipid nanoparticle archisuch as Miglyol tecture is well documented in the literature.11 ParticR 812 has the ability to increase the ularly, Miglyol loading capacity of lipid nanoparticles for a number of active compounds, avoiding or minimizing potential expulsion phenomena during storage.11 R The scientific literature reports that Miglyol 812 contributes to modify the release of active compounds from the lipid matrix of lipid nanoparticles,14 but this feature still remains an object of scientific debate. Some researchers speculate that this finding could R depend on a physical interaction between Miglyol 812 and the active compounds, which decreases the release from the lipid matrix.28

In vitro Photostability Studies and OMC–SLN were observed (p < 0.05), whereas OMC–SLN, OMC–NLC, and OMC-ME formulations showed flux values considerably lower than OMCGEL formulation (p < 0.01). The results of in vitro permeation experiments could be explained considR 812 both as main ering the key role of Miglyol lipophilic ingredient of OMC-ME and as a constituent R 812 is a caprylic/capric of OMC–NLC. Miglyol triglyceride characterized by medium length acyl chains that can interact with surfactant molecules of the formulations by means of hydrophobic interactions. In the case of OMC-ME formulation, this interaction, as reported by others,7 could create a closer packing of the surfactant layer, decreasing OMC difR 812 droplets and, consequently, fusion from Miglyol its release from this formulation. OMC-loaded NLC suspension formulation was characterized by a lower flux as compared with OMC– SLN (Fig. 5). As previously reported, differences between SLN and NLC are related to their inner structure. Particularly, the latter is organized in a solid

Figure 5. Steady-state fluxes of OMC through excised human skin from different formulations. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012

The spectral behavior of OMC-loaded formulations is reported in Figure 6. From the figures, one can directly observe that the spectral profile is the same for all four formulations, both before and after UVA irradiation. However, differences can be noted in the levels of UV absorbance reached after UVA exposure. A 37% decrease in absorbance can be observed for both OMC–NLC and OMC-ME formulations measured at 300 nm. This decrease is, however, more important for OMC–SLN (50%) and OMC-GEL (61%). The spectral decrease is attributed to the well-documented cis/ trans photoisomerization of OMC, where the resulting cis-isomer has the same spectral profile as the transisomer, but a lower extinction coefficient.38,39 The decrease in spectral absorbance may be also possibly due to [2 + 2] cycloaddition of OMC as a consequence of its photodegradation, as previously reported.39 From these results, it appears that OMC is more photoR 812 (OMstable in formulations containing Miglyol C–NLC and OMC–ME). This suggests that loading R -containing formulations appears of OMC in Miglyol to influence its photoisomerization/degradation because there is minor loss in absorbance following UVA exposure as compared with SLN. Considering the R 812, the high affinity between OMC and Miglyol greater OMC loading in NLC with respect to that in SLN (95% vs. 81%, respectively) may explain the above results. In fact, by loading a greater amount of OMC, NLC may be able to preserve and, consequently, protect, within the inner lipid shell, a larger quantity of the UV filter from UVA-induced modifications with respect to the theoretical value initially loaded in both particle systems. A similar mechanism may also explain the interesting result obtained in the R 812 OMC-ME formulation. In this case too, Miglyol plays a central role in stabilization and in increasing the amount of UV filter incorporated. DOI 10.1002/jps

LIPID NANOPARTICLES AS CARRIERS FOR OMC

309

Figure 6. UV absorption spectra of OMC-loaded formulations before and after UVA exposure, followed by extraction with ethyl acetate.

In addition, it was observed that the presence of alcohol appears to influence the photostability of OMC, as a remarkable decrease in absorbance was observed after UVA irradiation (Fig. 6d). This may have important implications for the use of alcohol-based spray sunscreens in which OMC is present because the photoprotective potential of this UV filter may be compromised. Concomitant to measuring the spectral behavior of OMC in the formulations before and after UVA exposure, the lipid peroxidation level of the formulations were also measured. UVA wavelengths (320–400 nm), which are the principal UV component of sunlight (>95%), are well known to promote and propagate lipid peroxidation (290–320 nm) via generation of ROS.40,41 Because the formulations tested are all lipid-based, any UVA and/or UV-filter-induced ROS generated within the formulations will lead to an increase in lipid peroxidation and, hence, in TBARS levels.42 Figure 7 shows the TBARS levels obtained before and after UVA exposure of different formulations loaded or not loaded with OMC (obviously OMC-GEL was not assessed due to the absence of lipids). In the absence of OMC, there is a significant increase in TBARS levels following UVA exposure, relative to the unexposed control in all three formulations. However, when the lipid-carrier formulations are loaded with OMC, TBARS levels are significantly reduced to DOI 10.1002/jps

similar extents after UVA exposure, compared with the respective control. In OMC-ME, these levels remain the same as that of the unexposed one. These results indicate that ROS are unlikely to be generated in the presence of this UV filter in the tested formulations. Secondly, the data suggest that in lipid-carrier formulations, the ordered structure of the nanoparticle system probably interferes with the propagation of lipid peroxidation, leading to reduced TBARS formation after UVA exposure, with respect to the control formulation. These results appeared to be very interesting because, on one hand, NLC preserve UV spectral absorbance of OMC better than SLN and, on the other hand, OMC–NLC appears to protect UVA-induced lipid peroxidation, compared with the OMC-ME form.

CONCLUSION The results of the present work have pointed out interesting applications of lipid nanoparticles in the cosmetic field as carriers of chemical UV filters such as OMC. Compared with SLN, NLC have shown improved active loading capacity and smaller dimensions. Furthermore, NLC demonstrated in vitro a significant reduction in OMC penetration and an elevated efficiency to preserve the UV filter from UV-mediated photodegradation. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012

310

PUGLIA ET AL.

Figure 7. Concentration of TBARS determined in formulations loaded or not loaded with OMC after extraction with ethyl acetate, followed by subsequent treatment for detection of TBARS (error bars represent SD; n = 3). ∗ p < 0.05 versus respective unexposed control–OMC; p < 0.05 versus respective unexposed control + OMC.

REFERENCES 1. Luckey TD. 1999. Nurture with ionizing radiation: A provocative hypothesis. Nutr Cancer 34:1–11. 2. Butt ST, Christensen T. 2001. Toxicity and phototoxicity of chemical sun filters. Radiat Prot Dosim 91:283–286. 3. Kimura K, Katoh T. 1995. Photo allergic contact dermatitis from the sunscreen ethylhexyl-p-methoxycinnamate (Parsol MCX). Contact Dermatitis 32:304–305. 4. Pattanaargson S, Limphong P. 2001. Stability of octyl methoxycinnamate and identification of its photo-degradation product. Int J Cosmet Sci 23:153–160. 5. Jiang R, Roberts MS, Collins DM, Benson HA. 1999. Absorption of sunscreens across human skin: An evaluation of commercial products for children and adults. Br J Clin Pharmacol 48:635–637. 6. Puglia C, Bonina F, Castelli F, Micieli D, Sarpietro MG. 2009. Evaluation of percutaneous absorption of the repellent DEET and the sunscreen octyl methoxy cinnamate (OMC) loaded solid lipid nanoparticles: An in vitro study. J Pharm Pharmacol 61:1013–1019. 7. Montenegro L, Carbone C, Puglisi G. 2011. Vehicle effects on in vitro release and skin permeation of octylmethoxycinnamate from microemulsions. Int J Pharm 405:162–168. ¨ T, Yener N. 2003. Importance of using solid 8. Yener G, Incegul lipid microspheres as carriers for UV filters on the example octyl methoxy cinnamate. Int J Pharm 258:203–207. 9. Golmohammadzadeh S, Jaafarixx MR, Khalili N. 2008. Evaluation of liposomal and conventional formulations of octyl methoxycinnamate on human percutaneous absorption using the stripping method. J Cosmet Sci 59:385–398. ¨ ¨ 10. Muller RH, Mader K, Gohla S. 2000. Solid lipid nanoparticles (SLN) for controlled drug delivery—A review of the state of the art. Eur J Pharm Biopharm 50:161–177. ¨ 11. Muller RH, Radtke M, Wissing SA. 2002. Nanostructured lipid matrices for improved microencapsulation of drugs. Int J Pharm 242:121–128. ¨ 12. Pardeike J, Hommoss A, Muller RH. 2009. Lipid nanoparticles (SLN, NLC) in cosmetic and pharmaceutical dermal products. Int J Pharm 366:170–184. ¨ 13. Wissing SA, Muller RH. 2001. Solid lipid nanoparticles (SLN)—A novel carrier for UV blockers. Pharmazie 56:783–786. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012

¨ 14. Muller RH, Radtke M, Wissing SA. 2002. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations. Adv Drug Deliv Rev 54:S131–S155. 15. Puglia C, Sarpietro MG, Bonina F, Castelli F, Zammataro M, Chiechio S. 2011. Development, characterization, and in vitro and in vivo evaluation of benzocaine- and lidocaine-loaded nanostructrured lipid carriers. J Pharm Sci 100:1892–1899. 16. Bunjes H, Unruh T. 2007. Characterization of lipid nanoparticles by differential calorimetry, X-ray and neutron scattering. Adv Drug Deliv Rev 59:379–402. 17. Kligman AM, Christophers E. 1963. Preparation of isolated sheets of human stratum corneum. Arch Dermatol 88:702–705. 18. Swarbrick J, Lee G, Brom J. 1982. Drug permeation through human skin. I. Effects of storage conditions of skin. J Invest Dermatol 78:63–66. 19. Bronaugh RL, Stewart RF, Simon M. 1986. Methods for in vitro percutaneous absorption studies VII: Use of excised human skin. J Pharm Sci 75:1094–1097. 20. Toitou E, Fabin B. 1988. Altered skin permeation of highly lipophilic molecule: Tetrahydrocannabinol. Int J Pharm 43:17–22. 21. Puglia C, Tropea S, Rizza L, Santagati NA, Bonina F. 2005. In vitro percutaneous absorption studies and in vivo evaluation of anti-inflammatory activity of essential fatty acids (EFA) from fish oil extracts. Int J Pharm 299:41–48. 22. Damiani E, Astolfi P, Giesinger J, Ehlis T, Herzog B, Greci L, Baschong W. 2010. Assessment of the photo-degradation of UV-filters and radical-induced peroxidation in cosmetic sunscreen formulations. Free Radic Res 44:304–312. 23. Seite S, Moyal D, Richard S, de Rigal J, Leveque JL, Hourseau C, Fourtanier AJ. 1998. Mexoryl SX: A broad absorption UVA filter protects human skin from the effects of repeated suberythemal doses of UVA. Photochem Photobiol B 44:69–76. 24. Buege JA, Aust SD. 1978. Microsomal lipid peroxidation. Methods Enzymol 52:302–310. 25. Puglia C, Filosa R, Peduto A, de Caprariis P, Rizza L, Bonina F, Blasi P. 2006. Evaluation of alternative strategies to optimize ketorolac transdermal delivery. AAPS PharmSciTech 7:64. 26. Puglia C, Blasi P, Rizza L, Schoubben A, Bonina F, Rossi C, Ricci M. 2008. Lipid nanoparticles for prolonged topical DOI 10.1002/jps

LIPID NANOPARTICLES AS CARRIERS FOR OMC

27.

28.

29.

30. 31.

32.

33.

34.

delivery: An in vitro and in vivo investigation. Int J Pharm 357:295–304. Ricci M, Puglia C, Bonina F, Di Giovanni C, Giovagnoli S, Rossi C. 2005. Evaluation of indomethacin percutaneous absorption from nanostructured lipid carriers (NLC): In vitro and in vivo studies. J Pharm Sci 94:1149–1159. Castelli F, Puglia C, Sarpietro MG, Rizza L, Bonina F. 2005. Characterization of indomethacin loaded lipid nanoparticles by differential scanning calorimetry. Int J Pharm 304:231– 238. ¨ Xia Q, Saupe A, Muller RH, Souto EB. 2007. Nanostructured lipid carriers as novel carrier for sunscreen formulations. Int J Cosmet Sci 29:473–482. Hunter RJ. 1993. Foundations of colloid science. Vol. I. Oxford: Clarendon Press. Siekmann B, Westesen K. 1994. Thermoanalysis of the recrystallization process of melt-homogenized glyceride nanoparticles. Colloids Surf B Biointerfaces 3:159– 175. ¨ Jenning V, Thunemann AF, Gohla SH. 2000. Characterisation of a novel solid lipid nanoparticle carrier system based on binary mixtures of liquid and solid lipids. Int J Pharm 199:167–177. ¨ Zimmermann E, Souto EB, Muller RH. 2005. Physicochemical investigations on the structure of drug-free and drug-loaded solid lipid nanoparticles (SLN) by means of DSC and 1H NMR. Pharmazie 60:508–513. ¨ Freitas C, Muller RH. 1999. Correlation between longterm stability of solid lipid nanoparticles (SLN) and crystallinity of the lipid phase. Eur J Pharm Biopharm 47:125– 132.

DOI 10.1002/jps

311

35. Hamdani J, Mo¨es AJ, Amighi K. 2003. Physical and thermal characterisation of Precirol and Compritol as lipophilic glycerides used for the preparation of controlled-release-matrix pellets. Int J Pharm 260:47–57. ¨ 36. Wissing SA, Muller RH. 2003. The influence of solid lipid nanoparticles on skin hydration and viscoelasticity—In vivo study. Eur J Pharm Biopharm 56:67–72. ¨ 37. Wissing S, Lippacher A, Muller R. 2001. Investigations on the occlusive properties of solid lipid nanoparticles (SLN). J Cosmet Sci 52:313–324. 38. Huong SP, Andrieu V, Reynier JP, Rocher E, Fourneron JD. 2007. The photoisomerization of the sunscreen ethylhexyl p-methoxy cinnamate and its influence on the sun protection factor. J Photochem Photobiol A Chem 186:65–70. 39. Broadbent JK, Martincigh BS, Raynor MW, Salter LF, Moulder R, Sjoberg P, Markides KE. 1996. Capillary supercritical fluid chromatography combined with atmospheric pressure chemical ionization mass spectrometry for the investigation of photoproduct formation in the sunscreen absorber 2-ethylhexyl-p-methoxycinnamate. J Chromatogr A 732:101–110. 40. Morliere P, Moysan A, Tirache I. 1995. Action spectrum for UV-induced lipid peroxidation in cultured human skin fibroblasts. Free Radic Biol Med 19:365–371. 41. Polte T, Tyrrell RM. 2004. Involvement of lipid peroxidation and organic peroxides in UVA-induced matrix metalloproteinase-1 expression. Free Radic Biol Med 36:1566–1574. 42. Esterbauer H, Cheeseman KH. 1990. Determination of aldehydic lipid peroxidation products: Malodialdehyde and 4 hydroxynonenal. Methods Enzymol 186:407–421.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012