European Journal of Pharmaceutical Sciences 51 (2014) 211–217

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Evaluation of nanostructured lipid carriers (NLC) and nanoemulsions as carriers for UV-filters: Characterization, in vitro penetration and photostability studies Carmelo Puglia a,⇑, Elisabetta Damiani b, Alessia Offerta a, Luisa Rizza a, Giorgia Giusy Tirendi a, Maria Stella Tarico c, Sergio Curreri c, Francesco Bonina a, Rosario Emanuele Perrotta c a

Dipartimento di Scienze del Farmaco, Università di Catania, 95125 Catania, Italy Dipartimento Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, 60131 Ancona, Italy c Dipartimento di Specialità Medico Chirurgiche, Sezione di Chirurgia Plastica, Azienda Ospedaliera per l’Emergenza ‘‘Cannizzaro’’, 95126 Catania, Italy b

a r t i c l e

i n f o

Article history: Received 23 July 2013 Received in revised form 17 September 2013 Accepted 21 September 2013 Available online 21 October 2013 Keywords: Nanostructured lipid carriers Sun filters Human skin In vitro percutaneous absorption Photo-stability studies

a b s t r a c t The increased awareness of protection against UV radiation damages has led to a rise in the use of topically applied chemical sunscreen agents and to an increased need of innovative carriers designed to achieve the highest protective effect and reduce the toxicological risk resulting from the percutaneous absorption of these substances. In this paper, nanostructured lipid carriers (NLC) and nanoemulsions (NE) were formulated to optimize the topical application of different and widespread UVA or UVB sun filters (ethyl hexyltriazone (EHT), diethylamino hydroxybenzoyl hexyl benzoate (DHHB), bemotrizinol (Tinosorb S), octylmethoxycinnamate (OMC) and avobenzone (AVO)). The preparation and stability parameters of these nanocarriers have been investigated concerning particle size and zeta potential. The release pattern of the sunscreens from NLC and NE was evaluated in vitro, determining their percutaneous absorption through excised human skin. Additional in vitro studies were performed in order to evaluate, after UVA radiation treatment, the spectral stability of the sunfilters once formulated in NLC or NE. From the results obtained, when incorporated in NLC, the skin permeation abilities of the sun filter were drastically reduced, remaining mainly on the surface of the skin. The photostability studies showed that EHT, DHHB and Tinosorb S still retain their photostability when incorporated in these carriers, while OMC and AVO were not photostable as expected. However, no significant differences in terms of photoprotective efficacy between the two carriers were observed. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Overexposure of human skin to ultraviolet radiation (UV) is responsible for a wide variety of damage including photoaging and photocarcinogenesis (Nikolic´ et al., 2011; Ullrich, 2005). To protect the skin from these harmful effects, a wide assortment of topical sunscreen products have been developed. The most common active ingredients in these preparations are organic sunscreen agents which absorb UV radiations (Nohynek et al., 2010). Modern sunscreen products should provide broad-spectrum UV protection and therefore they should contain at least two UV filters, one with optimal performance in the UVA region (320–400 nm) and the other one in the UVB region

⇑ Corresponding author. Address: Department of Drug Science, University of Catania, Viale A. Doria n°6, 95125 Catania, Italy. Tel.: +39 957384209; fax: +39 95222239. E-mail address: [email protected] (C. Puglia). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.09.023

(290–320 nm). Generally, these substances are characterized by a high affinity for the stratum corneum (SC) and are designed to remain on the outermost skin layers, shielding the skin from harmful UV radiation as effectively as possible, and to reduce the toxicological risk resulting from their percutaneous absorption (Hayden et al., 1997; Maier et al., 2001). It is widely recognized that the performance of a sunscreen formulation relies not only on the physicochemical properties of the filters, but also on the carrier used to deliver them (Olvera-Martínez et al., 2005). Until now, most sun protection products have been based on emulsions, oils and gels that despite their advantages, they also have some important limitations such as water washability, instability and increased percutaneous absorption of sun filters. Several innovative carriers have been proposed to overcome these limitations and to ensure an adequate efficacy of the sun filters; they include nanoemulsions, microspheres, cyclodextrins, liposomes and nanoparticles (Olvera-Martínez et al., 2005; Shi et al.,

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2012; Monteiro et al., 2012; Morganti, 2010). These colloidal carriers have been demonstrated to enhance the accumulation of the sunscreen at the administration site and to increase water resistance, sun protection factor (SPF) and photostability of these agents (Shi et al., 2012; Vettor et al., 2008). Recently, lipid nanoparticles (SLN: solid lipid nanoparticles and NLC: nanostructured lipid carriers) have become increasingly interesting for scientists due to their potential to overcome topical drug delivery challenges (Müller et al., 2002a). Lipid nanoparticles (LN) are colloidal particles composed of a biocompatible/ biodegradable lipid matrix that is solid at body temperature and exhibits a size range between 100 and 400 nm. The main advantages associated with LN compared with other colloidal systems are high biocompatibility, good physical stability, possibility of controlled release of drug and active substances, easy largescale production and cheap raw materials. Among lipid nanoparticle typologies, NLC represent the latest innovation (Müller et al., 2002b). They are characterized by a blend of a solid lipid with a liquid lipid (oil) and are formulated with the original purpose to avoid lipid re-crystallization causing an expulsion of the enclosed active substances observed with SLNs (Puglia and Bonina, 2012; Xia et al., 2007). This new approach has also lead to significant improvements in promoting the dermal delivery of different drugs and cosmetic actives such as some sun filters (Puglia et al., 2008, 2011, 2012). Niculae et al. (2012) for instance, formulated nanostructured lipid carriers based on butyl-methoxydibenzoylmethane (BMDBM), a well known UVA filter, characterized by an enhanced UVA blocking effect and an erythemal UVA protection factor about four times higher than those specific to conventional emulsions. Nikolic´ et al. (2011) instead, formulated lipid nanocarriers containing a mixture of sunscreens (ethylhexyl triazone, bis-ethylhexyloxyphenol methoxyphenyl triazine, and ethylhexyl methoxycinnamate) characterized by an increased sun protection factor and by an efficacy strictly related to the solid state of the lipid and also to its type. Lastly, in a study performed in our research laboratories, we formulated octyl methoxycinnamate (OMC)-loaded NLC. In an in vitro experiment, these carriers showed a lower flux through excised human skin with respect to SLN and other conventional formulations. Furthermore, photostability studies revealed that NLC were the most efficient at preserving OMC from UV-mediated photodegradation (Puglia et al., 2012). Based on our previous evidence, in this study we evaluated NLC as a carrier system to optimize the topical application of new sunscreen actives. The most important basic requirement on sunscreen actives is efficacy: an efficient UV filter must show good absorption in the relevant UV range (290–400 nm), must be easily incorporated in any kind of formulation, therefore soluble in different emollients in order to be cosmetically acceptable and thirdly, must be photostable. Unstable UV filters lose efficacy and may lead to safety concerns upon irradiation. The new sunscreen actives tested were: (a) Bemotrizinol (Tinosorb S), a new oil-soluble filter with strong broad-spectrum protection in the UVA and UVB regions, high molecular mass (629 Da) and extinction coefficient at 310 nm = 46,800 mol1 cm1. Due to its outstanding filter efficacy, combined with its inherent photostability and compatibility with all types of cosmetic filters as well as other cosmetic ingredients, Tinosorb S represents a new, efficient generation of cosmetic UV filters. Its only drawback is its relatively high cost compared to other sunscreen actives. (b) Ethyl hexyltriazone (EHT) has a very high extinction coefficient (119,500 mol1 cm1 at 314 nm) and a high molecular mass (823 Da) which makes it an extremely efficient UVB absorber. Its only drawback is its limited solubility although it can be incorporated in sunscreen formulations in substantial amounts. (c) Diethylamino hydroxybenzoyl hexyl benzoate (DHHB, 398 Da, 35,900 mol1 cm1 at

354 nm) designed on classical benzophenone chemistry, is a successor of BMDBM, without the drawback of photoinstability. It also has good solubility properties (Herzog et al., 2005). Furthermore, a NLC based formulation containing both the UVB filter OMC and the UVA filter avobenzone (BMDBM) was prepared and characterized as well. Both OMC and avobenzone dominate the ranking of market shares in most countries because of their low cost and good compatibility with cosmetic formulations. With this aim, we investigated the influence of NLC on in vitro percutaneous absorption of the previous reported sun filters compared with nanoemulsion (NE) based formulations, which were chosen as reference vehicle since they represent nanocarriers of growing interest and utilization. Lastly, photostability measurements of sun filters were performed in order to determine the photoprotective efficacy of the nanocarriers containing these active substances. 2. Materials and methods 2.1. Materials CompritolÓ 888 ATO (glyceryl behenate, tribehenin), a mixture of mono, di, and triglycerides of behenic acid (C22), was a gift from Gattefossè (Milan, Italy). MiglyolÓ 812 (caprylic/capric triglycerides) was provided by Eigenmann & Veronelli S.p.A (Milan, Italy). LutrolÓ F68, ethyl hexyltriazone (EHT), diethylamino hydroxybenzoyl hexyl benzoate (DHHB), bemotrizinol (TinosorbÓ S) and avobenzone were provided by BASF Chem-Trade GmbH (Burgbernheim, Germany). OMC was purchased from Cognis S.p.A. (Milan, Italy). Xanthan gum was purchased from Sigma Chemicals (Milan, Italy). 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. 2.2. NLC and NE preparation In Table 1 the composition of NLCs prepared by ultrasonication method are reported. Briefly, a weighted amount of CompritolÓ 888 ATO was melted at 80 °C, and MiglyolÓ812 and the sun filters (EHT, OMC, DHHB, Tinosorb S or Avobenzone) were added. The melted lipid phase was dispersed in a hot (80 °C) aqueous solution containing LutrolÓ F68 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 high-speed 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 nanoemulsions were prepared following the same procedure described above, by replacing CompritolÓ 888 ATO with MiglyolÓ 812 (Table 1). For in vitro studies, NLCs and NEs were formulated into hydrogel using glycerol (10% w/w) and xanthan gum (1% w/w) as excipients. The final concentration of sun filters in the hydrogel formulations was 1% w/w. All the formulations were stored at 4 °C before use. 2.3. Particle size distribution and zeta potential measurements Mean particle size and zeta potential of the lipid dispersions were measured by PCS. A Zetamaster (Malvern Instrument Ltd., Sparing Lane South, Worcs, England), equipped with a solid state

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Avobenzone Compritol 888 ATO DHHB EHT Miglyol 812 OMC Pluronic F68 Tinosorb S Water

Formulation code Empty NLC

NLC OMC

NLC AVO

NLC EHT

NLC TINO

NLC DHHB

NLC UVMIX

Empty NE

NE OMC

NE AVO

NE EHT

NE TINO

NE DHHB

NE UVMIX

– 3.36

– 2.24

1.12 2.24

– 2.24

– 2.24

– 2.24

1.12 2.24

– –

– –

1.12 –

– –

– –

– –

1.12 –

– – 1.12 – 0.95 – 94.57

– – 1.12 1.12 0.95 – 94.57

– – 1.12 – 0.95 – 94.57

– 1.12 1.12 – 0.95 – 94.57

– – 1.12 – 0.95 1.12 94.57

1.12 – 1.12 – 0.95 – 94.57

– – 1.12 1.12 0.95 – 93.45

– – 4.48 – 0.95 – 94.57

– – 3.36 1.12 0.95 – 94.57

– – 3.36 – 0.95 – 94.57

– 1.12 3.36 – 0.95 – 94.57

– – 3.36 – 0.95 1.12 94.57

1.12 – 3.36 – 0.95 – 94.57

– – 3.36 1.12 0.95 – 93.45

laser having a nominal power of 4.5 mW with a maximum output of 5 mW 670 nm, was employed. Analyses were performed using a 90° scattering angle at 20 ± 0.2 °C. Samples were prepared diluting 10 ll of the suspension with 2 ml of deionized water previously filtered through a 0.2 lm Acrodisc LC 13 PVDF filter (Pall-Gelman Laboratory, Ann Harbor, MI, USA). During the experiment, the refractive index of the samples always matched the liquid (toluene) to avoid stray light. The Zeta (n) potential was automatically calculated from the electrophoretic mobility based on Smoluchowski’s equation (Eq. (1)):

 V¼



eE n m

ð1Þ

where m is the measured electrophoretic velocity, g is the viscosity, e is the electrical permittivity of the electrolytic solution and n is the electric field. The accuracy was 0.12 lm cm/V s for the aqueous systems. Samples were suspended in distilled water and the measurements were recorded at 25 °C. 2.4. In vitro studies 2.4.1. 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 (Kligman and Christophers, 1963), after which SCE (stratum corneum/epidermis) was removed from the dermis using a dull scalpel blade. Epidermal membranes were dried in a desiccator at 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 under the reported conditions (Swarbrick et al., 1982). Besides, preliminary experiments were carried out in order to assess the barrier integrity of SCE samples by measuring the in vitro permeability of [3H] water through the membranes using the Franz cell method described below. The value of the calculated permeability coefficient (Pm) for [3H]water agreed well with those previously reported (Bronaugh et al., 1986). 2.4.2. 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 Franztype diffusion cells supplied by LGA (Berkeley, CA). 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 the sink conditions and to sustain permeant solubilization (Toitou and Fabin, 1988).

Furthermore, it was stirred at 500 rpm and thermostated at 32 ± 1 °C throughout all the experiments (Puglia et al., 2005). A weighted amount of each formulation was placed on the skin surface in the donor compartment and the latter was covered with ParafilmÓ. Each experiment was run in duplicate for 24 h using three different donors (n = 6). At predetermined intervals, samples (200 ll) of receiving solution were withdrawn and replaced with fresh solution. The samples were analyzed for active content by HPLC as described below. The fluxes of each sun filter 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 v-intercept values (lag time) by linear regression analysis. The sun filter fluxes (lg/ cm2 h1), 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. 2.4.3. In vitro photostability studies 2.4.3.1. UVA exposure. To determine the photostability of lipid nanoparticles loaded with sun filters against UVA radiation, the method reported in Damiani et al. (2010) was adopted. Briefly, each formulation was spread using a gloved finger, onto a 5  5 cm glass plate at a quantity equal to 2 mg/cm2. The plates were left to dry in the dark for 20 min, before placing them on a brass block embedded on ice at a distance of 20 cm from the light source (commercial UVA sun lamp equipped with a 400 W ozone-free Philips HPA lamp, UV type 3, Philips Original Home Solarium, model HB 406/A, Groningen, Holland). The output of UVA measured with a UV Power Pack Radiometer (EIT Inc, Sterling, USA) corresponded to 0.045 W/cm2. Samples were irradiated for 10 min corresponding to an incident dose of UVA of 270 kJ/m2 (i.e. the dose approximately equivalent to about 90 min of sunshine at the French Riviera (Nice) in summer at noon (Seite et al., 1998)). The nonirradiated control plates were kept in the dark at room temperature for 30 min. 2.4.3.2. Optical absorption spectra. After UVA exposure, irradiated and control plates were immersed for 30 min in a beaker containing 10 ml ethyl acetate for extraction of sun filters. From this organic solution, 50 ll were added to 2.45 ml ethyl acetate in a quartz cuvette and its absorption spectra was measured on a Shimadzu UV-2401PC spectrophotometer against a blank containing the solvent alone. 2.5. High-performance liquid chromatography The HPLC apparatus consisted of a Shimadzu LC10 AT Vp (Milan, Italy) equipped with a 20 ll loop injector and a SPD-M 10 A Vp Shimadzu photodiode array UV detector. Chromatography was

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performed using a Symmetry Shield Waters C18 RP column (particle size, 5 lm; 25 cm  4.6 mm i.d.; Waters S.P.A, Italy). The HPLC method consisted of a mobile phase composed of acetonitrile (80%) and a water solution (20%) containing 1% acetic acid (Wharton et al., 2011). The flow rate was set at 1 ml/min, the detection was carried out at 310 nm for OMC and 360 nm for Avobenzone, DHHB and EHT. The retention times were 11.5, 12.0, 11.6 and 10.2 min for OMC, Avobenzone, EHT and DHHB respectively. HPLC method for Tinosorb S was characterized by a mobile phase composed of methanol and acetonitrile (90:10, v/v) (Smyrniotakis and Archontaki, 2004). The flow rate was set at 2 ml/min, the detection was carried out at 310 nm and the retention time was 19 min.

Table 2 Particle size, polydispersivity index (PDI) and zeta potential values of different NLC based formulations. Sample

Particle size (nm, mean ± SD)

Polidispersivity index (PDI, mean ± SD)

Zeta potential (-mV, mean ± SD)

NLC-OMC NLC-AVO NLC-EHT NLC-TINO NLC-DHHB NLC-UVMIX Unloaded NLC

318.8 ± 25.4 175.6 ± 9.3 186.2 ± 11.5 163.8 ± 18.2 194.7 ± 8.2 121.2 ± 7.3 250.6 ± 10

0.25 ± 0.02 0.34 ± 0.04 0.21 ± 0.03 0.25 ± 0.04 0.23 ± 0.02 0.23 ± 0.01 0.29 ± 0.04

26.5 ± 1.8 28.8 ± 1.3 24.7 ± 2.1 44.3 ± 1.5 27.5 ± 1.6 28.8 ± 0.9 25.4 ± 0.4

2.6. Statistical analysis Statistical analysis of in vitro data was performed using the Mann–Whitney U-test. A probability P of less than 0.05 was considered significant.

Table 3 Particle size, polydispersivity index (PDI) and zeta potential values of different NE based formulations. Sample

Particle size (nm, mean ± SD)

Polidispersivity index (PDI, mean ± SD)

Zeta potential (-mV, mean ± SD)

NE-OMC NE-AVO NE-EHT NE-TINO NE-DHHB NE-UVMIX Unloaded NE

190.6 ± 20.1 170.4 ± 14.3 198.4 ± 7.2 191.2 ± 11.9 186.5 ± 20.3 118.2 ± 4.4 290.4 ± 18.7

0.26 ± 0.02 0.23 ± 0.03 0.23 ± 0.03 0.22 ± 0.01 0.23 ± 0.01 0.22 ± 0.03 0.39 ± 0.04

13.3 ± 0.5 22.8 ± 1.1 37.7 ± 1.5 28.6 ± 1.2 27.2 ± 1.9 20.5 ± 1.8 24.9 ± 0.6

3. Results and discussion 3.1. Vehicle characterization NLC formulations were prepared using Compritol 888 ATO as solid lipid and Miglyol 812 as liquid lipid (oil). These ingredients were chosen taking into account that the successful entrapment of a drug or a cosmetic active substance into an NLC system is its adequate solubility or miscibility with the lipid. After heating to a temperature 5 °C higher than Compritol’s melting point, all the solutions were clear and no crystals were detected, indicating complete solubilization of the sun filters in the lipid matrix. Both NLC and NE were prepared by coupling high shear homogenization (HSH) with an ultrasound treatment (US). This method has many advantages compared to preparations performed using only one of these techniques, such as high homogeneity and reduced dimensions of nanoparticle dispersions (Puglia et al., 2013). Tables 2 and 3 report mean diameter, PDI and zeta potenzial values of NLC and NE based suspensions. From the evidence obtained, HSH/US method resulted suitable to produce nanocarriers characterized by a mean diameter around 200 nm and a polydispersivity index below 0.25. The latter parameter in particular, gave important indications concerning sample homogeneity; in fact, a PDI below 0.25 reflects relatively homogeneous nanoparticles, with minimum predisposition to aggregation (Mitri et al., 2011). By observing the difference between loaded and unloaded vehicles, it can be noted that both empty NLC and empty NE exhibit higher average diameters. This result can probably be explained considering the oily characteristics of the sun filters loaded in the nanocarriers which reduce the viscosity of the lipid mixture and, therefore, decrease the surface tension, thus forming nanocarriers with smaller dimensions compared to empty ones. The zeta potential is an important parameter that allows predictions on the physical stability of colloidal dispersions. In theory, higher values of zeta potential, either positive or negative, tend to stabilize the suspension. Usually, aggregation phenomena are less likely to occur for charged particles with pronounced zeta potential (>|20|), due to the electrostatic repulsion between particles with the same electrical charge (Gonzalez-Mira et al., 2010). In this study, the zeta potential mean value registered for all formulations was around 30 mV, which predicts a good long-term stability. 3.2. In vitro photostability studies The spectral behavior of the sun-filter-loaded NLC and NE formulations before and after UVA irradiation is reported in Fig. 1

(panels A and B, respectively). Their profiles are in accordance with those observed directly in ethyl acetate reported elsewhere (Damiani et al., 2007). From the spectra recorded, one can observe the differences in spectral profiles for the different UV filters according to whether they are UVA (avobenzone, DHHB), UVB (OMC, EHT) or broadband UVA/B (Tinosorb S) filters. Reported is also the spectral profile of the formulation containing both OMC and avobenzone (UVMIX). From the profiles obtained, one can deduce that some sun filters are less photostable than others following UVA irradiation, notably avobenzone whose instability due to keto-enol isomerization and photo-cleavage is well-known and documented (Cantrell and McGarvey, 2001; Schwack and Rudolph, 1995). OMC is also recognized to undergo a certain degree of photoinstability following UVA irradiation, attributed to its cis/trans photoisomerization (Huong et al., 2007; Broadbent et al., 1996) and this can be observed in Fig. 1 where there is roughly a 35% decrease in absorbance. The combination of these two filters, which is a popular one on the cosmetic market for ensuring UVA/B coverage, also results unstable as has long been known if adequate photostabilizing molecules, such as octocrylene, are not present (Damiani et al., 2007; Dondi et al., 2006; Gonzales et al., 2007). For the other sun filters tested, there is no significant decrease in absorbance after UVA irradiation, as expected, since they are known to be reliably photostable (Herzog et al., 2005). No significant differences in spectral profile and behavior were observed between the NLC formulations and the NE ones (Fig. 1A versus B), and this is in line with our previous study where no differences were observed between OMC-NLC formulations and OMC-NE ones (Puglia et al., 2012). 3.3. In vitro percutaneous absorption studies This study was carried out using human SCE membranes instead of full-thickness skin since the dermis could distort in vitro evidence, acting as a further artificial barrier to the absorption of sun filters (van de Sandt et al., 2004). Plotting the cumulative

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0.20 0.18

0.20 noUVA UVA

NLC-AVO

0.14

0.12

0.12

0.10

0.10

0.08

0.08

0.06

0.06

0.04

0.04

0.02

0.02

0.00 280

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noUVA UVA

NLC-DHHB

0.16

0.14

ABS

ABS

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440

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340

0.20

0.18

0.16

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0.06 0.04

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nm

noUVA UVA

NLC-EHT

300

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0.20 noUVA UVA

NLC-UVMIX

0.16

0.18

0.14

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0.08

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0.04

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noUVA UVA

NLC-TINO

0.10

0.08

300

440

0.16

ABS

ABS

440

nm

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0.00 280

420

0.08

0.04

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400

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noUVA UVA

NLC-OMC

ABS

ABS

0.18

360

nm

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440

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nm

Fig. 1. Panel A – UV absorption spectra of NLC loaded with sun filters before and after UVA exposure, followed by extraction with ethyl acetate; Panel B – UV absorption spectra of NE loaded with sun filters before and after UVA exposure, followed by extraction with ethyl acetate (see Section 2.4.3 for details).

amounts of the sun filters permeated through SCE membranes as a function of time, flux values at the steady state from NLC and NE based formulations were obtained (Fig. 2). As can be seen from the results reported in Fig. 1, the lowest fluxes of sun filters through SCE membranes after 24 h were obtained in the case of NLC based formulations; this result is in accordance with other evidence concerning the accumulation of sunscreen filters encapsulated into NLCs (Lacerda et al., 2011; Sanad et al., 2010; Durand et al., 2010). Fig. 2 also shows different profiles for the tested formulations, in terms of amount of sun filter permeated after 24 h. No penetrated amount of EHT and Tinosorb S was observed in the receptor phase in Franz diffusion cells, both for NLC and for NE based formulations. This result could be explained considering the high ‘‘substantivity’’ of EHT and Tinosorb S towards keratin, intended as the ability of a substance to be ad- or absorbed by keratin substrates of epidermis, thus resulting in a minimization of its percutaneous absorption (Varvaresou, 2006; Monti et al., 1993). The different affinity of the sunscreen filters towards the stratum corneum can also explain the different results obtained for OMC, DHHB and avobenzone. Worthy of interest is the analysis of avobenzone flux values from NLC and NE. In fact when incorporated in NLC, the skin permeation ability of the sun filter is drastically

reduced, remaining mainly on the surface of the skin, where ‘‘in vivo’’ it is intended to act. Modern suncare cosmetics are often formulated using two or more sun filters to increase the protective effect against UV radiations by covering both the UVA and UVB regions of the solar spectrum. This concurrent application is not riskless and could produce harmful side-effects due to a potentially increased percutaneous absorption of the sun filters. Therefore, in this study we also evaluated the effect of NLC and NE in the attempt to optimize a concurrent application of sun filters, choosing the UVA filter avobenzone and the UVB filter OMC, since they are the most popular and widespread sun filters on the cosmetic market. Fig. 3 reports the flux values at the steady state of OMC and avobenzone from NLC-UVMIX and NE-UVMIX formulations. The results show that when the sun filters are incorporated in NLC dispersions, they exhibit a lower flux with respect to the nanoemulsion formulation containing the same amount of substances. From a comparison between the flux values obtained from NLCOMC and NLC-AVO with respect to NLC-UVMIX formulation, it seems that the concurrent application of sun filters did not produce any appreciable increase in the amount of sun filters penetrating through SCE membranes (p > 0.05).

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0.20

0.20

noUVA

NE-AVO

0.18

UVA

0.16

0.16

0.14

0.14

0.12

0.12

ABS

ABS

0.18

0.10 0.08

0.08 0.06

0.04

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0.10

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NE-OMC

noUVA

0.20

UVA

0.18

0.16

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noUVA

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UVA

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NE-EHT

noUVA UVA

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420

440

noUVA UVA

NE-TINO

0.10

0.08

0.00 280

420

nm

ABS

ABS

0.18

400

0.08

0.04

0.20

380

0.10

0.06

0.00 280

360

nm

nm

0.00 280

300

320

340

360

380

400

420

440

nm

nm Fig. 1 (continued)

Fig. 2. Sun filter fluxes at steady state. The sun filters were from nanostructured lipid carriers (NLC) and from nanoemulsions (NE) formulations.

These differences become significant however, when comparing the flux values of OMC and avobenzone from NE-OMC and NE-AVO versus the NE-UVMIX formulation (p < 0.05). In particular, the flux increase for avobenzone obtained by dividing avobenzone flux

Fig. 3. Fluxes at steady state of OMC and Avobenzone from nanostructured lipid carriers (NLC-UVMIX) and from nanoemulsions (NE-UVMIX) formulations.

value from NE-UVMIX for the one obtained from NE-AVO, resulted to be 1.21, while the flux increase for OMC was as high as 1.75.

C. Puglia et al. / European Journal of Pharmaceutical Sciences 51 (2014) 211–217

Although, NLC and NE show a different behavior with respect to the permeation profile of the two sun filters, the values of epidermal penetration, obtained from these in vitro studies, are low for all the formulations tested and below the minimum limit accepted for safe products. In conclusion, these results demonstrate the capability of NLC to reduce permeation through the skin of all the sun filters tested with respect to NE, leading to their accumulation in the horny layer.

4. Conclusions From the results obtained, both NLC and NE vehicles showed submicron dimensions and a similar capability to protect the included actives from photo-instability phenomena. Nevertheless, only NLC were able to drastically reduce the skin permeation abilities of the sun filters, which remained mainly located on the surface of the skin. Therefore, the results of the present work confirm the high potential of NLC as carriers for chemical UV filters.

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