http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.920359

RESEARCH ARTICLE

Improving photoprotection: 4-methylbenzylidene camphor microspheres Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Nyu Medical Center on 05/14/15 For personal use only.

Marisanna Centini1,2, Giovanna Miraglia2, Valeria Quaranta2, Anna Buonocore1,2, and Cecilia Anselmi1,2 1

Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, Siena, Italy and 2Centro Interdipartimentale di Scienza e Tecnologia Cosmetiche, University of Siena, Siena, Italy Abstract

Keywords

We propose a new approach for photoprotection. 4-Methylbenzylidene camphor (4-MBC), one of the most widely used UV filters, was encapsulated in microspheres, with a view to overcoming problems (percutaneous absorption, photodegradation and lack of lasting effect) arising with organic sunscreens, and to achieve safe photoprotection. We focused on this filter in the light of the Cosmetics Europe opinion concerning its possible effects on the thyroid gland. Microspheres were prepared by emulsification–solvent evaporation, using different amounts of 4-MBC and characterized for morphology, encapsulation efficiency and particle size. The particles were then mixed in O/W emulsions. The in vitro sun protection factors, in vitro release and photostability were investigated and compared with emulsions containing the free sunscreen. The new microspheres offer good morphology and loading (up to 40%), and the same photoprotection as the free filter while at the same time protecting it from photodegradation. The systems also give a slower release from the emulsions.

4-Methylbenzylidene camphor, microspheres, photoprotection, photostability, release, sunscreens

Introduction Solar radiation causes acute and chronic reactions in and on human skin. UVA and UVB rays induce DNA damage both directly and indirectly, due to oxidative stress. Exposure to UV radiation has an important causal role in several pathologies of human skin like inflammation, immune system alterations and skin tumors, besides skin aging1–3. Topical products containing sunscreens are among the most widely employed methods to protect the skin from radiation1,4,5. However, phototoxic and photo-allergic reactions arising with increased use of these products have fostered the development of new products that are safer for human use. Scientific research has come up with many delivery systems that have been employed by pharmaceutical companies for a long time but are only now starting to expand in cosmetics, as ‘‘new cosmetic delivery systems’’6. As demand becomes more and more sophisticated, especially in relation to skin care and anti-aging, the cosmetic industry and scientific research are both interested in developing innovative technologies respecting current cosmetic product regulations (EEC Council Directive, 1976, Regulation EC No 1223/2009), which stress that a cosmetic product is intended to be placed in contact with the outer parts of the human body. Photostability is the most important feature of an effective sunscreen. The photochemical decomposition of the sun filter reduces its photoprotective properties and can give rise to

Address for correspondence: Cecilia Anselmi, Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, Via della Diana 2, 53100 Siena, Italy. Tel: +39 0577 232039. Fax: +39 0577 232070. E-mail: [email protected]

History Received 17 March 2014 Revised 15 April 2014 Accepted 17 April 2014 Published online 22 May 2014

phototoxic and photo-allergenic degradation products7. The substantivity (i.e. the adhesive properties) of a molecule towards keratin minimizes percutaneous absorption, which is a very important consideration in relation to cosmetic safety8. Microencapsulation of sunscreens has been considered a promising approach in photoprotection because it is safer (due to the lack of percutaneous absorption and the reduced photodegradation) and more effective (due to the lasting effect on the skin and stability of the sunscreen). Microspheres9,10, micro- and nanocapsules10–13, lipid particles14–17, hydrotalcitelike anionic clays18 and inclusion complexes19–21 have all attracted interest in recent years as vehicles for sunscreens. Encapsulated sunscreens offer various advantages: better photostability and substantivity, ease of formulation, less contact with skin and homogeneous skin distribution. Our studies were aimed at achieving functional and safe systems; improving photoprotection microspheres were designed to act only on the outermost part of the stratum corneum (SC) thanks to their polymeric nature; at the same time, they should not be absorbed by the skin. These systems protect both the sunscreens and the skin, avoiding degradation and oxidation, loss of protection and allergies or irritation. In the light of a recent EU regulation (Regulation EC No 1223/ 2009), which lays much emphasis on the safety evaluation of cosmetic products, it seemed interesting to study in depth the commercial sunscreen 4-methylbenzylidene camphor (4-MBC), which is the subject of a COSMETICS EUROPE – The Personal Care Association (ex- COLIPA, Comite´ de liaison de la parfumerie) opinion regarding possible effects on the thyroid gland22–24. 4-MBC (Figure 1) is a widely used sunscreen agent which absorbs most efficiently in the 290–320 nm region (UVB, lmax

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O

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Figure 1. Chemical structure of 4-MBC. CAS number: 36861-47-9; EINECS number: 253-242-6; white powder with weak camphor-like odor; molar formula: C18H22O; molecular weight: 254.37 g mol1; melting point: 66–68  C; refractive index (20  C): 1.543–1.547; lmax ¼ 295–300 nm; " ¼ 53 600 M1; solubility: poorly soluble in water (0.00013 g/100 mL; 20  C), slightly soluble in ethanol and vegetable oils, very slightly soluble in chloroform.

295–300 nm) of UV spectrum. It is included in the list of authorized UV filters in Europe25, USA and Australia. Although 4-MBC was long considered fairly photostable26,27, recent studies have indicated that it undergoes marked degradation under exposure to sunlight28,29. 4-MBC has estrogenic activity in vitro and in the uterotrophic assay, and is a preferential estrogen receptor (ER)-beta ligand. However, its actions in vivo are more complex, since it can also interact with the thyroid axis30. Sub-acute and sub-chronic studies in rats suggest marked interference in thyroid hormone metabolism as evidenced by changes in thyroid weight, levels of circulating thyroid hormones and histological evidence of thyroid stimulation. In addition, interference with thyroid function may affect other parameters, such as red blood cell turnover. Fortunately, these studies did not suggest the same results in human beings and tests for skin irritation, sensitization, phototoxicity, photosensitization and photo-contact allergy were negative. The compound very rarely caused contact allergy in humans31. Nevertheless, the findings in rats cannot be disregarded without a proper understanding of the mechanisms involved and disturbances of the thyroid hormone axis must be considered with great caution. 4-MBC was systemically absorbed after wholebody topical application to human volunteers32. The increase in 4-MBC retention on the skin surface achieved by its inclusion in microparticle systems should enhance its efficacy and reduce the potential toxicological risks associated with skin penetration. Scalia et al.33 studied the influence of microencapsulation in solid lipid microparticles on the percutaneous penetration of the sunscreen. The penetration of 4-MBC into the SC was lower from the emulsion containing the sunscreenloaded microparticles than the formulation prepared with the free UV filter. The safety offered by ‘‘novel cosmetic delivery systems’’ thus acquired a more important role in the light of the COSMETICS EUROPE (ex-COLIPA) opinion and the recent review34 highlighting the few human studies on the potential side effects of UV filters, including disturbance of the hypothalamic–pituitary– thyroid axis (HPT). In this study, we prepared microspheres containing 4-MBC with the aim of improving photoprotection achieving safe functional systems without the drawbacks of traditional formulations. The microspheres loaded with 4-MBC were then incorporated in cosmetic formulations and we investigated their influence on the sun protection factor (SPF), light-induced degradation and release of the UV filter.

Materials and methods Materials Materials were obtained from commercial suppliers and used as received. The following were used to prepare microspheres: dichloromethane (DCM; Riedel-de Haen, Seelze,

Germany), ethyl acetate (EA; Sigma-Aldrich, Milan, Italy), co-polymers of poly(ethylacrylate, methyl methacrylate) and trimethyl aminoethyl methacrylate chloride (Eudragits RS 100, Mol. wt. 150 000, Ro¨hm Pharma Polymers, Darmstadt, Germany), 4-MBC (Parsol 5000, DSM, Heerlen, The Netherlands), polyvinyl alcohol (PVA: Mol. wt. 13–23 000 and 22 000, 87–89%, Sigma-Aldrich, Milan, Italy). To prepare emulsions, we used: tri-C12–13 alkyl citrate (Cosmacol ECI, SASOL ITALY S.p.A., Milan, Italy), caprylic/capric triglyceride (Myritol 318, Cognis, Monheim, Germany), cetearyl glucoside, cetearyl alcohol (Montanov 68, Seppic Italia Srl, Milan, Italy), potassium cetyl phosphate (Amphisol K, DSM, Heerlen, The Netherlands) and deionized water. All other chemicals and solvents, such as tetrahydrofuran (THF) and ethanol, were of analytical reagent grade. Preparation of blank microspheres Microspheres were prepared using an emulsification–solvent evaporation technique35, employing a synthetic co-polymer of poly(ethylacrylate, methyl methacrylate) and trimethyl aminoethyl methacrylate chloride. This polymer was chosen for several reasons such as its solubility in DCM and EA, which were used in the preparations, and its insolubility in water which allowed the dispersion in aqueous medium. Another factor was independence from the pH, which allows it to remain undissolved at skin pH and not affected by individual physiological changes. Also, this polymer, with its quaternary ammonium groups, is substantive and the microspheres remain tied on the skin surface. First, to control and optimize the method, unloaded microspheres were formulated and the formulations were optimized on the basis of process variables such as the devices used for emulsification, stirring rate and temperature. The polymer (1 g) was dissolved in 10 mL of DCM (10% v/v) or in EA. Separately, an aqueous solution was prepared dissolving PVA (1% w/v) in 100 mL of water. The organic phase was slowly injected, using a syringe, into an aqueous phase containing PVA as dispersing agent, and emulsified. The O/W emulsion was maintained under continuous magnetic stirring for about 24 h, to ensure complete evaporation of the organic solvent and formation of the microspheres. At the beginning, the critical step of emulsification was carried out at room temperature but subsequently, to avoid premature solvent evaporation, we used an ice-bath at 4–5  C. At different intervals during preparation of the microparticles, microphotographs were taken using an optical microscope equipped with a digital camera. Finally, the hardened microspheres were recovered by centrifugation (3600 rpm, 30 min) and washed three times with deionized water. The microspheres, suspended in a small amount of deionized water, were frozen and lyophilized, or collected by filtration under vacuum and dried at room temperature in a desiccator under low pressure for 48 h. 4-Methylbenzylidene camphor-loaded microspheres After a preliminary study with unloaded microspheres, we prepared 4-MBC-loaded microspheres by emulsification–solvent evaporation, following the procedure described above. Different amounts of 4-MBC (5, 10, 20, 40, 80% filter:polymer w/w) were dissolved in the organic phase. All batches of microparticles were produced at least in triplicate. We investigated some process parameters to improve the encapsulation efficiency, especially for the samples prepared with a large amount of sunscreen. The aim was to find operating conditions in which the filter was most soluble in the organic phase so that higher loading could be reached. Both the type and volume of solvent were varied.

Photoprotection of 4-MBC microspheres

DOI: 10.3109/10837450.2014.920359

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Table 1. Variables investigated in the microsphere preparation; 4-MBC microsphere loading and encapsulation efficiency.

Sample

Filter/polymer (%)

Solvent

Stirring (rpm)

Loading (%)

Encapsulation efficiency (%)

5 10 20 40 80 80 5 80 80

10 mL CH2Cl2 10 mL CH2Cl2 10 mL CH2Cl2 10 mL CH2Cl2 10 mL CH2Cl2 10 mL C2H5CO C2H5 2.5 mL CH2Cl2 2.5 mL CH2Cl2 15 mL CH2Cl2

1000–2500 1000–2500 1000–2500 1000–2500 1000–6000 1000–6000 1000–6000 1000–6000 1000–6000

3.70 ± 0.15 9.14 ± 0.25 16.54 ± 0.69 24.24 ± 0.64 39.39 ± 0.68 43.03 ± 0.44 4.12 ± 0.17 38.71 ± 1.22 40.56 ± 0.65

74.00 ± 3.00 91.40 ± 2.50 82.70 ± 3.45 61.35 ± 1.63 49.24 ± 0.84 53.79 ± 0.55 82.40 ± 3.40 48.38 ± 1.52 50.70 ± 0.81

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A B C D E F G H I

Type of solvent DCM was replaced by EA in sample F (Table 1). For the evaporation phase, we employed a Rotavapor (at 35  C and 250 mbar) to speed up the process.

Table 2. Composition of the O/W emulsions (%) and their sun protection factors (SPF). Emulsions Ingredient (INCI NAME)

Volume of solvent We used 15 mL or 2.5 mL of DCM in the total emulsion (samples G, H, I). Morphological analysis Microparticle morphology was examined by optical and scanning electron microscopy (SEM). An optical microscope was used (Olympus 1  71) to monitor the formation of microspheres from emulsions and for preliminary screening of samples to be analyzed with a scanning electron microscope (SEM). Detailed information on the shape, surface and porosity of the particles was obtained using an XL 20 SEM (Philips, Amsterdam, The Netherlands) and XL 30 SEM (Philips, CDU Leap Detector). Samples were prepared by placing a droplet of an aqueous suspension of microspheres on an aluminium specimen stub. The samples were dried overnight and were sputter-coated with gold before imaging (Emitech K-550X sputter-coater, Ashford, Kent, UK). Coating was done at 20 mA for 4 min.

1a

1b

2a

2b

Tri-C12–14 alkyl citrate 15.00 15.00 – – Cetearyl glucoside, cetearyl 5.00 5.00 5.00 5.00 alcohol Caprylic/capric triglyceride – – 15.00 15.00 4-Methylbenzylidene camphor 3.00 – 3.00 – a a Microspheresa – – Potassium cetyl phosphate 0.50 0.50 0.50 0.50 Aqua 76.50 76.50 76.50 76.50 SPF 4.82 ± 0.45 4.25 ± 0.26 5.00 ± 0.34 3.85 ± 0.30 a

The microspheres (sample E) were prepared in emulsions containing 3% of the filter.

Preparation of O/W emulsions

The particle size of the various microsphere preparations was analyzed with an Accusizer 770 (Particle Sizing System, Santa Barbara, CA), using the Single Particle Optical Sensing technique. The particles were suspended in deionized water (a small amount of Tween 80Õ was added to prevent aggregation, if necessary). Analyses were done in triplicate and the size was expressed as mean diameter.

Two types of non-ionic/anionic O/W emulsions (Table 2), using different emollients, were prepared to examine the influence of the formulation on sunscreen SPF. Both contained the same amount of the filter (3% w/w), either free or in microspheres, and the same emulsifiers (emulsifying couple): cetearyl glucoside, cetearyl alcohol (Montanov 68) as the non-ionic non-ethoxylated surfactant and potassium cetyl phosphate (Amphisol K) as the anionic surfactant. The only difference was the emollient: tri-C12–13 alkyl citrate (Cosmacol ECI), in ‘‘type 1’’ emulsion and caprylic/capric triglyceride (Myritol 318), in ‘‘type 2’’ emulsion. The emulsions were prepared by inversion phase method adding aqueous phase (containing Amphisol K) to oil phase (containing Montanov 68)36. The UV filter was dissolved in the oil phase, and the sunscreenloaded microspheres were dispersed in water and added during the cooling phase of the emulsion at about 40  C.

Microspheres 4-MBC content

Sun protection factor

Microsphere loading was investigated by UV analysis: 2.5 mg accurately weighed 4-MBC-loaded microspheres were dissolved in a mixture of THF/H2O (9:1 v/v), under sonication, to obtain filter concentrations in the range of 5–30 mg/mL. A calibration standard curve ranging from 2 to 20 mg/mL of 4-MBC dissolved in THF/H2O 9:1 v/v was used as reference. The sunscreen concentration was determined by measuring the absorbance at 297 nm (Varian Cary 1E ver. 3.03) in quartz cuvettes (path length 1 cm). Unloaded microspheres gave insignificant absorbance at the same wavelength. The amount of the filter in the microparticles was expressed as a percentage of the total weight of the sample. Each sample was analyzed in triplicate. Encapsulation efficiency was calculated as the percentage ratio between the amount of 4-MBC entrapped in the microspheres and the amount of sunscreen added to the preparation.

We compared the ability to protect against UV rays of 4-MBCloaded microspheres and the free filter in the O/W emulsion. The O/W emulsions containing 3% (w/w) of filter either free or included in microspheres were prepared. The protective efficacy was examined by measuring the in vitro SPF37,38, using a Labsphere spectrophotometer (UV-1000S Ultraviolet Transmittance Analyzer), a quality control tool designed specifically for this purpose. According to the COSMETICS EUROPE (ex-COLIPA) protocol39, 2 mg/cm2 of the emulsion were spread on TransporeÔ tape and the SPF was measured after 15 min, according to the Diffey and Robson equation reported in Figure 2, where T is the sunscreen transmittance at wavelength l, E is the erythemal spectral effectiveness and S is the spectral irradiance of terrestrial sunlight at wavelength l. The UV-1000S calculates the SPF of the sunscreen sample by measuring the spectral

Particle size measurement

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Figure 2. Diffey equation. E is the spectral irradiance of ‘‘standard sun’’ corresponding to COLIPA ‘‘SPF method’’ (sunlight expected for a clear sky at noon in midsummer for a latitude of 40 N and solar altitude 70 ); S is the erythema action spectrum corresponding to CIE publication No 90 (1991); T is the spectral transmittance of the sunscreen.

transmittance of UV radiation (290–400 nm) through the TransporeÔ tape substrate before and after application of the sunscreen product. The term transmittance refers to the percentage of the radiant flux transmitted through the sample, relative to the incident flux. Five measurements were made for each sample and the mean and standard deviations were calculated. Photostability We compared the photostability of 4-MBC, free or encapsulated, incorporated into the two O/W emulsions. These cosmetic formulations were spectrophotometrically analyzed before and after irradiation. A portion of the test sample (7 mg, accurately weighed), containing the free filter (3% w/w) or the same amount of the sunscreen included in microspheres, was transferred to a quartz cuvette of 0.01 mm path length to simulate the film of sunscreen distributed on the skin and then exposed to the solar simulator equipped with a xenon lamp (Universal Arc Lamp Housing model 66000 and Arc Lamp Power Supply model 68805, LOT ORIEL Italia, Milan, Italy). Before each measurement the xenon arc lamp was calibrated with a radiometer (Goldilux Smart Meter model 70234, LOT ORIEL Italia, Milan, Italy) equipped with a UVB probe. Samples were placed 40 cm from the lamp, irradiated with 300 mJ/cm2, equivalent to 10 MED (Minimal Erythemal Dose: 1 MED ¼ 25 mJ/cm2 for skin phototype II)40 and air-cooled during irradiation. After the appropriate exposure time, the cuvette was removed and its content quantitatively transferred into a 10-mL calibrated flask, and diluted to volume with a mixture of THF/H2O (9:1 v/v). After sonication, the resulting solution was analyzed by UV spectrophotometry (Cary 1E, Varian, Milan, Italy). The operating conditions were the same as for loading determination. The degree of photodegradation was measured by comparing the filter amounts of irradiated samples with the non-exposed preparations. We also ran the stability test on both O/W emulsions, to check the influence of the emollient. The microspheres loaded with 80% of 4-MBC were irradiated at 10 MED in aqueous dispersion in the same conditions to evaluate morphological changes before and after irradiation. 4-MBC release We studied the free and encapsulated filter release from the vehicle, using two receiver fluids: hydrophilic, using phosphate buffer, pH 5.9 (NaH2PO4 + Na2HPO4), employing a dialysis bag41, and lipophilic, using caprylic/capric triglyceride and Strainer cells42. Hydrophilic receiver fluid The hydrophilic receiver fluid was a phosphate buffered solution pH 5.9 prepared as reported by Gennaro43: the free filter or microspheres were exactly weighed and placed in a cellulose dialysis bag (cut-off 12 000–14 000, Medicell International, London, UK), previously hydrated for 24 h and washed with distilled water. The bag was immersed in 100 mL of the buffered

Pharm Dev Technol, Early Online: 1–9

solution, under magnetic stirring. At appropriate intervals (0, 5, 15, 30, 60, 120, 240, 420 and 1440 min) 1-mL aliquots of the receptor phase were withdrawn and replaced with an equal volume of fresh receiving buffer. The samples were filtered (0.45 mm), diluted (1:1) with ethanol and assayed spectrophotometrically. A calibration standard curve ranging from 1 to 5 mg/mL of 4-MBC dissolved in phosphate buffer/ethanol 1:1 v/v was used as reference. Analyses were done on 100 mg of free filter and, in the same way, on two 50-mg samples of 4-MBC-loaded microspheres, prepared with 5 and 80% of sunscreen: (samples A and E). After analysis of the release, the microspheres were recovered and spectrophotometrically assayed in the same operating conditions as for loading determination for further confirmation of the results. Lipophilic receiver fluid The lipophilic receiver fluid was caprylic/capric triglyceride (Myritol 318) because 4-MBC is soluble in it, while the wall polymer of microspheres remains intact. The two O/W emulsions were employed as vehicles for the free or encapsulated filter. Release was evaluated using a modification of the method proposed by Casolaro et al.42. Briefly, 2.0 g of the sample was layered on the bottom of a Strainer cell, in close contact with the surface of 20 mL of Myritol 318 at room temperature, under magnetic stirring (150 rpm) for max 24 h. Strainer cells are sterile sieves made of strong 100-micron nylon mesh. One hundred microliter of the receiving fluid were taken at each time (0, 5, 15, 30, 60, 120, 240, 420 and 1440 min), diluted with 900 mL ethanol (v/v) and analyzed by UV spectrophotometry. A standard calibration curve ranging from 2 to 20 mg/mL of 4-MBC dissolved in Myritol 318:ethanol 1:9 v/v was used as reference. After each withdrawal 100 mL of fresh receiver fluid was added to keep the volume constant. Analyses were run in triplicate; means and standard deviations were calculated. Statistical analysis Findings were compared by analysis of variance (ANOVA). p50.05 was considered statistically significant.

Results and discussion Preparation and characterization of microspheres Microspheres containing 4-MBC were prepared using the emulsification–solvent evaporation method. To investigate how some process parameters influenced the particle characteristics, we first studied blank microspheres, using different emulsifier devices, stirring rates and temperatures to prepare systems suitable for encapsulating a sunscreen agent. The following devices and rates were used for emulsification: magnetic stirrer at 150 rpm, Vortex at 1500 rpm, homogenizing mixer at 1000 rpm for 2 min then at 2500 rpm for 30 min. The equipment was selected mainly to achieve good emulsification of the two phases in a short time. Speed, in fact, is fundamental so the solvent does not evaporate already in this first critical step of the preparation. Microspheres were characterized for shape and surface morphology. Figure 3 shows SEM photographs of samples prepared with the different processes. The particles obtained with magnetic stirring at 150 rpm are spherical but with a rough surface (Figure 3a). The preparations produced by vortexing were not uniformly distributed in size, and were irregularly shaped with a porous, wrinkled surface with pores of various sizes that could harbor small particles (Figure 3b). At room temperature heterogeneous particles were obtained, measuring up to 10 mm, not perfectly spherical, with a non-porous surface (Figure 3c).

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DOI: 10.3109/10837450.2014.920359

Photoprotection of 4-MBC microspheres

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Figure 3. SEM photographs of unloaded microspheres, emulsified with a magnetic stirrer (a), with a Vortex (b) and with a homogenizing mixer at room temperature (c) or at 4–5  C (d).

Figure 4. SEM photographs of shape (a) and internal matrix (b) of 4-MBC-loaded microspheres (sample A), emulsified with a homogenizing mixer at 1000 rpm at 4–5  C.

The best results were obtained using the homogenizing mixer, at 4–5  C (Figure 3d): the particles were more homogeneous, spherical, with a porous surface. 4-MBC-loaded microspheres were therefore prepared in these conditions. Spherical particles with a porous surface (Figure 4a, sample A) were obtained with the addition of 5% of filter, and their internal matrix was compact, as observed in cryoscopic section (Figure 4b). The surface pores might be caused by rapid evaporation of the solvent due to the thermal shock between the two emulsifying phases (4–5  C) and during evaporation (room temperature).

Later, we prepared microspheres containing a larger amount of filter (10–80% w/w in relation to polymer). The aim was to obtain particle systems containing more of the encapsulated sunscreen, which would therefore be more suitable for use in cosmetic products. The preparation with 80% of the filter was obtained by operating at 1000 rpm for 2 min and then at 6000 rpm for 30 min to improve the emulsification. Samples B, C and D, prepared with larger amounts of filter (10, 20 and 40% filter/polymer), had very similar morphology to sample A. Sample E (80% filter/polymer w/w) had some external particles, because the filter was not encapsulated (Figure 5).

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Microsphere loading ranged from 3.70 ± 0.15% to 39.39 ± 0.62% (Table 1). To improve the morphological and loading characteristics, particularly for the sample with a large amount of sunscreen, we modified some of the process parameters. Sample F was prepared using EA as solvent, but the resulting particles had an irregular shape (Figure 6a). In other preparations, we employed different amounts of DCM. Samples G (5% filter/polymer w/w) and H (80% filter/polymer w/w) (Figures 6b and 6c) were prepared with a smaller volume of solvent (510 mL), while in sample I (80% filter/polymer w/w) (Figure 6d) we used 15 mL of dichloromethane. With sample G the particles were spherical, with a smooth surface but with different sizes. Samples H and I gave irregularly shaped microparticles. Sample I irradiated at 10 MED in aqueous dispersion showed no change in its morphology (Figure 6e). Loading data were satisfactory and ranged from 4.12 ± 0.17% to 43.03 ± 0.44%. In all cases, the particle size was between 5

Pharm Dev Technol, Early Online: 1–9

and 100 mm, with the majority in the 20–40 mm range, which is appropriate for topical application when percutaneous absorption should be prevented, as for sunscreen agents44,45. Encapsulation efficiency was good enough (75–90%) for microspheres prepared with a low ratio of sunscreen to polymer (from 5% to 20%), but low (50%) for microspheres prepared with the largest amounts of sunscreen. The loss of encapsulation efficiency was due to the high ratio of filter to polymer46. Raising the amount of 4-MBC:polymer from 5% to 80%, the encapsulation efficiency decreases but the loading increases up to 40%. This is very important because the aim of the study was to obtain microspheres containing as much sunscreen as possible. The maximum concentration of 4-MBC allowed by the Regulation is 4%, and this could be achieved by introducing about 10% of microspheres (40% loaded) into the cosmetic formulation. In view of the best morphological results, subsequent analyses were done only on samples A and E. Sun protection factor

Figure 5. SEM photographs of shape and surface of 4-MBC-loaded microspheres (sample E), emulsified with a homogenizing mixer at 1000 rpm at 4–5  C.

To assess the photoprotective effectiveness of the system, we compared the in vitro SPF of O/W emulsions (1b and 2b) containing the 4-MBC-loaded microspheres (sample E) and of emulsions prepared with the free filter (1a and 2a). SPF were measured using the Diffey and Robson equation38 based on integrating sphere-equipped spectrophotometer, which can read the transmittance of opaque samples such as sun-care products. The substrate used was TransporeÔ tape, a surgical tape manufactured by the 3M Company, which simulates the porosity and texture of human skin, is inexpensive, readily available and easy to use. Table 2 shows the results. The values were very similar for the sunscreen loaded in microspheres or free. Differences were nonsignificant between the in vitro SPF of the emulsions containing the sunscreen free (1a: 4.82 ± 0.45) or microencapsulated (1b: 4.25 ± 0.26) (p50.05, ANOVA). These results indicate that the inclusion of 4-MBC in the microspheres did not affect its efficacy and the preparations based on microparticles were effective in protecting the skin against UV rays.

Figure 6. SEM photographs of 4-MBC-loaded microspheres: (a) sample F prepared with ethyl acetate; (b) sample G prepared with 2.5 mL of dichloromethane and 5% (w/w filter/polymer) 4-MBC; (c) sample H prepared with 2.5 mL of dichloromethane and 80% (w/w filter/polymer) 4-MBC; (d) sample I prepared with 15 mL of dichloromethane and 80% (w/w filter/polymer) 4-MBC; (e) sample I irradiated at 10 MED.

Photoprotection of 4-MBC microspheres

DOI: 10.3109/10837450.2014.920359

The influence of the emollient can be seen for the emulsions containing 4-MBC-loaded microspheres, 1b and 2b, whose SPF values were 4.25 ± 0.26 and 3.85 ± 0.30, respectively. The best results were obtained using tri-C12-13 alkyl citrate (Table 2). Photostability To verify how the polymeric carriers influenced the photochemical behavior of 4-MBC, photostability experiments were done on

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Table 3. 4-MBC content (free or encapsulated in microspheres) in O/W emulsions before and after 10 MED irradiation.

Emulsion 1a 1b 2a 2b

4-MBC before irradiation (%)

4-MBC after irradiation (%)

Filter degradation (%)

3.15 ± 0.15 3.29 ± 0.06 3.11 ± 0.04 3.36 ± 0.08

2.67 ± 0.12 3.08 ± 0.08 2.64 ± 0.06 3.32 ± 0.11

15.23 ± 0.22 6.39 ± 0.72 15.11 ± 0.84 1.20 ± 0.92

MED, Minimal erythemal dose. Table 4. Microsphere loading before and after release in hydrophilic medium. Loading Samples A E

Filter/polymer (%)

Before release

After release

5 80

3.70 ± 0.68% 39.39 ± 0.68%

3.68 ± 0.65% 38.98 ± 0.70%

Table 5. 4-MBC released, free or loaded in microspheres, from the two O/W emulsions. Filter released (%) Time (min) T0 T1 T2 T3 T4 T5 T6 T7 T8

(0) (5) (15) (30) (60) (120) (240) (420) (1440)

Emulsion 1a

Emulsion 1b

Emulsion 2a

Emulsion 2b

0.0 0.16 ± 0.17 0.95 ± 0.23 1.29 ± 0.79 2.01 ± 1.31 3.75 ± 2.07 5.30 ± 0.63 5.95 ± 0.37 9.66 ± 0.06

0.0 0.0 0.07 ± 0.09 0.12 ± 0.05 0.25 ± 0.10 0.69 ± 0.22 1.18 ± 0.20 1.64 ± 0.40 4.59 ± 1.25

0.0 0.0 0.01 ± 0.00 0.29 ± 0.23 0.85 ± 0.00 2.17 ± 0.14 4.01 ± 0.79 5.05 ± 1.37 7.37 ± 0.46

0.0 0.0 0.07 ± 0.00 0.17 ± 0.07 0.51 ± 0.19 0.93 ± 0.40 1.92 ± 0.63 2.13 ± 0.63 4.88 ± 1.29

Figure 7. 4-MBC release profiles from samples of O/W emulsions, containing 3% (w/w) of free (1a and 2a) or encapsulated filter (1b and 2b), in the caprylic/capric triglyceride.

7

the two O/W emulsions. These were selected as model formulations as they are the most common types of sunscreen preparation, thus simulating real conditions of use. 4-MBC (3% w/w) alone or encapsulated in microspheres was incorporated into the emulsion and exposed to the solar simulator. The microspheres protected the sunscreen, with about 15% degradation in both formulations containing free 4-MBC (1a and 2a), while in emulsion 1b (with tri-C12–13-alkyl citrate as emollient) containing the sunscreen-loaded microspheres the photodegradation was 6% and in emulsion 2b (with caprylic/ capric triglyceride as emollient) it was 1%. Thus, the sunscreen was clearly protected by the microspheres and the vehicle had substantial effect (Table 3). The results agree with those reported by Scalia et al.19 concerning 4-MBC in RM-b-CD. 4-MBC release Various techniques, such as the sample-and-separate method, the membrane barrier method, in situ methods and continuous-flow methods are used to characterize in vitro drug transport kinetics from emulsions. This study employed a modification of the membrane barrier method, which includes dialysis bag equilibrium and cell diffusion methods. We used two different media to investigate the release of 4-MBC in a hydrophilic or lipophilic environment. The hydrophilic receiver fluid, used for dialysis bag equilibrium (a phosphate buffered solution at pH 5.9), was chosen to mimic the skin pH; while the lipophilic receiver fluid, used for Strainer cell (caprylic/capric triglyceride), was chosen to simulate the lipophilic characteristics of SC. Moreover, sunscreens can be formulated as oil or water lotion, hydrophilic gel or lipogel and emulsion. Thus, we report the experimental results in different vehicles and compared the behavior filter-free or included in microspheres both in water lotion and emulsion. No release was observed after 24 h in hydrophilic receiver fluid probably due to the lack of solubility in water of the filter. As expected, the microsphere loading values were the same before and after the release (Table 4). This was confirmed by spectrophotometric analysis on the microspheres recovered. Table 5 reports the percentages of sunscreen released, free or included in microspheres, from the two emulsions in the lipophilic medium. Figure 7 shows 4-MBC release profiles from emulsions containing 3% (w/w) of free or encapsulated filter, in Strainer cells. The sunscreen diffused to the lipophilic receiving fluid. For both preparations containing microspheres, release was slow,

8

M. Centini et al.

reaching 5% in 24 h, whereas it was faster from free sunscreens, reaching a peak of 10% in 24 h. The encapsulated filter release was independent of the emollient.

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Conclusions This study shows that these new microspheres offer good morphological and loading results. The photoprotective ability of 4-MBC is not affected by the polymeric matrix, which protects the filter from photodegradation, but it does depend on the emollients used in the formulation. Microspheres give less release of the filter; in fact the release is very low for encapsulated filter in microspheres if we consider the real time of exposure to the sun as around 7 h/day (release was 1.64 and 2.13% in 1b and 2b emulsions, respectively). 4-MBC in substantive microspheres is no longer in close contact with the skin, reducing the risk of percutaneous absorption and systemic effects such as interference with thyroid function. These results hold promise for future in vivo studies and for the development of innovative, bettertolerated sunscreen products for consumers.

Pharm Dev Technol, Early Online: 1–9

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Acknowledgements The authors would like to thank Dr. Paolo Blasi (Dept. of Pharmaceutical Science, University of Perugia) for help with the SEM and dimensional analysis.

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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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References 1. National Institute of Health consensus statement online. Sunlight UV Radiant Skin 1989;7:1–29. 2. Green A, Williams G, Neale R, et al. Daily sunscreen application and betacarotene supplementation in the prevention of basal-cell and squamous-cell carcinomas of the skin: a randomised controlled trial. Lancet 1999;354:723–729. 3. Ziegler A, Jonason AS, Leffel DJ, et al. Sunburn and p53 in the onset of skin cancer. Nature 1994;372:773–776. 4. Lens M, Dawes M. Global perspectives of contemporary epidemiology trends of cutaneous malignant melanoma. Br J Dermatol 2004;150:179–185. 5. Gasparro M, Mitchnick FP, Nash JF. A review of sunscreen safety and efficacy. Photochem Photobiol 1998;68:243–256. 6. Magdassi S, Touitou E. Novel cosmetic delivery systems. New York: Marcel Dekker Inc.; 1999. 7. Rieger MM. Photostability of cosmetic ingredients on the skin. Cosm Toil 1997;112:65–72. 8. Monti D, Saettone MF, Centini M, Anselmi C. Substantivity of sunscreens. ‘‘In vitro’’ evaluation of the transdermal permeation characteristics of some benzophenone derivatives. Int J Cosm Sci 1993;15:45–52. 9. Gogna D, Jain SK, Yadav AK, Agrawal GP. Microsphere-based improved sunscreen formulation of ethylhexyl methoxycinnamate. Cur Drug Del 2007;4:153–159. 10. Anselmi C, Centini M, Rossi C, et al. New microencapsulated sunscreens: technology and comparative evaluation. Int J Pharm 2002;242:207–211. 11. Hanno I, Anselmi C, Bouchemal K. Polyamide nanocapsules and nano-emulsion containing Parsol MCX and Parsol 1789: in vitro release, ex vivo skin penetration and photo-stability studies. Pharm Res 2012;29:559–573. 12. Lapidot N, Gans O, Biagini F, et al. Advanced sunscreens: UV absorbers encapsulated in sol-gel glass microcapsules. J Sol-Gel Sci Technol 2003;26:67–72. 13. Weiss-Angel V, Bourgeois S, Pelletier J, et al. Development of an original method to study drug release from polymeric nanocapsules in the skin. J Pharm Pharmacol 2010;62:35–45. 14. Albertini B, Mezzena M, Passerini N, et al. Evaluation of spray congealing as a technique for the preparation of highly loaded solid

27. 28. 29. 30.

31.

32.

33.

34. 35.

36. 37.

lipid microparticles containing the sunscreen agent Avobenzone. J Pharm Sci 2009;98:2759–2769. Nikolic S, Keck CM, Anselmi C, Muller RH. Skin photoprotection improvement: synergistic interaction between lipid nanoparticles and organic UV filters. Int J Pharm 2011;414:276–284. Scalia S, Mezzena M. Incorporation in lipid microparticles of UVA filter, butyl methoxy dibenzoylmethane combined with the UVB filter octocrylene: effect on photostability. AAPS PharmSciTech 2009;10:384–390. Wissing SA, Muller RH. Solid lipid nanoparticles as carrier for sunscreens: in vitro release and in vivo skin penetration. J Control Release 2002;81:225–233. Perioli L, Ambrogi V, Bertini B, et al. Anionic clays for sunscreen agent safe use: photoprotection, photostability and prevention of their skin penetration. Eur J Pharm Biopharm 2006;62:185–193. Scalia S, Tursilli R, Iannuccelli V. Complexation of the sunscreen agent, 4-methylbenzylidene camphor with cyclodextrins: effect on photostability and human stratum corneum penetration. J Pharm Biomed Anal 2007;44:29–34. Anselmi C, Centini M, Maggiore M, et al. Non-covalent inclusion of ferulic acid with a-cyclodextrin improves photo-stability and delivery: NMR and modeling studies. J Pharm Biomed Anal 2008; 46:645–652. Simeoni S, Scalia S, Benson HAE. Influence of cyclodextrins on in vitro human skin absorption of the sunscreen butyl-methoxydibenzoylmethane. Int J Pharm 2004;280:163–171. Scientific Committee on Consumer Non Food Products (SCCNFP). Opinion concerning 4-Methylbenzylidene Camphor. COLIPA No. S60; 2004. Scientific Committee on Consumer Products (SCCP). Opinion on 4-Methylbenzylidene Camphor. No. S60; 2006. Scientific Committee on Consumer Products (SCCP). Opinion on 4-Methylbenzylidene Camphor (4-MBC). COLIPA No. S60; 2008. EEC Council Directive. 76/768 Annex VII; 1976. Tarras-Wahlberg N, Stenhagen G, Larko¨ O, et al. Changes in ultraviolet absorption of sunscreens after ultraviolet irradiation. J Invest Dermatol 1999;113:547–553. Deflandre A, Lang G. Photostability assessment of sunscreens. Benzylidene camphor and dibenzoylmethane derivatives. Int J Cosmet Sci 1988;10:53–62. Hauri U, Lu¨tolf B, Schlegel U, Holhl C. Determination of photodegradation of UV filters in sunscreens by HPLC/DAD and HPLC/MS. Mitt Lebensm Hyg 2004;95:147–161. Gaspar LR, Maia Campos PMBG. Evaluation of the photostability of different UV filter combinations in a sunscreen. Int J Pharm 2006; 307:123–128. Maerkel K, Durrer S, Henseler M, et al. Sexually dimorphic gene regulation in brain as a target for endocrine disrupters: developmental exposure of rats to 4-methylbenzylidene camphor. Toxicol Appl Pharmacol 2007;218:152–165. Schmidt T, Ring J, Abeck D. Photoallergic contact dermatitis due to combined UVB (4-methylbenzylidene camphor/octyl methoxycinnamate) and UVA (benzophenone-3/butylmethoxydibenzoylmethane) absorber sensitization. Dermatol 1998;196:354–357. Janjua NR, Mogensen B, Andersson AM, et al. Systemic absorption of the sunscreens benzophenone-3, octylmethoxycinnamate, and 3-(4-methyl-benzylidene) camphor after whole-body topical application and reproductive hormone levels in humans. J Invest Dermatol 2004;123:57–61. Scalia S, Mezzena M, Iannuccelli V. Influence of solid lipid microparticle carriers on skin penetration of the sunscreen agent, 4-methylbenzylidene camphor. J Pharm Pharmacol 2007;59: 1621–1627. Krause M, Klit A, Blomberg Jensen M, et al. Sunscreens: are they beneficial for health? An overview of endocrine disrupting properties of UV-filters. Int J Androl 2012;35:424–436. Benoit J-P, Marchais H, Rolland H, Velde VV. Biodegradable microspheres: advances in production technology. In: Benita S, ed. Microencapsulation. methods and industrial applications. New York: Marcel Dekker Inc.; 1996:35–72. Courtney DL. Emulsifier selection/HLB. In: Rieger MM, Rhein LD, eds. Surfactants in cosmetics. 2nd ed. [Revised and Expanded]. New York: Marcel Dekker Inc.; 1997:127–138. Diffey BL, Robson J. A new substrate to measure sunscreen protection factors throughout the ultraviolet spectrum. J Soc Cosmet Chem 1989;40:127–133.

Photoprotection of 4-MBC microspheres

DOI: 10.3109/10837450.2014.920359

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38. Labsphere Technical Note. SPF analysis of sunscreens using the Labsphere UV-1000S Ultraviolet Transmittance Analyzer; 1998. 39. COLIPA GUIDELINES – CTFA-SA, JCIA, International Sun Protection Factor (SPF) Test Method; 2006. 40. Pathak MA. Photoprotection against harmful effects of solar UVB and UVA radiation: an update. In: Lowe NJ, Shaath NA, Pathak MA, eds. Sunscreens development, evaluation, and regulatory aspects. New York: Marcel Dekker Inc.; 1997:59–79. 41. Chidambaram N, Burgess DJ. A novel in vitro release method for submicron-sized dispersed systems. AAPS PharmSci 1999;1:32–40. 42. Casolaro M, Bottari S, Ito Y. Vinyl polymers based on L-histidine residues. Part 2. Swelling and electric behavior of smart

43. 44. 45. 46.

9

poly(ampholyte) hydrogels for biomedical applications. Biomacromolecules 2006;7:1439–1448. Gennaro AR. Remington’s pharmaceutical science. 18th ed. Easton (PA): Mack Publishing Company; 1990. Wiechers JW. Avoiding transdermal cosmetic delivery. Cosmet Toil 2000;115:39–46. Toll R, Jacobi U, Richter H, et al. Penetration profile of microspheres in follicular targeting of terminal hair follicles. J Invest Dermatol 2004;121:168–176. Jyothi NVN, Prasanna PM, Sakarkar SN, et al. Microencapsulation techniques, factors influencing encapsulation efficiency. J Microencapsul 2010;27:102–116.