Delivered by Ingenta to:UNIVERSIDADE SAO PAULO IFIP : 143.107.228.70Thu, 31 Mar 2011 12:09:18RESEARCHARTICLECopyright 2011 American Scientic PublishersAll rights reservedPrinted in the United States of AmericaJournal ofNanoscience and NanotechnologyVol. 11, 18801886, 2011Nanostructured Polymer and Lipid Carriers forSunscreen. Biological Effects and Skin PermeationP. D. Marcato1 , J. Caverzan1, B. Rossi-Bergmann2, E. F. Pinto2, D. Machado3,R. A. Silva3, G. Z. Justo3 4, C. V. Ferreira3, and N. Durn11Institute of Chemistry, Biological Chemistry Laboratory, Universidade Estadual de Campinas,P.O. Box 6154, Campinas-SP, CEP 13083-970, Brazil2Institute of Biophysical Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902, Brazil3Institute of Biology, Universidade Estadual de Campinas, 13083-940, Brazil4Department of Biochemistry, Universidade Federal de So Paulo, 04024-000, BrazilThe interest in developing new sunscreens is increasing due to the harmful effects of UV radia-tion on the skin, such as erythema, accelerated skin ageing (photoageing) and the induction ofskin cancer. However, many molecular sunscreens penetrate into the skin causing photoallergies,phototoxic reactions and skin irritation. Thus, the aim of this work was the preparation and char-acterization of polymeric and solid lipid nanoparticles to act carriers of benzophenone-3 (BZ3),aiming to improve the safety of sunscreen products by increasing the sun protection factor (SPF),decreasing BZ3 skin penetration and decreasing BZ3 concentration in sunscreen formulation. BZ3was encapsulated in poly(-caprolactone) (PCL) nanoparticles by the nanoprecipitation method andin solid lipid nanoparticles (SLN) by the hot high pressure homogenization method. The particleswere stable for 40 days. The BZ3 encapsulated in PCL nanoparticles was released faster than BZ3encapsulated in SLN. The sun protection factor increased when BZ3 was encapsulated in bothnanostructures. However, BZ3 encapsulated in PCL nanoparticles decreased its skin permeationmore than SLN-BZ3. Furthermore, BZ3 encapsulated in SLN did not exhibit cytotoxic or phototoxiceffects in human keratinocytes (HaCaT cells) and BABL/c 3T3 broblasts, whereas PCL nanoparti-cles with BZ3 showed phototoxic potential in HaCaT cells. Nevertheless, BZ3 free and encapsulatedin PCL nanoparticles or in SLN did not show allergic reactions in mice. Our results suggest thatthese nanostructures are interesting carriers for sunscreen.Keywords: Sunscreen, Benzophenone-3, Solid Lipid Nanoparticles, PLC Nanoparticles.1. INTRODUCTIONPhotoprotection is an essential topic in daily life due to theworldwide decrease of the ozone layer and the consequentincrease in skin cancer, erythema and photoageing.1Sun-screens were initially developed to protect against UVBradiation. In the 1990s, compounds with UVA-absorbingability also became available. UVB (mid wavelength, theso-called erythemal) radiation (290 to 320 nm) can causeskin burn and skin cancer and the UVA (long wavelengthUV, the so-called black light) (320 to 400 nm) radiationcan cause the skin to lose elasticity and interferes with theimmune system.2Thus, sunscreens with broad spectrumcoverage are necessary. Due to the long-term and frequentuse of sunscreen, particular attention has been given totheir efciency and, espectally, their safety.Author to whom correspondence should be addressed.Benzophenone-3 (BZ3) (Fig. 1) is used in sunscreen inconcentrations up to 10% alone or in combination withother UV lters. However, it has been shown that aftertopical application, BZ3 can pass through the skin andreach the circulatory system. Janjua et al.3studied the skinpenetration of three sunscreens in humans (BZ3, octyl-methoxycinnamate and 3-(4-methylbenzylidene) camphor).In this study all these sunscreens reached the systemic cir-culation, being detected in plasma and urine within twohours after the application of sunscreen containing 10%of them. This characteristic may leave the skin unpro-tected as well as increase the risk of phototoxic andphotoallergic reactions.4, 5Berne and Ross6veried that7.9% of 355 patients exhibited 42 types of allergic reac-tions when using sunscreens. Most patients were aller-gic to benzophenone-3. Furthermore, BZ3 was detectedin urine and breast milk after its topical application.3, 91880 J. Nanosci. Nanotechnol. 2011, Vol. 11, No. 3 1533-4880/2011/11/1880/007 doi:10.1166/jnn.2011.3135Delivered by Ingenta to:UNIVERSIDADE SAO PAULO IFIP : 143.107.228.70Thu, 31 Mar 2011 12:09:18RESEARCHARTICLEMarcato et al. Nanostructured Polymer and Lipid Carriers for Sunscreen. Biological Effects and Skin PermeationCH3OOH OFig. 1. Benzophenone-3 structure.Therefore, sunscreens with reduced concentrations ofchemical UV lter, but maintaining their effectivenesshave been studied.7Another investigated strategy is pen-etration reduction of the chemical UV lter. For this, theviscosity of the sunscreen formulation can be increasedor the UV lter can be incorporated in a micro ornanostructure.5Several nanostructures have been studied,such as cyclodextrins, polymeric nanoparticles and solidlipid nanoparticles.8BZ3 was encapsulated in modied poly(vinyl alco-hol) (PVA) with fatty acids (FAs). These nanoparticlesdecreased percutaneous absorption of BZ3, indicatingthem to be good sunscreen carriers.10Other nanostructures,such as solid lipid nanoparticles (SLN) were investigated.Wissing and Mller10studied the encapsulation of BZ3 innanostructured lipid carriers (NLC) prepared with tripalmi-tate and Miglyol 812. A synergistic effect in the incorpo-ration of sunscreen in NCL was observed, verifying thatthe amount of molecular sunscreen could be decreasedup to 50% while maintaining UV protection efcacy.Similar results were obtained with SLN and NCL usingcarnauba wax.11, 12Furthermore, the photodegradation ofa sunscreen (trans-2-ethylhexyl-p-methoxycinnamate) wasreduced by its encapsulation in nanostructures (ethylcel-lulose and poly(D,L-lactide-co-glycolide) nanoparticles asdescribed by Perugini et al.13Similar results were ver-ied by Paese et al.14when BZ3 was encapsulated inPCL nanocapsules. Thus, nanoparticles associated withBZ3 can increase the protection factor and decrease theagents concentration in sunscreen formulations, therebyincreasing the safety of these products. Therefore, the aimof this work was to prepare and to characterize poly-meric nanoparticles with polymer PCL and SLN with lipidcetyl palmitate containing benzophenone-3. The in vitrocytotoxicity and phototoxicity and in vivo allergenicityof these particles was evaluated. High encapsulation ef-ciency (99%) of BZ3 in PCL nanoparticles and in SLNwas obtained. The sun protection factor was increasedwhen BZ3 was nanoencapsulated in both nanostructuresand the skin permeation was reduced, demonstrating thatit is possible to maintain BZ3 on the surface of the skinfor a longer time with larger SPF. The particles alone orwith BZ3 did not exhibited allergic reactions in mice andSLN did not show any toxic effects up to the highest testedconcentrations in cell types (3T3 and HaCaT). The resultsdemonstrated that these nanoparticles are interesting carri-ers of sunscreen to produce a safer sunscreen.2. METHODS2.1. Solid Lipid Nanoparticles (SLN) PreparationSLN were produced by hot high pressure homo-genization.15The solid lipid (cetyl palmitate) used waskindly donated by Croda (Brazil). The solid lipid, with orwithout BZ3, was heated to around 10 C above its meltingpoint. Afterwards, the mixture was added to a hot aque-ous solution of Pluronic F68 (65 C) under high agitationin an Ultra-turraxT18 to form a pre-emulsion. The pre-emulsion was homogenized using a Panda 2k (Niro Soavi,Italy), applying two homogenization cycles at 600 bar andcooled to form the SLNs.2.2. Polymeric Nanoparticles PreparationPolymeric nanoparticles were prepared by the nanopre-cipitation method with poly(a-caprolactone) (PCL) (MW65000 g/mol, Sigma) as covering material.16The polymer(125 mg) and BZ3 were rst dissolved in acetone (25 mL).The resulting organic solution was poured into an aqueoussolution of Tween 80 (0.3%) under high agitation in anUltra-turraxT18. Then, the acetone was removed underreduced pressure at 30 C and PCL nanoparticles wereobtained.2.3. Atomic Force Microscopy (AFM)The morphology of the SLN and PCL nanoparticles wasevaluated by Atomic Force Microscopy (AFM) (SPM-9600, Shimadzu, Kyoto, Japan). The samples were pre-pared by depositing dilute particle dispersions on freshlycleaved mica plates, followed by drying overnight at 25 C.The images were collected in a dynamic mode, usingcommercial silicon cantilevers. The resonance frequencieswere 210230 kHz.2.4. Particle Size and Zeta PotentialThe average particle size (number average size) and sizedistribution were measured by photon correlation spec-troscopy (PCS) (Nano ZS Zetasizer, Malvern InstrumentsCorp, UK) at 25 C in polystyrene cuvettes with a pathlength of 10 mm. The zeta potential was measured in cap-illary cells with path lengths of 10 mm, using the Nano ZSZetasizer. Measurements were performed in distilled wateradjusted with a solution of 0.1 mM sodium chloride.2.5. Encapsulation EfciencyThe encapsulation efciency of BZ3 encapsulated inSLN and in PCL nanoparticles was determined byHigh Performance Liquid Chromatography (HPLC) on aJ. Nanosci. Nanotechnol. 11, 18801886, 2011 1881Delivered by Ingenta to:UNIVERSIDADE SAO PAULO IFIP : 143.107.228.70Thu, 31 Mar 2011 12:09:18RESEARCHARTICLENanostructured Polymer and Lipid Carriers for Sunscreen. Biological Effects and Skin Permeation Marcato et al.Shimadzu (CBM 20A) system with a multi-wavelengthUV-VIS detector Shimadzu (SPD-20AV).17The analysiswas carried out at 25 C on a C18 column (4.6 mm250 mm i.d., Varian) with a similar pre-column. Themobile phase consisted of methanolwater (80:20), lteredthrough a 0.45 jm membrane lter (Millipore, USA) inthe isocratic ow. The mobile phase was continuouslydegassed before and during the use. The ow rate was1.0 ml/min and the detector was set at 380 nm. For theencapsulation efciency determination, 500 mL of parti-cle dispersion were ltered in a Microcon centrifugal l-ter device containing ultraltration membranes (MWCO100,000, Millipore). The ltrate was assayed to determinethe concentration of the non-encapsulated drug. Encapsu-lation efciency (%) and loading capacity (LC%) werecalculated using the following equations:18(%) =(mass of BZ3 encapsulated/mass of BZ3 total) 100(LC%) =(mass of BZ3 in the nanoparticles/mass of particles recovered) 1002.6. In Vitro ReleaseBZ3 (100 jg/mL) free or nanoencapsulated in SLN orin PCL nanoparticles was added in falcon tubes withphosphate buffer (PBS, 50 mM, pH 7.4) with 5% ofTween 80. The tubes were maintained at 36.5 0.5 Cand 120 rpm. At predetermined time intervals, samples(0.5 mL) were collected and analyzed through HPLCaccording to the method described above. The volumeremoved was replaced with an equal volume of phosphatebuffer with 5% of Tween 80. All the in vitro release wascarried out at sink conditions.19, 202.7. Cytotoxicity and Phototoxicity AssayThe BALB/c 3T3 mouse embryo broblast cell line,obtained from the National Institutes of Health (Baltimore,MD) and HaCaT human keratinocyte cell line, kindly pro-vided by Dr. Liudmila Kodach (Academic Medical Center,Amsterdam University), were maintained in Dulbeccosmodied Eagles medium (DMEM) supplemented with100 U/mL penicillin, 100 jg/mL streptomycin and 10%fetal bovine serum. Cells were grown in a monolayer at37 C in a humidied atmosphere containing 5% CO2.21, 22Cells (7 104HaCaT cells/mL) were seeded in 96-wellplates and incubated for 24 h, when they formed half-conuent monolayers. For each cell type, two plates wereprepared: one for determination of cytotoxicity (UVA),and the other for determination of phototoxicity (+UVA).The cells were washed twice with phosphate bufferedsaline (PBS), and treated with different particle disper-sion concentrations for 1 h. One plate was then exposedto a dose of 5 J/cm2UVA (+UVA), at room temperaturefor 50 minutes, whereas the other plate was kept in thedark (UVA), under the same conditions. The radiationsource was a 100 W Bellarium S UVA lamp (Wolff Sys-tem Technology Corporation; Kennesaw, GA) that has themajority of its energy output between 300 and 420 nm.The emitted dose was calculated using a UVA radiome-ter photodetector (Cole-Parmer Instrument Co.). At theend of the irradiation period, cells were washed twicewith PBS and incubated in culture medium for 22 h. Cellviability was then determined by the neutral red uptake(NRU) as described by Borenfreund and Puerner.23Controlcells were treated with PBS only. Thiourea and hemato-porphyrin IX were used as negative and positive controls,respectively. Neither cytotoxicity nor phototoxicity wasobserved for thiourea, whereas hematoporphyrin presentedonly phototoxicity, with an IC50 value of 1.16 jmol L1.For prediction of phototoxic potential the concentra-tion response curves concurrently obtained in the presence(+UVA) and in the absence (UVA) of UVA irradiationwere compared, usually at the IC50, as described by Spiel-mann et al.242.8. Preparation of Gels Containing Free andEncapsulated BZ3 in NanostructuresCarbopol 940gels containing free or encapsulated BZ3 inSLN or PCL nanoparticles were developed. At rst, Car-bopol gel (0.1%) with propyleneglycol (5%) was prepared.Free BZ3 (1.6% w/w) or encapsulated BZ3 was then addedto the Carbopol gel.2.9. Sun Protection Factor (SPF)Gels with free or nanoencapsulated sunscreen were dis-persed in ethanol (25 mL) to obtain a benzphenone-3concentration of 23 jg/mL. Then, the dispersion washomogenized in an ultrasonic bath for 5 minutes. After,the dispersion was analyzed by UV-Vis spectroscopy(Agilent 8453). The SPF was calculated by the followingequation25:SPF =FC290

320EE(\) I(\) abs(\)where, FC = correction factor (=10), EE(\) =erythematogenic effect of radiation at the given \, I(\) =intensity of solar light at the given \, abs(\) =absorbanceof sample at the specied \. The EE(\) I(\) aretabulated values for each wavelength (Table I).25, 262.10. Human Skin Permeation ExperimentsThe studies of skin permeation were made over 24 h ina Franz diffusion cell with human skin, donated fromplastic surgery. Prior to use, the skin specimens were1882 J. Nanosci. Nanotechnol. 11, 18801886, 2011Delivered by Ingenta to:UNIVERSIDADE SAO PAULO IFIP : 143.107.228.70Thu, 31 Mar 2011 12:09:18RESEARCHARTICLEMarcato et al. Nanostructured Polymer and Lipid Carriers for Sunscreen. Biological Effects and Skin PermeationTable I. Values of EE (\) I(\) in function of \ (nm).\ (nm) EE(\) I(\)290 0.0150295 0.0817300 0.2874305 0.3278310 0.1864315 0.0839320 0.0180defrosted and the full-thickness human skin or epidermiswere then placed in Franz-type diffusion cells. This cellhas a nominal area of 2.52 cm2and a receptor compart-ment. In this last compartment 10 mL of phosphate buffersolution (pH 7.4) containing 5% of Tween 80 was added.Gels with BZ3 (0.5 g of gel) of each formulation; BZ3-loaded SLN and BZ3-loaded in PCL nanoparticles, wereused. The cells were kept at 32 C for 24 h. After this, theBZ3 amounts were measured in epidermis/dermis by thetapping stripping method, as described in the literature,16and in the receptor uid by HPLC.2.11. Allergenicity AssayIn this study BALB/c mice with ages between 8 and12 weeks were used and mouse ear swelling was evaluated(n =4).27, 14The mice were sensitized on 3 alternating days(D-0, D-2 and D-4) by topical application of 100 jL offormulation (Carbopol gel with free or encapsulated BZ3)on dorsal skin. The animals were challenged 5 days afterthe last sensitization dose with 25 jL of formulation oneach dorsum ear. Oxazolone (0.5%) was used as positivecontrol. The ear thickness (ear swelling) was measuredwith a micrometer (Mitutoyo, Tokyo, Japan) after 24 and48 h of application. An increase in ear thickness of over10% was considered a positive result. Percent ear swellingwas calculated by the following equation:% increase in ear thickness=|(post-treatment ear thicknesspre-treatment ear thickness)/pre-treatment ear thickness]1003. RESULTSTable II shows the results of SLN and PCL nanoparti-cles with and without BZ3. The preparation method ofTable II. Results of size, polydispersity index, zeta potential, encapsulation efciency and loading capacity of PCL and solid lipid nanoparticles.Size Polydispersity Zeta Encapsulation LoadingNanostructure (nm) index potential (mV) efciency (%) capacity (%)PCL nanoparticles without BZ3 117.5 0.149 9.8 PCL nanoparticles with BZ3 137.5 0.123 9.5 99.2 12.0SLN without BZ3 133.0 0.137 29.7 SLN par with BZ3 147 0.164 33.2 99.9 16.70 10 20 30 40 500255075100125150Time (days)Size (nm)0.000.080.160.240.32Polydispersity indexFig. 2. Comparative graph of particles stability at 4 C: PCLnanoparticles-BZ3 (white bars and -- line) and SLN (striped bars and-- lines).13.20[nm]0.001.00 um 2.502.50 um(B)6.13[nm]0.00500.00 nm1.501.50 um(A)Fig. 3. (A) AFM topographic image of PCL nanoparticles (scan area1.51.5 jm2), (B) AFM topographic image of SLN (scan area 2.50 2.50 jm2).both particles (SLN and PCL nanoparticles) showed to bereproducible. Furthermore, the polydispersity index of allparticles was smaller than 0.2 indicating low polydisper-sity. The zeta potential of all the preparations was negativebut SLN exhibited a larger supercial charge (33.2 mV)than PCL nanoparticles (9.5 mV). This difference canbe related with the surfactant type.28However, signi-cant changes in the size and zeta potential of both nano-structures over a period of 40 days at 4 C were notobserved (Fig. 2). This result demonstrates that both theseparticles are good carrier systems.The AFM image of the PCL nanoparticles (Fig. 3(A))and SLN (Fig. 3(B)) showed spherical particles.J. Nanosci. Nanotechnol. 11, 18801886, 2011 1883Delivered by Ingenta to:UNIVERSIDADE SAO PAULO IFIP : 143.107.228.70Thu, 31 Mar 2011 12:09:18RESEARCHARTICLENanostructured Polymer and Lipid Carriers for Sunscreen. Biological Effects and Skin Permeation Marcato et al.Fig. 4. Release prole of: free BZ3 (), PCL-BZ3 (....) and SLN-BZ3 (--).The average size of PCL nanoparticles and SLN,calculated by the AFM software, was 100 nm and 120 nm.These values were similar to the sizes calculated by photoncorrelation spectroscopy (PCS). Some small particles can(a) (b)(c) (d)Fig. 5. Cytotoxicity (gray bars) and phototoxicity (white bars) of: (A) 3T3 cells treated with PCL nanoparticles, (B) HaCaT cells treated with PCLnanoparticles, (C) 3T3 cells treated with SLN, (D) HaCaT cells treated with SLN.be seen in the AFM images, demonstrating that the methodproduced particles with different sizes. However, in thePCS analysis only one peak was observed demonstrating amonomodal, but wide, size distribution. The encapsulationefciency was high (around 99%) independent of the nano-structure. The high encapsulation efciency is due to thehigh afnity between BZ3 and the covering material dueto the hydrophobicity characteristic of these materials (UVlter, lipid and polymer). The encapsulation efciency wasnot signicantly altered at least 40 days after the prepa-ration. This result shows that BZ3 was not expulsed fromthe particles during this period.3.1. In Vitro ReleaseIn the in vitro release study it was observed that BZ3encapsulated in PCL nanoparticles was released slightlyfaster than BZ3 encapsulated in SLN (Fig. 4). This differ-ence can be due to differences in nanoparticle crystallinity.Low crystallinity in the particles can lead to an enhancedrate of release due, probably, to less resistance to drugdiffusion from the nanoparticles.29, 30Furthermore, bothrelease proles (BZ3 in PCL nanoparticles and in SLN)1884 J. Nanosci. Nanotechnol. 11, 18801886, 2011Delivered by Ingenta to:UNIVERSIDADE SAO PAULO IFIP : 143.107.228.70Thu, 31 Mar 2011 12:09:18RESEARCHARTICLEMarcato et al. Nanostructured Polymer and Lipid Carriers for Sunscreen. Biological Effects and Skin Permeationwere different from BZ3 dissolution, indicating that BZ3is partially encapsulated in the nanoparticles.3.2. Phototoxicity AssayThe photo-irritancy factor (PIF) can be calculated only ifthe concentration-response curves obtained in the presenceand the absence of UV-light drop below 50% of the con-trols, because only in these cases two IC50 values (UVAand +UVA) can be determined with a cut-off value ofPIF =5 for phototoxic potential.24PCL nanoparticles did not show phototoxic potential in3T3 and HaCaT cells, as predicted by the PIF values of 1.0and 1.3, respectively (Figs. 5(A, B)). In 3T3 cells, no pho-totoxic potential was also predicted for BZ3 encapsulatedin PCL nanoparticles, as a PIF value of 4.0 was obtained(Fig. 5(A)). In contrast, in HaCaT cells an IC50 value forencapsulated BZ3 was only obtained in the presence ofUVA (410 jmol L1) (Fig. 5(B)). When a chemical is onlycytotoxic +UVA and is not cytotoxic when tested UVA,the PIF cannot be calculated, although this is a result indi-cating phototoxic potential. In these cases, the highest testconcentration [Cmax(UVA)] is used for calculation of the>PIF, and a >PIF value >1 predicts phototoxic poten-tial. This value is calculated by the following equation:PIF = Cmax(UVA)IC50(+UVA)Therefore, BZ3 encapsulated in PCL nanoparticles pre-sented a phototoxic potential in HaCaT cells, as predictedby a >PIF value of 1.4.In the case of BZ3 encapsulated in SLN, both IC50(UVA) and IC50 (+UVA) could not be calculated dueto the fact that the test samples did not show any cyto-toxicity up to the highest test concentrations in both 3T3and HaCaT cells (Figs. 5(C, D)). This result shows thatSLN, with or without BZ3, can be used in cosmetic for-mulations without phototoxic effects when exposed to UVradiation and without cytotoxicity, as indicated by testswith the BALB/c 3T3 broblast and HaCaT human ker-atinocyte cell lines.3.3. Skin PermeationIn the skin permeation study it was observed that BZ3encapsulation decreased its penetration into the skin. PCLnanoparticles decreased BZ3 skin permeation by 70% inthe epidermis and dermis and 80% in the receptor uid.However, the skin permeation of SLN-BZ3 was not sig-nicantly different from free BZ3 (Fig. 6). This differencecan be due to particle exibility. Borgia et al.31veriedthat SLN increased the skin permeation of nile red dyemore than CLN. Bhalekar et al.32also observed that SLNenhanced skin permeation of miconazole nitrate. However,the sun protection factor (SPF) was higher when BZ3was encapsulated in SLN than when encapsulated in PCLEpidermis Dermis Receptor fluid02468101214161820Retained amount (g/cm2)Free BZ3SLN-BZ3PCL-BZ3Fig. 6. Skin permeation of BZ3 free (squared bars) and encapsulated inSLN (white bars) and in PCL nanoparticles (striped bars).nanoparticles. The SPF of no-encapsulated BZ3 was 16while the SPF of BZ3 encapsulated in PCL nanoparticlesor in SLN were 18 and 21, respectively. This synergisticeffect is due to particle crystallinity. Particles with highcrystallinity are able to scatter/reect more incoming UVradiation increasing the SPF more.12The degree of crys-tallinity, measured by DSC, of SLN was 78.4% while forPCL nanoparticles it was 71.2%. This difference in thedegree of crystallinity can explain the difference in theSPF. This result is in agreement with the release proleof encapsulated BZ3 as discussed above. Therefore, it ispossible to use less BZ3 while maintaining the same SPF.In this case, a safer sunscreen formulation can be obtained.3.4. Allergenicity AssaysIn the study of allergenicity using mouse ear swelling itwas observed that after three topical applications on three Carbopol gel BZ3 SLN-BZ3 PCL-BZ3 Oxazole010203040506070% of swelling ear24 h48 hFig. 7. Increase in ear thickness at 24 h (white bars) and 48 h (stripedbars) post application of formulation (Carbopol Gel, free BZ3 in Car-pobol gel (Free BZ3), BZ3 encapsulated in PCL nanoparticles or SLN,both in Carbopol gel).J. Nanosci. Nanotechnol. 11, 18801886, 2011 1885Delivered by Ingenta to:UNIVERSIDADE SAO PAULO IFIP : 143.107.228.70Thu, 31 Mar 2011 12:09:18RESEARCHARTICLENanostructured Polymer and Lipid Carriers for Sunscreen. Biological Effects and Skin Permeation Marcato et al.alternate days, all formulations (Carbopol gel with BZ3free or encapsulated in PCL nanoparticles or in SLN)exhibited less than 10% of ear swelling after 24 hours and48 hours, while oxazolone (as positive control) exhibited65.7% increased swelling after 24 hours and 46.1% after48 hours (Fig. 7). This result demonstrated that none ofthe formulations induced cutaneous sensitivity, indicatingthat they can be used in cosmetic products. Similar resultswere described in the literature with PCL nanocapsuleswith BZ3.144. CONCLUSIONSLN and PCL nanoparticles were homogeneous and spher-ical as observed by atomic force microscopy and scan-ning electron microscopy. In 40 days, the SLN and PCLnanoparticle sizes as well as the encapsulation efciencychanged only slightly, showing the relative long-term sta-bility of these particles. The encapsulation efciency ofBZ3 in the nanoparticles was high (around 99%) inde-pendent of the particle type. BZ3 encapsulation in thePCL nanostructure decreased its skin permeation morethan SLN-BZ3 and both nanostructures increased the sunprotection factor. Therefore, BZ3 will be on the surface ofthe skin for a longer time, where it is intended to act withlarger SPF.BZ3 encapsulated in PCL nanoparticles showed photo-toxic potential only in HaCaT cells while BZ3 encapsu-lated in SLN did not exhibit any toxic effects up to thehighest test concentrations in both cell types (3T3 andHaCaT). Furthermore, the BZ3, free or encapsulated inPCL nanoparticles or in SLN, did not show allergic reac-tions in mice.Our results suggest that these nanoparticles are inter-esting carriers of sunscreen, as demonstrated by the goodstability, lower toxicity, lack of phototoxic effect in cellsand no allergic reaction in mice. In addition these parti-cles, due to their crystallinity, can scatter/reect incomingUV radiation, increasing the SPF.Acknowledgments: Support from CNPq, FAPESP, theBrazilian Nanobiotechnology Network (MCT/CNPq) andthe Brazilian Nanocosmetic Network (MCT/CNPq) areacknowledged.References and Notes1. J. J. Finlay-Jones and P. H. Hart, Mutat. Res.-Fundam. Mol. Mech.Mutagen. 422, 155 (1998).2. P. J. Matts, Dermatol. Clin. 24, 1 (2006).3. N. R. Janjua, B. Kongshoj, A. M. Andersson, and H. C. Wulf, J. Eur.Acad. Dermatol. Venereol. 22, 456 (2008).4. S. Simeoni, S. Scalia, R. Tursilli, and H. Benson, J. Incl. Phenom.Macrocycl. Chem. 54, 275 (2006).5. S. A. Wissing and R. H. Mller, Int. J. Pharm. 242, 373 (2002).6. B. Berne and A. Ros, Contact Dermatitis 38, 61 (1998).7. M. V. R. Velasco, F. D. Sarruf, I. M. N. Salgado-Santos, C. A.Haroutiounian-Filho, T. M. Kaneko, and A. R. Baby, Int. J. Pharm.363, 50 (2008).8. P. Marcato and N. Durn, J. Nanosci. Nanotechnol. 8, 2216 (2008).9. T. Felix, B. J. 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