Inorganic PolysilsesquioxaneâSilica Particles as

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New Hybrid Organic/Inorganic PolysilsesquioxaneSilica Particles as Sunscreens Stephanie H Tolbert, Peter D McFadden, and Douglas A. Loy ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b10472 • Publication Date (Web): 05 Jan 2016 Downloaded from http://pubs.acs.org on January 24, 2016

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ACS Applied Materials & Interfaces

New Hybrid Organic/Inorganic Polysilsesquioxane-Silica Particles as Sunscreens Stephanie H. Tolbert, Peter D. McFadden, and Douglas A. Loy Departments of Materials Science and Engineering and Chemistry and Biochemistry The University of Arizona, Tucson, AZ 85721

Abstract: Effectiveness of organic sunscreens is limited by phototoxicity and degradation. Both of which can be significantly reduced by encapsulation in hollow particles or covalent incorporation into the solid structure of particles, but direct comparisons of the two methods have not been reported. In this study, physical encapsulation and covalent incorporation of sunscreens were compared with 1mol% salicylate and curcumeroid sunscreens. 2-Ethylhexyl salicylate was physically encapsulated in hollow silica nanoparticles prepared by oil-in-water (O/W) microemulsion polymerizations (E-Sal). Some of these particles were coated with an additional shell or cap of silica to reduce leaking of sunscreen (Cap E-Sal). Covalent incorporation involved copolymerizing tetraethoxysilane (TEOS) with 0.2 mol% of new salicylate and curcumin sunscreen monomers with triethoxsilyl groups. Particles were prepared with the salicylate attached to the silica matrix through single silsesquioxane groups (pendant; P-Sal) and two silsesquioxane groups (bridged; B-Sal). Particles based on a new curcuminoid bridged monomer were also prepared (B-Curc). Sunscreen leaching, photodegradation and sunscreen performance was determined for the E-Sal, Cap E-Sal, P-Sal, B-Sal, and B-Curc particles. Covalent attachment, particularly with bridged sunscreen monomers, reduced leaching and photodegradation over physical encapsulation, even with capping. Keywords: Sunscreen, hybrid organic inorganic particles, pendant versus bridged polysilsesquioxanes.

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1. Introduction Sunscreens are commonly used to mitigate sunburn,1-3 accelerated skin ageing,4-7 and skin cancer8, 9 from UV exposure. Organic sunscreens, such as salicylates, absorb UV radiation when applied directly to skin, but have limited lifetime due to photodegradation1 and may be photo-toxic10-12 or photo-allergenic.13, 14 The facility with which topical sunscreens absorb through skin15-17 also raises concerns over their estrogenicity.16, 18, 19 One method to improve stability and reduce transdermal transfer is to encapsulate sunscreens in matrices, such as gelatin,20 polymers,21-23 lipids,24 cyclodextrins,25-27 silica (SiO2),28-31 or alumina (Al2O3)30, to be dispersed in lotions or cosmetics. Sunscreens can be physically encapsulated in30-32 or covalently attached to33-35 silica particles. Hydrophobic sunscreens as the core of hollow silica particles can be prepared by oil-in-water (O/W) microemulsion polymerizations (Scheme 1).30 Alternatively, water soluble sunscreens are encapsulated by a reverse microemulsion process (water-in-oil, or W/O).36, 37 In both cases, inherent porosity or mechanical damage allow sunscreen to leak out.32 Covalent incorporation has increasingly superseded physical encapsulation as a means to minimize sunscreen leaching.33-35 Hybrid organic-inorganic particles of this nature are prepared via sol-gel polymerization,38 most commonly by a modified Stöber procedure.39 This method involves co-condensation of a silyl-modified organic monomer (organotrialkoxysilane) with a silica monomer such as tetraethoxysilane (TEOS) in ammoniacal ethanol to afford a dilute suspension of organically modified silica particles. Sunscreen is less likely to leach from the silica matrix as it is attached to the silica

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particles via covalent bonds and, if in the interior of the particle, physical encapsulation, as well (Scheme 2). In addition, immobilization in the silica matrix decreases diffusion of reactive oxygen species and increases energy dissipation, thereby improving UV stability of organic sunscreens.40 Previously, we compared the retention and photostability of physically encapsulated and covalently incorporated 1 mol% fluorescent dansyl amide dyes in silica particles.41 Covalently attached dansyl dyes showed only minimal leaching and significantly increased lifetimes over dansyl amides physically encapsulated in hollow silica particles through O/W emulsion polymerizations. We also determined that dansyl amide dye attached through two silsesquioxane groups showed less leaching and greater stability than a dansyl amide dye attached through a single silsesquioxane group. The objective of the present study is to determine if the results with the dansyl amide dyes can be expanded to hybrid sunscreen silica particles as well. Here, leaching, photochemical stability, and sunscreen performance of salicylate and curcumin sunscreens encapsulated in silica particles are evaluated as a function of physical encapsulation (E-Sal), physical encapsulation with capping (Cap-E-Sal), covalent attachment of sunscreens to the silica particles as pendant (P-Sal) and as bridged monomers (B-Sal and B-Curc). Salicylates are UVB absorbing sunscreens that can cause allergic reactions and skin inflammation.14, 42, 43 2-Ethylhexyl salicylate was physically encapsulated in hollow silica particles (E-Sal) by O/W microemulsion polymerization of TEOS. Some of the hollow salicylate particles were reinforced with an additional layer of silica (Cap-E-Sal) as an alternative approach to reducing sunscreen leakage (Scheme 3) that has been successfully applied to reducing fluorescent dye44 and drug45 leakage, but

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has not been reported for sunscreens. Covalent salicylate incorporation was achieved by Stöber polymerization of TEOS with salicylate monomer 2 with a single triethoxysilyl group (P-Sal, Scheme 2A) or 4 with two triethoxysilyl groups (B-Sal, Scheme 2B). Lastly, particles based on a second bridged sunscreen were prepared from curcuminoid 6 with three triethoxysilyl groups (B-Curc, Scheme 2B). Curcumin has been proposed as a UVA/UVB sunscreen due to significant reduction in thymine dimerization levels seen after topical application in presence of UVB radiation.46 To date, no curcuminoid-bridged silsesquioxane has been used as an ingredient for sunscreens.

2. Experimental Procedures 2.1 Materials All reagents were used as received without further purification unless otherwise stated. Sunscreen monomers were stored by freezing in benzene solutions. Salicylate monomers. Salicylic acid (99%), 2-hydroxyterephthalic acid (97%), 3aminopropyltriethoxysilane (APTES, 99%), N,N-dimethylformamide (DMF, anhydrous), triethylamine (Et3N, 99.5%), and benzene (99.8%), were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Thionyl chloride (SOCl2, 98%) was purchased from Pfaltz & Bauer (Waterbury, CT, USA). 3-Aminopropyltriethoxysilane was distilled from calcium hydride before use.

Curcuminoid Monomer. Curcumin (70%) was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), and potassium carbonate (K2CO3, anhydrous, 99%) was

purchased

from

EMD

Chemicals,

4

Inc.

(San


Diego,

CA,

USA).

3-

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Isocyanatopropyltriethoxysilane (IPTES, 95%) was purchased from Gelest, Inc. (Morrisville, PA, USA) and distilled from calcium hydride before use.

Silica Particles. Tetraethoxysilane (TEOS, 98%), EtOH (anhydrous 99.5%), and cetyltrimethylammonium bromide (CTAB, ≥ 99%), were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Ammonium hydroxide (NH4OH, 28-30%) was purchased from EMD Chemicals, Inc. (San Diego, CA, USA). 2-Ethylhexyl salicylate (>98 %) was purchased from TCI America (Portland, OR, USA). TEOS was distilled from calcium hydride before use, and ethanol was refluxed over and distilled from magnesium turnings before use. NH4OH (14.845 M) was titrated with hydrochloric acid (HCl), which was previously standardized with sodium carbonate (Na2CO3).

2.2 Materials Characterization Column chromatography was performed on silica gel (60 Å, 70-230 mesh, 63200 µm) purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). For silsesquioxane monomers, silica gel was treated before using with a 1:150 TEOS:hexanes solution (v/v) followed with hexanes to remove residual TEOS. Then column was flushed with the eluent stated in the detailed experimental procedures below. Reaction progress was monitored by thin layer chromatography on Baker-flex® Precoated Flexible Sheets (IB-F). Melting points were measured on a SRS EZ-Melt Automated Melting Point Apparatus and are uncorrected. Infrared Spectroscopy (IR) was performed on a Thermo Scientific NicoletTM iSTM5 FT-IR Spectrometer. Solid samples were prepared by grinding with KBr into a pellet,

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while liquid samples were sandwiched between two NaCl plates. 1H and Magnetic Resonance Spectroscopy (1H NMR and

13

13

C Nuclear

C NMR, respectively) were

performed on a Bruker Avance-III 400 MHz Spectrometer at 298 K. Chemical shifts are reported in parts per million (ppm), by reference to the hydrogen residues of deuterated solvents as internal standard. CDCl3: δ = 7.26 ppm (1H NMR) and δ = 77.16 ppm (13C NMR) Signals are described as s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), sex. (sextet), sep. (septet), dd (doublet of doublets), dt (doublet of triplets), and m (multiplet). All coupling constants (J) are in hertz (Hz). High resolution and low resolution mass spectrometry (MS) were performed on a Bruker 9.4 T Apex-Qh hybrid Fourier transfer ion-cyclotron resonance (FT-ICR) instrument equipped with a dual ESI/MALDI source. UV-Vis absorbance spectroscopy was obtained on a Shimadzu UV-Probe PC2501 UV-Vis Double Beam Spectrophotometer. Fluorescence was measured at 445 nm for the salicylates and 500 nm for on a PTI Quanta Master 40 Steady State Spectrofluorimeter. Excitation wavelengths of 324 nm (salicylate) and 420 nm (curcuminoid) were used. Dynamic Light Scattering (DLS) of ethanolic suspensions of particles (3 mg/mL) was performed using a Malvern Zetasizer Nano ZS90 at 20 °C and 173°. Scanning Electron Microscopy (SEM) was performed on gold-sputter coated samples with an FEI Inspec-S electron microscope with an accelerating voltage of 30 keV. Ethanol suspensions of particles (3 mg/mL) were dropcast onto a clean silicon wafer and evaporated to dryness before gold coating and analysis. Transmission Electron Microscopy (TEM) of particle samples dispersed on a grid were analyzed with a Hitachi

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H8100 electron microscope with an accelerating voltage of 200kV and a high brightness LaB6 electron source. Nitrogen adsorption-desorption isotherms of dry particles were measured with Nitrogen Adsorption Porosimetry on a Quantachome Autosorb-1 Porosimeter at 77.35 K. Particle samples were degassed at 100 °C for 24 h under vacuum. Specific surface areas were determined by the Multipoint Brunauer-Emmett-Teller (BET) method.47 Pore size distributions were determined by the Barrett-Joyner-Halenda (BJH) model to the desorption isotherm.48

2.3 Monomer Synthesis 2-Hydroxybenzoyl chloride (1) (Scheme 4). Synthesis was modified from Agwada, et. al.49 A flame-dried, single-neck round-bottom flask fitted with a reflux condenser and magnetic stir bar was charged with salicylic acid (3.41 g, 22.73 mmol), SOCl2 (8.62 mL) and DMF (8 drops) under argon and the reaction mixture was stirred at reflux (90 °C) for 48 h. The acid dissolved to a clear pale yellow solution upon addition of DMF. After cooling to room temperature, SOCl2 was removed by alternately adding toluene and removing volatiles in vacuo 4x to afford a slightly translucent pale yellow oil (3.54 g, 99%), which was used in the synthesis of 2 without further purification.

2-Hydroxy-N-(3-(triethoxysilyl)propyl)benzamide (2) (Scheme 4).

Synthesis was

modified from Gounko, et. al.50 A single-neck round-bottom flask fitted with a magnetic stir bar was charged with APTES (5.33 mL, 22.6 mmol) and Et3N (3.64 mL, 26.14 mmol) in benzene (25 mL). To this, a separate solution of 1 (3.54 g, 22.63 mmol) in

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benzene (20 mL) was added dropwise at 0 °C to minimize the evolution of a white fog, using excess benzene (5 mL) to facilitate transfer. The reaction mixture was then stirred at 24 °C until consumption of starting material (48h), monitoring by TLC (5:1 dichloromethane:ethyl acetate). Solid white triethylammonium chloride was removed via vacuum filtration, and volatiles removed in vacuo. The clear, pale yellow oil was purified by running through a silica plug (5:1 dichloromethane:ethyl acetate) and concentrating in vacuo to afford a clear, colorless oil (7.10 g, 92%). 1H NMR (400 MHz, CDCl3, 25°C): δ 0.70 (m, 2H, NHCH2CH2CH2Si), 1.22 (t, J = 7.0 Hz, 9H, OCH2CH3), 1.76 (m, 2H, NHCH2CH2CH2Si), 3.44 (m, 2H, NHCH2CH2CH2Si), 3.82 (q, J = 7.0 Hz, 6H, OCH2CH3), 6.80 (ddd, 1H, J = 8.0, 7.0, 1.2 Hz, C(5)H), 6.92 (br s, 1H, NH), 6.94 (ddd, J = 8.4, 1.2, 0.4 Hz, 1H, C(3)H), 7.35 (ddd, J = 8.4, 7.1, 1.6 Hz, 1H, C(4)H), 7.39 (dd, J = 8, 1.6, 1H, C(6)H), 12.48 (s, 1H, ArOH); (NHCH2CH2CH2Si),

18.35

13

C NMR (101 MHz, CDCl3, 25°C): δ 7.88

(OCH2CH3),

22.72

(NHCH2CH2CH2Si),

41.75

(NHCH2CH2CH2Si), 58.65, (OCH2CH3), 114.60, (C(1)-CO), 118.53, (C(3)-H), 118.55 (C(5)-H), 125.56 (C(6)-H), 134.03 (C(4)-H), 161.62, (C(2)-OH), 170.08 (C=O). IR (νmax, cm-1): 3382, 3019, 2976, 2928, 2400, 1641, 1596, 1539, 1490, 1447, 1363, 1304, 1215, 1169, 1075, 929, 758, 668. HR-MS (ESI, [M+]) m/z: Calcd for C16H27 NO5Si: 341.1658 Found: 341.1569.

2-Hydroxyterephthaloyl dichloride (3) (Scheme 5). Synthesis was modified from Kauffman, et. al.51 A flame-dried single-neck round-bottom flask fitted with a reflux condenser and magnetic stir bar was charged with 2-hydroxyterephthalic acid (0.706 g, 3.76 mmol), SOCl2 (8.62 mL) and DMF (8 drops) under argon and the reaction mixture

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was stirred at reflux (90 °C) for 48 h. The acid dissolved to a clear yellow solution upon addition of DMF. After cooling to room temperature, SOCl2 was removed by repeatedly adding toluene and removing volatiles in vacuo (4x) to afford a yellow solid (0.823 g, 97%), which was used in the synthesis of 4 without further purification. 2-Hydroxy-N',N''-bis(3-triethoxysilylpropyl)terephthalamide

(4)

(Scheme

5).

Synthesis was modified from Gounko, et. al.50 A single-neck round-bottom flask fitted with a magnetic stir bar was charged with APTES (1.8 mL, 7.52 mmol) and Et3N (1.11 mL, 7.896 mmol) in benzene (25 mL). To this, a separate solution of 3 (0.823 g, 3.76 mmol) in benzene (20 mL) was added dropwise at 0 °C to minimize the evolution of a white fog, using excess benzene (5 mL) to facilitate transfer. The reaction mixture was stirred at 24 °C until consumption of starting material (48 h), monitoring by thin layer chromatography (TLC) on silica plates (2:1 ethyl acetate:dichloromethane). White triethylammonium chloride was filtered off and solvent was removed in vacuo. The clear, dark yellow oil was purified by running through a silica plug (2:1 ethyl acetate:dichloromethane) and concentrating in vacuo to afford a clear, dark yellow, tacky solid (2.13 g, 96%). 1H NMR (400 MHz, CDCl3) δ 0.62 (m, 4H, NHCH2CH2CH2Si), 1.134 (t, J = 7.0 Hz, 9H, OCH2CH3), 1.142 (t, J = 7.0 Hz, 9H, OCH2CH3), 1.68 (m, 4H, NHCH2CH2CH2Si), 3.37 (m, 4H, NHCH2CH2CH2Si), 3.74 (q, J = 7.0 Hz, 6H, OCH2CH3), 3.75 (q, J = 7.0 Hz, 6H, OCH2CH3) 6.55 (t, J = 5.6 Hz, 1H, NH), 7.06 (t, J = 5.6 Hz, 1H, NH), 7.15-7.17 (m, 2H, C(3)H and C(5)H), 7.39 (d, J = 8.2 Hz, 1H, C(6)H), 12.44 (s, 1H, ArOH); (OCH2CH3),

22.65

13

C NMR (101 MHz, CDCl3) δ 7.98 (NHCH2CH2CH2Si), 18.39 (NHCH2CH2CH2Si),

22.86

(NHCH2CH2CH2Si),

41.87

(NHCH2CH2CH2Si), 42.40 (NHCH2CH2CH2Si), 58.65 (OCH2CH3), 58.72 (OCH2CH3),

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114.26 (C(1)C=O), 116.22 (C(3)H), 117.28 (C(5)H), 125.88 (C(6)H), 139.94 (C(4)C=O), 161.52 (C(2)OH), 166.39(C(7)=O), 169.29 (C(8)=O). IR (νmax, cm-1): 3683, 3619, 3019, 2976, 2400, 1647, 1528, 1423, 1215, 1075, 929, 759, 669. HR-MS (ESI, [M+ H+]) m/z: Calcd for C26H49N2O9Si2: 589.2977 Found: 589.2991.

2-Methoxy-4-((1E,6E)-7-(3-methoxy-4-(((3-(triethoxysilyl)propyl)carbamoyl)oxy) phenyl)-3-oxo-5-(3-(triethoxysilyl)propoxy)hepta-1,6-dien-1-yl)phenyl(3(triethoxysilyl) propyl)carbamate (6). Synthesis was adapted from Shughart, et. al.52 A single-neck round-bottom flask fitted with a magnetic stir bar was charged with curcumin (1.0 g, 1.90 mmol) in anhydrous tetrahydrofuran (25 mL). To this, K2CO3 (0.663 g, 4.75 mmol) was added and the orange solution immediately turned dark red. The reaction mixture was allowed to stir 24h at 24 °C. Afterwards, freshly distilled IPTES (0.941 mL, 3.80 mmol) was added. The mixture was stirred at 24 °C, monitoring by TLC (2:1 ethyl acetate:dichloromethane) until consumption of starting material (48 h). The red solution containing suspended solids was vacuum filtered, concentrated, and purified through silica plug (2:1 ethyl acetate:dichloromethane) to afford a clear red oil which solidified to a translucent tacky solid upon standing (1.174 g, 56%). 1H NMR (400 MHz, CDCl3) δ 0.68 (m, 6H, 3 x NHCH2CH2CH2Si), 1.22 (dt, J = 7.0, 4.4 Hz, 27H, OCH2CH3), 1.65 – 1.77 (m, 6H, 3 x NHCH2CH2CH2Si), 3.27 (m, 6H, 3 x NHCH2CH2CH2Si), 3.74 – 3.86 (m, 24H, 3 x OCH2CH3 and 1 OCH3 ), 5.41 (t, 1.7H, NH), 5.47 (t, 0.3H, NH), 5.82 (s, 1H, C(4)-H), 6.52 (d, J = 15.8 Hz, 2H, (C(2,6)H), 7.10 (m, 6H, C(9,9’,12,12’,13,13’)H), 7.567.66 (m & d, J = 15.7 Hz, 2H, );

13

C NMR (101 MHz, CD2Cl2) δ 7.56, 7.65, 7.76

(NHCH2CH2CH2Si), 18.32 (OCH2CH3), 23.09, 23.16, 25.12 (NHCH2CH2CH2Si), 43.59,

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43.72, 45.39 (NHCH2CH2CH2Si), 55.97 (OCH3), 58.47, 58.50, 58.53 (OCH2CH3), 101.69 (C(4)-H), 111.47 (C(13,13’)H), 121.10 (C(9,9’)H), 122.01 (C(5)-O), 123.68 (C(12,12’)H, 123.99 (C(2,6)H), 129.12 (small amount of isomer), 133.36 (C(8,8’)H), 140.10 (C(1,7)H), 141.7 (C(10, 10’)OCH3), 151.99 (2 C(11,11’)-OC=O), 153.98 (3 NHC=O), 183.14 (C=O). IR (νmax, cm-1): 3346, 3053, 2975, 2928, 1740, 1628, 1587, 1510, 1464, 1390, 1265, 1206, 1165, 1124, 1077, 1034, 961, 909, 848, 739, 704, 667. HR-MS (ESI, [M+ Na+]) m/z: Calcd for C51H83N3NaO18Si3: 1132.4877; Found: 1132.4878.

2.4 Particle Preparation Procedures Unmodified surfactant templated silica particles. A 250 mL single-neck round-bottom flask fitted with a magnetic stir bar was charged with CTAB (0.32 g, 0.89 mmol) in anhydrous ethanol (60 mL) and distilled water (102 mL). To this, TEOS (2.0 mL, 8.96 mmol) was added and the clear solution stirred 5 min at 24 °C. Finally, aq. NH4OH (13.4 M, 2.0 mL) was added and the solution stirred 3h at 24 °C (700 RPM). Sphere evolution was indicated by an increasing opalescence of the mixture beginning 2 min after adding the NH4OH solution. The transition to a turbid white suspension occurred within a few more minutes. After 3h, the product was centrifuged to remove excess catalyst and starting material, then washed 3x with water (0.581 g). Hydrodynamic diameter (DLS) = 800 ± 126 nm. Diameter (SEM) = 760 ± 57 nm. BET surface area = 6 m2/g. BJH pore size = 31 Å.

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Physically encapsulated salicylate particles (E-Sal). Particles were prepared according to Teng, et. al.53 A 250 mL single-neck round-bottom flask fitted with a magnetic stir bar was charged with CTAB (0.32 g, 0.89 mmol) in anhydrous ethanol (40 mL) and distilled water (102 mL). To this, a previously prepared solution of 2-ethylhexyl salicylate (0.023 g, 0.089 mmol) in anhydrous ethanol (20 mL) was added. Afterwards, TEOS (2.0 mL, 8.96 mmol) was added and the clear solution stirred 5 min at 24 °C. Finally, aq. NH4OH (13.4 M, 2.0 mL) was added and the solution was stirred 3h at 24 °C (700 RPM). Sphere evolution was indicated by an increasing opalescence of the mixture beginning 2 min after adding the NH4OH solution. The transition to a turbid white suspension occurred within a few more minutes. After 3h, the product was centrifuged to remove excess catalyst and starting material, then washed 3x with water (0.724 g, yield = 135 % based on silica only). Hydrodynamic diameter (DLS) = 830 ± 56 nm. Diameter (SEM) = 657 ± 71 nm. BET surface area = 9 m2/g. BJH pore size = 31 Å.

Capped physically encapsulated salicylate particles (cap E-Sal).54 A 50 mL singleneck round-bottom flask equipped with a magnetic stir bar was charged with a suspension of E-Sal (0.272 g) in anhydrous ethanol (20 mL). To this, NH4OH (13.4 M, 1.6 mL) and then freshly distilled TEOS (0.30 mL, 1.34 mmol) were added, and the suspension was allowed to stir at 24 °C for 24h. Afterwards, the product was centrifuged to remove excess catalyst and starting material, then washed 3x with ethanol (0.292 g, 83% based on full conversion and addition of silica from TEOS). Hydrodynamic diameter (DLS) = 868 ± 119 nm. Diameter (SEM) = 809 ± 80 nm. BET surface area = 6 m2/g. BJH pore size = 16 Å.

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Un-modified Stöber silica particles. Particles were prepared according to a modified methodology of Walenzyk, et. al.40 A 100 mL, 3-neck, round-bottom flask was equipped with a thermometer and magnetic stir bar. The flask was charged with anhydrous ethanol (33.1 mL) and aq. NH4OH (5.154 M, 20.1 mL) and the solution stirred at 30 °C. In a scintillation vial in the same water bath, TEOS (5 mL) was heated to 30 °C. Once the temperatures of both solutions had equilibrated at 30 °C, TEOS was added to the reaction flask and the resultant solution was allowed to stir 15s. Afterwards, stirring was stopped and the reaction mixture was allowed to stand at 30 °C for 2h. Sphere evolution was indicated by an increasing opalescence of the mixture beginning 1–5 minutes after adding TEOS. The transition to a turbid white suspension occurred within a few more minutes. After 2 h, the product was centrifuged to remove excess catalyst and starting material, then washed 3x with ethanol (1.569 g, yield = 117 % based on fully condensed structure). Hydrodynamic diameter (DLS) = 556 ± 34 nm. Diameter (SEM) = 520 ± 58 nm. BET surface area = 12 m2/g. BJH pore size = 3 Å.

Pendant salicylate particles (P-Sal). Particles were prepared by a procedure based on that of Walenzyk, et. al.40 A 100 mL, 3-neck, round-bottom flask was equipped with a thermometer and magnetic stir bar. The flask was charged with anhydrous ethanol (33.1 mL) and aq. NH4OH (5.154 M, 20.1 mL) and the solution stirred at 30° C. In a scintillation vial in the same water bath, a previously prepared 1 mole% monomer solution of 2 in TEOS (5 mL) was heated to 30 °C. Once the temperatures of both solutions had equilibrated at 30° C, monomer solution was added to the reaction flask and

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the resultant solution was allowed to stir 15s. Afterwards, stirring was stopped and the reaction mixture was allowed to stand at 30 °C for 2h. Sphere evolution was indicated by an increasing opalescence of the mixture beginning 1-5 min after adding the monomer solution. The transition to a turbid white suspension occurred within a few more minutes. After 2h, the product was centrifuged to remove excess catalyst and starting material, then washed 3x with ethanol (1.539 g, yield = 114 % based on fully condensed structure). Hydrodynamic diameter (DLS) = 781 ± 42 nm. Diameter (SEM) = 657 ± 71 nm. BET surface area = 13 m2/g. BJH pore size = 31 Å.

Bridged salicylate particles (B-Sal). A 100 mL, 3-neck, round-bottom flask was equipped with a thermometer, reflux condenser, and magnetic stir bar. The flask was charged with anhydrous ethanol (33.1 mL) and aq. NH4OH (5.154 M, 20.1 mL) and the solution stirred at 30 °C. In a scintillation vial in the same water bath, a previously prepared 1 mole% monomer solution of 4 (0.131 g, 0.224 mmol) in TEOS (5 mL) was heated to 30 °C. Once the temperatures of both solutions had equilibrated at 30 °C, monomer solution was added to the reaction flask and the resultant solution was allowed to stir 15s. Afterwards, stirring was stopped and the reaction mixture was allowed to stand at 30 °C for 2h. Sphere evolution was indicated by an increasing opalescence of the mixture beginning 1-5 min after adding the monomer solution. The transition to a turbid white suspension occurred within a few more minutes. After 2h, the product was centrifuged to remove excess catalyst and starting material, then washed 3x with ethanol (1.608 g, yield = 120 % based on fully condensed structure). Hydrodynamic diameter

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(DLS) = 795 ± 33 nm. Diameter (SEM) = 668 ± 74 nm. BET surface area = 7 m2/g. BJH pore size = 19.5 Å.

Bridged curcuminoid particles (B-Curc). A 100 mL, round-bottom flask was equipped with a thermometer and magnetic stir bar. The flask was charged with anhydrous ethanol (33.1 mL) and aq. NH4OH (5.154 M, 20.1 mL) and the solution stirred at 30 °C. In a scintillation vial in the same water bath, a previously prepared monomer solution of 6 (0.196 g, 0.175 mmol) in TEOS (5 mL, 4.70 g, 22.56 mmol) was heated to 30 °C. Once the temperatures of both solutions had equilibrated at 30 °C, monomer solution was added to the reaction flask and the resultant solution was allowed to stir 15s. Afterwards, stirring was stopped and the reaction mixture was allowed to stand at 30 °C for 2 h. Sphere evolution was indicated by an increasing opalescence of the mixture beginning 15 minutes after adding the monomer solution. The transition to a turbid white suspension occurred within a few more minutes. After 2 h, the product was centrifuged to remove excess catalyst and starting material, then washed 3x with ethanol. After drying, a creamy white powder was obtained (1.585 g, yield = 118 % based on fully condensed structure). Hydrodynamic diameter (DLS) = 688 ± 56 nm. Diameter (SEM) = 665 ± 70 nm. BET surface area = 5 m2/g. BJH pore size = 22 Å.

2.5 Leaching Analysis Sunscreen particle suspensions in anhydrous ethanol (3 mg/mL) were stirred 24h at 24 °C. To remove particles, each sample was filtered (6x) through a 0.20µm nylon syringe filter obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). Efficacy

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of removal of particles from suspensions was checked with DLS and SEM analyses of the filtered and un-filtered solutions. Leaching was assessed with fluorescence spectroscopy before and after filtration. Sunscreen concentrations were calculated from prepared fluorescence vs. concentration calibration plots. Excitation wavelengths of 324 nm (salicylate) and 420 nm (curcuminoid) were used.

2.6 UV Stability Analysis UV stability analysis was performed according to COLIPA standards.55 Sunscreen particle suspensions were dispersed in 70/30 ethanol/glycerin (10 wt%). Ethylhexyl salicylate solutions (10 wt%) in 70/30ethanol/glycerin were prepared as experimental controls. The suspensions/solutions (18.5 mg) were dropcast onto a clean quartz slide (2.55 cm × 3.63 cm) to ensure coverage of 2.0 mg/cm2. A second quartz slide of the same dimensions was placed above the sample to sandwich it. Each sample was then irradiated with a Solar Simulator (Xe arc lamp, UVB = 0.039 W/cm2, UVA = 0.052 W/cm2) for 6h, monitoring by UV-Vis absorbance spectroscopy at specified time intervals. After 6h, the irradiated free sunscreen samples were washed from the quartz slide and analyzed by NMR or GC-MS. The criteria stipulated and chosen for the stability experiments are listed in Table 1S in supporting materials.

2.7 In vitro SPF and UVAPF Determinations Incremental UV absorbance data for both the salicylate and curcuminoid particles (obtained during UV stability analysis) was utilized for these experiments. In vitro SPF values were then determined according to the methodology of Mansur, et. al,56 using

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erythemal effect (EE) and solar intensity (I) constants derived by Sayre, et. al.57 In vitro UVA protection efficacy (UVAPF), namely critical wavelength58 and pre- and postirradiation UVA/UVB ratios59, 60 as well as Boot's Star Ratings,61-64 were also determined from the UV stability spectral data.

3. Results and Discussion 3.1 Monomer Syntheses Chemical structures of each sunscreen monomer were designed to ensure the absorbance and emission profiles were similar to 2-ethylhexyl salicylate and curcumin. Monomer 2 is the amide of salicyclic acid and 3-aminopropyltriethoxysilane (APTES) (Scheme 4). Bridged salicylate 4 is the bis-amide prepared from 2-hydroxyterephthalic acid and APTES (Scheme 5). The salicylate monomers are both o-hydroxybenzamides, which ensures intramolecular hydrogen bonding and retention of UV absorbance from 267 – 340 nm. Curcuminoid monomer 6, which is the tris-carbamate of curcumin and 3isocyanatopropyltriethoxysilane (IPTES; Scheme 6), retains the electron donating functionalities in the meta and para positions.

3.2.1. Synthesis of Salicylate sunscreens Salicylate monomers 2 and 4 were successfully synthesized in high yields via condensation of APTES with salicyloyl chloride 1 and 2-hydroxyterephthaloyl chloride 3, respectively (Schemes 4 & 5). The acid chlorides were synthesized from salicyclic acid and 2-hydroxyterephthalic acid in near quantitative yields using modified literature procedures.49,

51

Pendant monomer 2 had been previously prepared by condensation of

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ethyl salicylate with APTES by Gounko, et. al. without experimental details.50 Bridged monomer 4 has not been previously reported.

Synthesis of Bridged Curcuminoid sunscreen Curcumin is composed of a mixture of isomers that contain an average of two methoxy and two phenol hydroxyl groups as seen in Scheme 6. Commercial samples are typically purified to only 75 – 85%, meaning the position and extent of methoxylation of the phenol hydroxyl groups varies. As such, the proposed curcuminoid monomer includes all natural derivatives of the parent curcumin. Since the major constituents possess a bisphenol residue, preparation of the bridged silsesquioxane analogue simply entailed (1) deprotonation with K2CO3 followed by (2) condensation with freshly distilled IPTES to afford not the bis-triethoxysilylcarbamate 5, but the tris-triethoxysilylcarbamate 6 (Scheme 6).52 Synthesis of an analogue of 5 has been patented by Arkles, et. al., and involves hydrosilylation of 4,4’-bisallyloxycurcumin with HSi(OEt)3 (74% crude).65 The carbamate route was a more direct and less costly approach to preparing a bridged curcumin, and good yields were obtained (72%). The yield would have been higher save we had only anticipated two equivalents of the isocyanatopropyltriethoxysilane reacting with each curcumin, instead of three.

For the purposes of this work, the tris-

triethoxysilylcarbamate monomer 6 is better suited as a sunscreen than 5, as the monomer is locked into the enol configuration with its extended conjugation and strong absorption, whereas 5 would be a mixture of the enol and diketone isomers.

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3.2 Spherical Particle Preparation All sunscreen particle samples were prepared in triplicate with 1 mole% of the sunscreen with respect to TEOS. Unmodified silica particles were also prepared to examine the effect of sunscreen incorporation on resultant particle size. Particles are described in terms of their diameter determined by SEM rather than their hydrodynamic diameter determined by dynamic light scattering. Particle yields greater than 100% are common in sol-gel polymerization reactions and were expected, as the final product generally contains un-reacted alkoxysilane and silanol groups. Physical encapsulation of 2-ethylhexyl salicylate via microemulsion polymerization Silica particles containing 2-ethylhexyl salicylate, physically encapsulated within the hollow core, (E-Sal) were successfully prepared via a modified O/W microemulsion technique.53 CTAB assembles into micelles containing TEOS and the hydrophobic sunscreen. Hydrolysis of the TEOS results in hydrophilic intermediates that react to form silica at the interface between the micelle and the water phase leaving the sunscreen to remain in the hollow interior of the particles. Teng, et. al. showed that hollow particles were obtained with if the ethanol/water ratio 0.37 – 0.7.53 In our case, an ethanol/water ratio of 0.59 afforded salicylate filled particles with an average diameter of 743 ± 78 nm (Figure 1a), compared with hollow particles prepared without sunscreen exhibiting diameters of 761 ± 57 nm (Figure 1b). The SEM clearly reveals a ruptured particle (circled in red) highlighting the problem of release of sunscreen with mechanical damage to the silica shells. TEM micrographs demonstrate that the particles are hollow with a silica shell thickness of 150 nm (Figure 2a).

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Particle capping, i.e. fortifying of the particle surface with an additional silica layer,54 was performed on the E-Sal particles to determine if leaking of sunscreen from the hollow particles could be reduced. The average diameters of the capped particles (Cap E-Sal) increased to 809 ± 81 nm by TEM (Figure 2b), implying an average cap thickness of 50 – 60 nm. Comparison of the size of the particles in the SEM’s for E-Sal and Cap E-Sal (Figure 1b & c) confirms the cap thickness. Adsorption-desorption isotherms of E-Sal, Cap E-Sal, and hollow SiO2 particles without sunscreen are reversible and convex over the entire pressure range, with an indistinct monolayer-multilayer N2 adsorption point consistent with non-porous particles with weak adsorbent-adsorbate interactions (Type III isotherm). This is evidenced by the small pore volumes and BET surface areas, the latter of which are consistent with the calculated geometric surface areas (Table 1).

Covalently incorporated salicylate particles via sol-gel polymerization Pendant salicylate (P-Sal) and bridged salicylate (B-Sal) particles were successfully prepared via co-condensation of sunscreen monomers 2 and 4 with TEOS. SEM and DLS analyses have confirmed that the P-Sal particles are spherical in shape and morphology with a mean diameter of 657 ± 71 nm (Figure 3b). The B-Sal particles, while mostly spherical, are more irregular in shape and slightly larger in diameter (668 ± 74 nm) than the pendant samples (Figure 3c). Additionally, some particles were fused together. Overall, salicylate incorporation resulted in larger particles compared to unmodified silica counterparts prepared only from TEOS (Figure 3a).

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Monomers with single triethoxysilyl groups, such as 2, are reported to segregate to the surface of silica prepared by sol-gel co-polymerizations with TEOS.66

This is

believed to arise from the steric bulk of the pendant organic group and the tendency to form cyclic siloxanes. Bridged monomers, such as 4 or 5, are generally believed to incorporate more uniformly through silica particles because the multiple silyl groups circumvents any steric hindrance to network formation.67 Energy Dispersive Spectroscopy (EDS), which allows for elemental surface analysis of substrates due to the shallow penetration depth of the electron probe, was used to examine the P-Sal and B-Sal particles (Figure 4). In the P-Sal particles, silicon (Si), oxygen (O), and small amounts of carbon (C) atoms were seen on the surface. In the B-Sal particles only Si and O were observed. These results are consistent with more of the pendant monomer (2) segregating to the particles’ surfaces than the bridged monomer (4) (Figure 4).

Like the particles

prepared via microemulsion polymerization, the adsorption-desorption isotherms for PSal, B-Sal, and unmodified silica are of type III consistent with non-porous particles (Table 1).

Covalently incorporated curcuminoid particles via sol-gel polymerization Bridged curcuminoid (B-Curc) particles are similar to the B-Sal particles with more irregular spherical shapes and fusing of particles together than with P-Sal particles (Figure 3d). Average particle diameters of B-Curc are approximately the same as the BSal particles, at 665 ± 70 nm, but larger than unmodified silica particles. The adsorptiondesorption isotherm for B-Curc particles is of type III with small pore volumes and BET surface area, the latter of which is consistent with the calculated geometric surface areas

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(Table 1). As such, it can be deduced that the B-Curc particles are non-porous like the previously prepared salicylate and unmodified silica particle samples.

3.3 Quantification of Sunscreen Content in Particles Fluorescence spectroscopy experiments were performed to quantify the sunscreen content (mg/g). Fluorescence was chosen due its greater sensitivity and less impact from scattering from the silica particles. Sunscreen loadings were calculated for each from the fluorescence spectra of 3.5 mL ethanolic particle suspensions (3.0 mg/mL) using standard calibration plots for 2-ethylhexyl salicylate and curcumin solutions. Compared to the theoretical 1.0 mole % sunscreen incorporation, the actual loading is approximately 5x less than theoretically calculated (Table 2). The fact that 80% of the sunscreen monomers failed to incorporate is likely due to the lower reactivity of the alkyl substituted triethoxysilyl groups under alkaline polymerization conditions compared with tetraethoxysilane. The remaining sunscreen monomers may have remained in solution or in suspension as smaller particles discarded during the centrifugation purification of the larger sunscreen particles. Given the Food and Drug Administration (FDA) recommended sunscreen application of 2.0 mg/cm2,68 and the average surface area of human males (1.9 m2) and females (1.71 m2),69 approximately 34 – 38 g of sunscreen should be applied for full body protection. Using this mass, the maximum FDA allowable 2-ethylhexyl salicylate concentration of 5%,68 and the calculated values listed in Table 2, the sunscreen formulation would require the use of 235.3 g, 171.4 g, and 92.8 g of E-Sal, P-Sal, and BSal particles, respectively. A circuminoid formulation (using a maximum concentration

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of 3%)70 would require the use of 41.7 g of B-Curc particles. This work is based on the particles prepared with 0.2 mol% covalently incorporated sunscreens.

3.4 Sunscreen Leaching Analysis To maintain high levels of protection over time and minimize skin contact, the sunscreen must not leach from the silica particles. The first mode of analysis defined in this study was to determine if covalent incorporation minimizes leaching over that of physical encapsulation. It was hypothesized that minimal leaching would be achieved with covalent incorporation, as the silica shell of the physical samples are fragile and can easily rupture, and the sunscreen is only retained by electrostatic forces (Figure 1a).28 If so, the second goal was to analyze which mode of covalent incorporation (bridged or pendant) minimizes leaching. The increased number of bonding sites in the bridged sample (6) as opposed to the pendant analogue (3) would imply that more bonds are required to break before the ingredient can leach from the particle, thereby resulting in less leach out.67, 71 The third and final goal was to determine if covalent incorporation would eliminate the need for an extra capping step. It was assumed that the magnitude of leaching from the covalent samples would be less than the capped physically encapsulated sample, thereby negating the need for capping. To test the above hypotheses, ethanolic salicylate particle suspensions (3.0 mg/mL) were stirred for 24 h at ambient temperature (24 °C), then filtered 6x times with a 0.2 µm microfilter to ensure fluorescence emission intensities were not the result of residual incorporated sunscreen. SEM results detailed the successful removal of particles from the ethanolic suspensions (Figure 5).

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Fluorescence spectra comparisons of the filtered and un-filtered particle suspensions (Figure 6 and Table 2) detail significant leaching (24.17%) when 2ethylhexylsalicylate is physically encapsulated within silica particles. Since all prepared particle samples were determined to be non-porous, this failure of dye retention can be explained due to shell fragility and rupture of the particles (Figure 1a), which can easily allow for leaching of 2-ethylhexyl salicylate from the particle core. While capping did reduce leach out to 6.78%, the capped samples were still outperformed by the covalent analogues. Covalent incorporation of salicylate silsesquioxane analogues 2 (P-Sal) and 4 (BSal) was seen to greatly enhance retention, as dye leach out was reduced to 0.80% and 0.04%, respectively. Regarding pendant versus bridged incorporation, more bonds are required to break before the sunscreen can leach from the particle. Of the four samples analyzed, bridged encapsulation was deemed to be the incorporation method of choice due to minimized overall leaching. Based on these results, bridged incorporation is necessary to ensure isolation of sunscreen from skin. This was also evidenced by analysis of the curcuminoid particle sample, for only 0.29 % leaching was seen (Figure 6e). The slightly greater amount of leaching of the bridged curcumonoid, compared with the bridged salicylate, may be due to the greater hydrolytic instability of the phenolic carbamate linkages compared to amides.

3.5 UV Stability of Incorporated Sunscreen UV stability of sunscreens is an important criteria in evaluation.1 Analysis typically involves exposing a sunscreen solution to a specified dosage of UV radiation for a given

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amount of time, and the effect on spectrophotometric response (typically UV absorbance or fluorescence) is monitored.55, 72 To adequately simulate the irradiance and dosimetry of natural sunlight exposure, Berset, et.al., in collaboration with COLIPA, developed a simple and reliable technique which involves the irradiation of a liquid film sample on glass with a Xenon lamp of a specified irradiance and dosage.55 The effect on UV absorbance before and after exposure is then analyzed. Essentially, sunscreen particle and liquid sunscreen samples were exposed to UV radiation via a full-spectrum solar simulator (1 sun output intensity), which is designed to mimic the spectrum and intensity of sunlight hitting the earth. The samples were exposed for a total of 6 h at irradiances of 0.039 W/cm2 (UVB) and 0.052 W/cm2 (UVA) per hour. This is equivalent to dosages of 140.4 J/cm2 (UVB) and 187.2 J/cm2 (UVA), or 1.0 MED/h. UV absorbance for each sample was monitored by UV-Vis spectroscopy initially (pre-irradiation) and at specified time intervals. After 6 h, the free sunscreen samples were washed from the quartz slides and photo-products were analyzed by GC-MS or NMR spectroscopy. Summaries of percent UV absorption ability for the irradiated salicylate samples are presented in Figure 7 and Table 3. As expected, the salicylate samples were relatively stable over the course of analysis (≥ 75 %),1 though UV-induced degradation over time was seen for each (Figure 7 and Table 3). Comparisons of maximum UV absorbance for each sample (303 – 315 nm) after 6 MED of exposure detailed that the B-Sal particles retained the highest UV absorption ability at 88.40 ± 0.63 %, which is increased over that of free 2-ethylhexyl salicylate (75.74 ± 0.41 %) and physically encapsulated 2-ethylhexyl salicylate (76.82 ± 0.01 %). Capping of the physically encapsulated 2-ethylhexyl salicylate improved

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stability (79.51 ± 0.11 %), though not as much as that afforded via pendant-covalent incorporation (82.64 ± 1.02 %). The curcuminoid samples were analyzed as above, with B-Curc and free curcumin both displaying UV-induced degradation over time. Attaching the curcumin to the silica particles only shifts the absorption a few nm to slightly longer wavelengths (Figure 8). This is not surprising since the attachment during significantly change the electronics of the pi conjugated system. However, attaching the curcumin to the B-Curc particles does result in (Table 3) a marked increase in UV stability (77.28 ± 0.09 %) over that of free curcumin (57.43 ± 1.66 %). These results indicate that the method of incorporation can have a marked effect on sunscreen photo-stability. Free sunscreens were examined after irradiation to give an indication of the photo-degradation pathways, as well as determine plausible structures of photo-products. From these results, it was determined that covalent incorporation is necessary to decrease levels of degradation and isolate any photoproducts from skin, and bridged-incorporation is the method of choice to adequately ensure this, thereby achieving long-lasting protection.

3.6 In vitro SPF and UVAPF Determination In vitro SPF and UVAPF determinations have been developed as fast and reasonable alternatives to in vivo testing due to the eliminated risk of UV exposure to human subjects. For SPF, the most common methods entail measuring UV absorption or transmission of sunscreen films within quartz plates,73 biomembranes,74 or synthetic membranes such as 3M Transpore tape.75

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Using the incremental UV absorbance data from UV stability analysis, SPF values for both salicylate and curcuminoid particles were determined via the methodologies of Sayre, et. al57 and Mansur, et. al.56 Sayre's calculations were developed according to Cosmetics Europe (COLIPA) protocols,55 which involve measuring the transmittance of a given sunscreen sample across the UVB spectrum (290 – 320 nm) weighted against the erythemal effect and solar intensity at each wavelength, as defined by Equation 1:57

SPFin vitro =

1 T 320

=

∑ EE (λ ) ∗ I (λ )

Equation 157

290

320

∑ EE (λ ) ∗ I (λ ) ∗ T (λ ) 290

where EE is the erythemal effect spectrum, I is the solar intensity spectrum, and T is the sunscreen transmittance. The normalized values of EE*I (See Table 2S in supporting materials), which are constants measured in 5 nm increments, define a sunscreen which transmits all light as having an SPF of 1.0, while one that absorbs all light as having an SPF of infinity.57

Equation 1 was further derived by Mansur, et. al. to improve method standardization by incorporating a correction factor, CF (Equation 2):56 320  EE (λ ) ∗ I (λ )   SPFin vitro = CF ∗ ∑  T (λ ) 290  

Equation 256

320

= CF ∗ ∑ EE (λ ) ∗ I (λ ) ∗ Abs (λ ) 290

where Abs is the sunscreen absorbance, and CF = 10 so that a standard 8% homosalate sunscreen would calculate to an SPF of 4.56

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Calculated SPF values for the un-irradiated particle samples are presented in Table 4. The salicylate values are relatively low when taking into account typical SPFs of commercial sunscreens, which would explain why other UVB absorbers in addition to salicylate ingredients are included within commercial formulations.61, 76 This is likely due to the low molar extinction coefficient (4900 M-1 cm-1 for 2-ethylhexyl salicylate), which arises from steric strain and slight deviation from planarity due to the ortho configuration of the hydroxyl and carboxylate residues.1 It is also important to remember that only small amounts of sunscreen (less than the theoretical 1.0 mole %) are incorporated for each sample (Table 2). Regarding physical versus covalent SPFs, a bathochromic shift in pre-irradiated UV absorbance was seen for the P-Sal and B-Sal samples (which contain incorporated salicylate silsesquioxanes) from that of E-Sal, which contains 2-ethylhexyl salicylate encapsulated within (Figure 9). This would imply a reduced ability to absorb UVB radiation, resulting in a lower SPF. More interesting, however, is the incremental SPF values after UV irradiation for each sample (Figure 9 and Table 4). After just 0.5 MED of exposure, SPF of the E-Sal particles was reduced by 10.8%, which is greater than that experienced by P-Sal (7.1%) and B-Sal (2.0%) particles. B-Curc particles experienced a comparable reduction of 3.0%. By 6 MED, the SPF of B-Sal was still only reduced by 13.5%, while the E-Sal and P-Sal samples display reductions of 22.9% and 18.4%, respectively. As before, the BCurc particles also maintained SPF well, with only 16.3% reduction seen. Maintenance of SPF can be attributed to the increased UV stability achieved with bridged incorporation, as the calculation correlates with the UV absorption ability over time.

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Since salicylates and curcuminoids also absorb UVA radiation, UVAPF, which delineates the efficacy of UVA protection, was also calculated for these samples. In vitro UVAPF determination is inherent with cross-laboratory variability such as substrate interactions, sunscreen film uniformity, sunscreen fluorescence, and sunscreen UV absorbance. To address these concerns, several methods have been developed that entail evaluating the breadth of UVA protection in comparison with UVB instead of absolute absorbance measurements. Two such methods were utilized in this study: the FDA recommended Critical Wavelength Method,58, 64 and the UK Boot’s Method.61-64 The Critical Wavelength Method58, 64 involves integrating the broad spectrum (290 nm – 400 nm) absorbance of a sunscreen film until the sum reaches 90% of the total area under the curve of this region. The wavelength that corresponds to this 90% sum is defined as the critical wavelength, and must lie between 290 nm – 320 nm for the sunscreen to provide appreciable UVB protection. The critical wavelength must be at least 370 nm for the sunscreen to be defined as broad-spectrum (UVA + UVB). The critical wavelength is defined according to Equation 3:

A(λ ) dλ = 0.9 ∫290 A(λ ) dλ 290

∫

λc

400

Equation 358, 64

where λc is the critical wavelength, A(λ) is the average absorbance at each wavelength, and dλ is the wavelength interval between measurements. Critical wavelengths for each sunscreen particle sample are > 370 nm (Table 5), implying none provide adequate UVB protection, as expected. Surprisingly, they can still be classified as broad-spectrum. The salicylate values are red-shifted from that of free 2-ethylhexyl salicylate at 331 nm.77 The Boots Method61-64 also involves measuring the integrated broad-spectrum UV absorbance of a sunscreen film before and after exposure to a controlled dose of UV

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radiation from a defined source. This data is then used to calculate UVA/UVB absorbance ratios according to Equations 4 and 5:

UVA / UVB ratio0 =

∫ ∫

400

320 320 290

∫ UVA / UVB ratio = ∫

400

320 320

290

A0 (λ )dλ / ∫320 dλ 400

A0 (λ )dλ / ∫290 dλ 320

A(λ )dλ / ∫320 dλ

Equation 461-64

400

A(λ )dλ / ∫290 dλ 320

Equation 561-64

where A0(λ) is the mean absorbance before UV exposure, A(λ) is the mean absorbance after UV exposure, and dλ is the incremental wavelength (1 nm). The closer each ratio is to unity, the higher the level of UVA protection. These pre- and post-irradiation ratios are then compared and utilized to classify the sunscreen into the five categories (or star levels) of protection of the Boots Star Rating System.61-64 Like SPF determination, UV stability absorbance data was utilized for all UVAPF calculations. Summaries of UVAPF data are presented in Table 6. Analysis of the pre-irradiation UVA/UVB ratios detail all salicylate samples are adequate UVA absorbers, with Boot Star Ratings of 3 stars for each. The curcuminoid particles rate "Ultra" for UVA protection with 5 stars. This pre-irradiation UVA protective ability directly correlates with UV absorbance profiles of the parent compounds, as curcuminoids absorb more strongly in the UVA region than salicylates. The E-Sal UVA/UVB ratio was un-affected after 6 MED, for the Boot's Star Rating was maintained at "good" (Table 6). Unfortunately, irradiation with 6 MED seems to affect UVA absorption ability for the P-Sal and B-Sal particles the most, as illustrated by an increased hypsochromic shifts in UV absorbance over that of E-Sal (Figure 7), resulting in reduced UVA/UVB ratios (Table 6). This was enough to reduce the Boot's Star Rating

30


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by one star from "good" to "moderate" for each. The B-Curc particles were comparable to the covalent salicylate particles, with a rating reduction from "ultra" to "superior".

4. Conclusions Hybrid

salicylate

sunscreen

particles

prepared

via

O/W

microemulsion

polymerization and a modified Stöber process provided an in depth analysis on the effect of incorporation method on dye leaching, UV stability, and UV protective ability. These results detail that the method of incorporation can have a marked effect on each of these characteristics. Overall, minimized leaching and decreased levels of photo-degradation were achieved with covalent incorporation, with bridged incorporation necessary to ensure isolation of the sunscreen and any photo-products from skin. Comparable results were also achieved with the bridged curcuminoid particles, despite the low concentration of the sunscreen monomer. Low SPF values of the salicylate particles also attributed to the small concentration of embedded sunscreen, but also the lower inherent UV absorption (i.e. low molar extinction coefficient) of the salicylates compared with the curcuminoid sunscreen. In addition a bathochromic shift in UV absorbance compared to encapsulated salicylate sunscreens may have also contributed to the lower the SPF and suggests a reduced ability to absorb UVB radiation. Despite this, the highest SPF maintenance after 6 MED of exposure was achieved with bridged incorporation, and is attributed to corollary UV stability. Both the salicylate and curcuminoid particles could be classified as broad-spectrum, and rate from moderate to superior in terms of UVA protective ability.

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These results have ultimately shed light on the effects of incorporation method by delineating the promise of bridged silsesquioxane incorporation, as an attractive method for sunscreen isolation and stabilization has been developed to eliminate the problems of photo-degradation and photo-toxicity associated with current sunscreens. Improvements in chemistry of sunscreen incorporation, such as using microemulsion polymerizations under acidic conditions, should raise SPF values to be at least comparable to commercial and encapsulated sunscreens with the added benefit of greater photostability and longer sunscreen lifetime. Acknowledgements S. Tolbert would like to thank the American Association of University Women for her dissertation fellowship.

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39. Stober, W.; Fink, A.; Bohn, E., Controlled Growth of Monodisperse Silica Spheres in Micron Size Range. Journal of Colloid and Interface Science 1968, 26, (1), 62-68. 40. Walenzyk, T.; Carola, C.; Buchholz, H.; König, B., Synthesis of mono-dispersed spherical silica particles containing covalently bonded chromophores. International Journal of Cosmetic Science 2005, 27, (3), 177-189. 41. Tolbert, S. H.; McFadden, P. D.; Loy, D. A., Fluorescent hybrid organicinorganic particles: Influence of physical encapsulation versus covalent attachment on leaching and UV stability. J Ceram Soc Jpn 2015, 123, (1441), 785-792. 42. Shaw, D. W., Allergic contact dermatitis from octisalate and cis-3-hexenyl salicylate. Dermatitis 2006, 17, (3), 152-155. 43. Singh, M.; Beck, M. H., Octyl salicylate: a new contact sensitivity. Contact Dermatitis 2007, 56, (1), 48-48. 44. Lewandowski, D.; Olejnik, A.; Schroeder, G., Adsorption studies and release of selected dyes from functionalized mesoporous MCM-41 silica. Cent Eur J Chem 2014, 12, (2), 233-241. 45. Slowing, I. I.; Vivero-Escoto, J. L.; Wu, C. W.; Lin, V. S. Y., Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv Drug Deliver Rev 2008, 60, (11), 1278-1288. 46. Tsai, K. D.; Lin, J. C.; Yang, S. M.; Tseng, M. J.; Hsu, J. D.; Lee, Y. J.; Cherng, J. M., Curcumin Protects against UVB-Induced Skin Cancers in SKH-1 Hairless Mouse: Analysis of Early Molecular Markers in Carcinogenesis. Evid-Based Compl Alt 2012. 47. Brunauer, S.; Emmett, P. H.; Teller, E., Adsorption of gases in multimolecular layers. J Am Chem Soc 1938, 60, 309-319. 48. Barrett, E. P.; Joyner, L. G.; Halenda, P. P., The Determination of Pore Volume and Area Distributions in Porous Substances .1. Computations from Nitrogen Isotherms. J Am Chem Soc 1951, 73, (1), 373-380. 49. Agwada, V. C., Potential Central Nervous-System Active Agents .1. Synthesis of Aromatic N-Benzyl Amides. J Chem Eng Data 1982, 27, (4), 479-481. 50. Gounko, I. K.; Rakovich, Y.; Volkov, Y.; Dongegan, J.; Kelleher, D.; Mitchell, S. Nanoparticle delivery systems. WO2005105035A2, 2005. 51. Kauffman, J. M.; Khalaj, A.; Litak, P. T.; Novinski, J. A.; Bajwa, G. S., Synthesis and Photophysical Properties of Fluorescent 2-Aryl-1,3-Dialkylbenzimidazolium Ions and a 1-Alkly-2-Arylbenzimidazole with Excited-State Intramolecular Proton-Transfer. J Heterocyclic Chem 1994, 31, (4), 957-965. 52. Shughart, E. L.; Ahsan, K.; Detty, M. R.; Bright, F. V., Site selectively templated and tagged xerogels for chemical sensors. Anal Chem 2006, 78, (9), 3165-3170. 53. Teng, Z. G.; Han, Y. D.; Li, J.; Yan, F.; Yang, W. S., Preparation of hollow mesoporous silica spheres by a sol-gel/emulsion approach. Micropor Mesopor Mat 2010, 127, (1-2), 67-72. 54. Zhang, L.; D'Acunzi, M.; Kappl, M.; Auernhammer, G. K.; Vollmer, D.; van Kats, C. M.; van Blaaderen, A., Hollow Silica Spheres: Synthesis and Mechanical Properties. Langmuir 2009, 25, (5), 2711-2717. 55. Berset, G.; Gonzenbach, H.; Christ, R.; Martin, R.; Deflandre, A.; Mascotto, R. E.; Jolley, J. D. R.; Lowell, W.; Pelzer, R.; Stiehm, T., Proposed protocol for

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determination of photostability Part I: cosmetic UV filters. International Journal of Cosmetic Science 1996, 18, (4), 167-177. 56. Mansur, J. S.; Breder, M. N. R.; Mansur, M. C. A.; Azulay, R. D., Determinação do fator de proteção solarpor espectrofotometria. Anais Brasileiros de Dermatologia 1986, 61, 121-124. 57. Sayre, R. M.; Agin, P. P.; Levee, G. J.; Marlowe, E., Comparison of Invivo and Invitro Testing of Sunscreening Formulas. Photochemistry and Photobiology 1979, 29, (3), 559-566. 58. Moyal, D.; Alard, V.; Bertin, C.; Boyer, F.; Brown, M. W.; Kolbe, L.; Matts, P.; Pissavini, M., The revised COLIPA in vitro UVA method. Int. J. Cosmet. Sci. 2013, 35, (1), 35-40. 59. Diffey, B. L., A method for broad spectrum classification of sunscreens. Int. J. Cosmet. Sci. 1994, 16, (2), 47-52. 60. Stokes, R.; Diffey, B., In vitro assessment of sunscreen photostability: the effect of radiation source, sunscreen application thickness and substrate. Int. J. Cosmet. Sci. 1999, 21, (5), 341-351. 61. Hojerova, J.; Medovcikova, A.; Mikula, M., Photoprotective efficacy and photostability of fifteen sunscreen products having the same label SPF subjected to natural sunlight. Int. J. Pharm. 2011, 408, (1-2), 27-38. 62. Springsteen, A.; Yurek, R.; Frazier, M.; Carr, K. F., In vitro measurement of sun protection factor of sunscreens by diffuse transmittance. Anal Chim Acta 1999, 380, (23), 155-164. 63. Khurana, A.; Ho, C. T., High-Performance Liquid-Chromatographic Analysis of Curcuminoids and Their Photo-Oxidative Decomposition Compounds in Curcuma-Longa L. J Liq Chromatogr 1988, 11, (11), 2295-2304. 64. Wang, S. Q.; Stanfield, J. W.; Osterwalder, U., In vitro assessments of UVA protection by popular sunscreens available in the United States. J Am Acad Dermatol 2008, 59, (6), 934-42. 65. Arkles, B. C. Curcumin and its derivatives for use as silicone colorants and a method of coloring a siliceous surface. US20070204412A1, 2007. 66. Husing, N.; Schubert, U., Organofunctional silica aerogels. J Sol-Gel Sci Techn 1997, 8, (1-3), 807-812. 67. Shea, K. J.; Loy, D. A., Bridged polysilsesquioxanes. Molecular-engineered hybrid organic-inorganic materials. Chem Mater 2001, 13, (10), 3306-3319. 68. Food and Drug Administration, H., Labeling and effectiveness testing; sunscreen drug products for over-the-counter human use. Final rule. In Federal Register, 2011; Vol. 76, pp 35620-65. 69. Sacco, J. J.; Botten, J.; Macbeth, F.; Bagust, A.; Clark, P., The Average Body Surface Area of Adult Cancer Patients in the UK: A Multicentre Retrospective Study. Plos One 2010, 5, (1). 70. Ogata, Y.; Takagi, K.; Takayana.Y, Photodecomposition of Alkyl Benzoates and S-Alkyl Thiobenzoates - Possibility of a Barton-Type Transition-State. J Chem Soc Perk T 1 1973, (12), 1244-1247. 71. Loy, D. A.; Shea, K. J., Bridged Polysilsesquioxanes - Highly Porous Hybrid Organic-Inorganic Materials. Chem Rev 1995, 95, (5), 1431-1442.

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72. Gonzenbach, H. U.; Schudel, P., 'Spectral stability' - a meaningful term? Int. J. Cosmet. Sci. 1987, 9, (6), 287-92. 73. Akrman, J.; Kubac, L.; Bendova, H.; Jirova, D.; Kejlova, K., Quartz plates for determining sun protection in vitro and testing photostability of commercial sunscreens. Int. J. Cosmet. Sci. 2009, 31, (2), 119-129. 74. Crovara Pescia, A.; Astolfi, P.; Puglia, C.; Bonina, F.; Perrotta, R.; Herzog, B.; Damiani, E., On the assessment of photostability of sunscreens exposed to UVA irradiation: From glass plates to pig/human skin, which is best? Int. J. Pharm. (Amsterdam, Neth.) 2012, 427, (2), 217-223. 75. Diffey, B. L.; Robson, J., A new substrate to measure sunscreen protection factors throughout the ultraviolet spectrum. J. Soc. Cosmet. Chem. 1989, 40, (3), 127-33. 76. Heinrich, U.; Tronnier, H.; Kockott, D.; Kuckuk, R.; Heise, H. M., Comparison of sun protection factors determined by an in vivo and different in vitro methodologies: a study with 58 different commercially available sunscreen products. Int. J. Cosmet. Sci. 2004, 26, (2), 79-89. 77. Osterwalder, U.; Sohn, M.; Kurth, N.; Herzog, B., Which sunscreens protect best? A theoretical and practical analysis to help improve compliance. Australas J Dermatol 2012, 53, 46-46.

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Captions

Figure 1. DLS spectra and SEM micrographs of particles prepared via microemulsion polymerization.53 (a) Physically encapsulated salicylate particles (E-Sal), and (b) hollow silica particles made without sunscreen. An example of shell rupturing is circled in red. DLS data represents the average of three trials. Diameters of 743 ± 78 nm and 761 ± 57 nm were seen for (a) and (b) according to SEM.

Figure 2. TEM micrographs of (a) E-Sal, (b) cap E-Sal particles, (c) unmodified silica particles prepared via microemulsion polymerization, and (d) unmodified silica particles prepared via sol-gel polymerization. The shell thicknesses for (a) and (b) are approximately 150 ± 20 nm and 203 ± 29 nm, respectively, implying a cap thickness of 53 nm according to TEM.

Figure 3. DLS spectra and SEM micrographs of (a) unmodified silica particles, (b) pendant salicylate particles (P-Sal), (c) bridged salicylate particles (B-Sal) and (d) bridged curcuminoid particles (B-Curc). DLS data represents the average of three trials.

Figure 4. EDS spectra of (a) P-Sal particles, and (b) B-Sal particles. The P-Sal spectrum details the presence of silicon (Si), oxygen (O), and small amounts of carbon (C) atoms on the particle surface, while the B-Sal spectrum details the presence of only Si and O atoms on the surface.

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Figure 5. SEM micrographs of P-Sal particle suspensions used in leaching analysis before and after filtration, detailing successful removal of particles. Micrographs are representative of all particle samples analyzed (data not shown).

Figure 6. Fluorescence spectra of salicylate particle samples before (blue) and after filtration (red). (a) E-Sal, (b) Cap E-Sal, (c) P-Sal, (d) B-Sal and (e) B-Curc particles. Overall, bridged incorporation was seen to minimize leaching the most.

Figure 7. UV-Vis Absorbance spectra for salicylate samples exposed for 6 h. Incremental percent reduction in UV absorbance at λmax (303 - 315 nm) is also shown for each.

Figure 8. UV-Vis Absorbance spectra for curcuminoid samples exposed for 6 MED, which detail UV-induced degradation over time for each. Incremental percent reduction in UV absorbance at λmax (382 - 402 nm) is also shown for each.

Figure 9. Incremental SPF reduction for salicylate and curcuminoid particles after UV irradiation over time.

Scheme 1. Physical encapsulation of hydrophobic sunscreens using an oil-in-water microemulsion polymerization of tetraethoxysilane.

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Scheme 2. Covalent encapsulation by copolymerization of sunscreen dyes bearing one (A) or two or more (B) triethoxysilyl groups with tetraethoxysilane in an oil in water microemulsion polymerization.

Scheme 3. Capping of physically encapsulated sunscreens by adding a second layer or cap of silica over the first.

Scheme 4. Protocol for synthesis of pendant (2) salicylate precursor. Condensation of salicylic acid with APTES via salicyloyl chloride 1.49,50

Scheme 5. Protocol for synthesis of bridged (4) salicylate precursor. Condensation of condensation of 2-hydroxyterephthalic acid with two equivalents of APTES via 2hydroxyterephthaloyl chloride 3.50,51

Scheme 6. Synthesis of bridged circuminoid 6.

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Table 1. Nitrogen Porosimetry data for salicylate particles prepared via emulsion polymerization. # Sample Description SBET Scalc *VBJH-d dBJH

E-Sal

(m2/g)

(m2/g)

(cm3/g)

(Å)

8.6

3.0

0.11

30.7

6.6

2.8

0.09

15.6

Stober silica particles

6.5

3.0

0.08

30.7

Salicylate attached via pendant

12.9

3.0

0.18

30.9

6.9

3.4

0.15

19.5

Stober silica particles

12.1

4.4

0.22

34.3

Curcuminoid attached via

5.3

3.4

0.08

22.0

Encapsulated salicylate in hollow silica particles

Cap E-Sal

Encapsulated and capped with more SiO2

SiO2 no sunscreen P-Sal

silsesquioxane B-Sal

Salicylate attached via bridged silsesquioxane

Unmodified SiO2 B-Curc

bridged silsesquioxane *BJH desorption cumulative pore volume of pores in the range of 0 - 5000.0 Å # Average pore diameter by BJH

Table 2. Sunscreen Loadings and percent leaching. Sample Theoretical (mg/g) Experimental (mg/g)

% Leached

E-Sal

41.67

8.50

24.17

Cap-E-Sal

32.92

8.64

6.78

P-Sal

56.83

11.67

0.80

B-Sal

98.01

21.56

0.04

B-Curc

143.66

28.77

0.29

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Table 3. Summary of sunscreen UV stability data after 6 MED exposure. Sample

UV absorption ability after 6 MED exposure*

2-Ethylhexyl salicylate

75.91 ± 0.26 %

E-Sal

76.82 ± 0.01 %

Cap E-Sal

79.51 ± 0.11 %

P-Sal

82.98 ± 0.63 %

B-Sal

88.40 ± 0.63 %

Free curcumin

57.43 ± 1.66 %

B-Curc

77.28 ± 0.09 %

*Data is the summary of three trials.

Table 4. Summary of in vitro SPF data for salicylate and curcuminoid particles before and after UV exposure. Sample SPF* % SPF maintained after % SPF maintained after 0.5 MED exposure

6.0 MED exposure

E-Sal

3.72

89.23

77.13

P-Sal

0.21

92.87

81.63

B-Sal

0.55

97.98

86.53

B-Curc

0.85

97.06

83.74

*Calculated from Equation 256

Table 5. Critical Wavelength of salicylate and curcuminoid particles. Sample E-Sal

Critical Wavelength (nm)* 384

P-Sal

384

B-Sal

379

B-Curc

389

*Calculated from Equation 358,64

42


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Table 6. UVA/UVB ratios and Boot's Star Ratings for salicylate and curcuminoid particles. Boot's Star UVA/UVB Ratio Boot's Star Rating Sample UVA/UVB * 61-64 (6 MED) Rating (6 MED)@ Ratio 0.66 ***(Good) 0.65 ***(Good) E-Sal P-Sal

0.65

***(Good)

0.53

**(Moderate)

B-Sal

0.62

***(Good)

0.54

**(Moderate)

B-Curc

0.93

***** (Ultra)

0.90

****(Superior)

*Calculated from Equation 461-64 Calculated from Equation 561-64

43



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Figure 1. DLS spectra and SEM micrographs of particles prepared via microemulsion polymerization.53 (a) Physically encapsulated salicylate particles (E-Sal), and (b) hollow silica particles made without sunscreen. An example of shell rupturing is circled in red. DLS data represents the average of three trials. Diameters of 743 ± 78 nm and 761 ± 57 nm were seen for (a) and (b) according to SEM. 254x338mm (72 x 72 DPI)


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Figure 2. TEM micrographs of (a) E-Sal, (b) cap E-Sal particles, (c) unmodified silica particles prepared via microemulsion polymerization, and (d) unmodified silica particles prepared via sol-gel polymerization. The shell thicknesses for (a) and (b) are approximately 150 ± 20 nm and 203 ± 29 nm, respectively, implying a cap thickness of 53 nm according to TEM. 254x190mm (72 x 72 DPI)



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Figure 3. DLS spectra and SEM micrographs of (a) unmodified silica particles, (b) pendant salicylate particles (P-Sal), (c) bridged salicylate particles (B-Sal) and (d) bridged curcuminoid particles (B-Curc). DLS data represents the average of three trials. 254x338mm (72 x 72 DPI)


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Figure 4. EDS spectra of (a) P-Sal particles, and (b) B-Sal particles. The P-Sal spectrum details the presence of silicon (Si), oxygen (O), and small amounts of carbon (C) atoms on the particle surface, while the B-Sal spectrum details the presence of only Si and O atoms on the surface. 352x264mm (72 x 72 DPI)



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Figure 5. SEM micrographs of P-Sal particle suspensions used in leaching analysis before and after filtration, detailing successful removal of particles. Micrographs are representative of all particle samples analyzed (data not shown). 352x470mm (72 x 72 DPI)


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Figure 6. Fluorescence spectra of salicylate particle samples before (blue) and after filtration (red). (a) ESal, (b) Cap E-Sal, (c) P-Sal, (d) B-Sal and (e) B-Curc particles. Overall, bridged incorporation was seen to minimize leaching the most. 254x190mm (72 x 72 DPI)



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Figure 7. UV-Vis Absorbance spectra for salicylate samples exposed for 6 h. Incremental percent reduction in UV absorbance at λmax (303 - 315 nm) is also shown for each. 352x470mm (72 x 72 DPI)


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Figure 8. UV-Vis Absorbance spectra for curcuminoid samples exposed for 6 MED, which detail UV-induced degradation over time for each. Incremental percent reduction in UV absorbance at λmax (382 - 402 nm) is also shown for each. 352x264mm (72 x 72 DPI)



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Figure 9. Incremental SPF reduction for salicylate and curcuminoid particles after UV irradiation over time. 352x264mm (72 x 72 DPI)


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Scheme 1. Physical encapsulation of hydrophobic sunscreens using an oil-in-water\r\nmicroemulsion polymerization of tetraethoxysilane. \r\n 254x190mm (72 x 72 DPI)



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Scheme 2. Covalent encapsulation by copolymerization of sunscreen dyes bearing one (A) or two or more (B) triethoxysilyl groups with tetraethoxysilane in an oil in water microemulsion polymerization. 352x264mm (72 x 72 DPI)


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Scheme 3. Capping of physically encapsulated sunscreens by adding a second layer or cap of silica over the first. 254x190mm (72 x 72 DPI)



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Scheme 4. Protocol for synthesis of pendant (2) salicylate precursor. Condensation of salicylic acid with APTES via salicyloyl chloride 1. 254x190mm (72 x 72 DPI)


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Scheme 5. Protocol for synthesis of bridged (4) salicylate precursor. Condensation of condensation of 2hydroxyterephthalic acid with two equivalents of APTES via 2-hydroxyterephthaloyl chloride 3.50,51 254x190mm (72 x 72 DPI)



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Scheme 6. Synthesis of bridged circuminoid 6. 254x190mm (72 x 72 DPI)


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