S i l i c ap a r t i c l e sa r e w i d e l yu s e d i n i n d u s t r y , m e d i c i n e ,a n dn a n o t e c h n o l o g y [ 1 , 2 ] . B u l ka n d s u r f a c ep r o p e r t i e sa r e m o d i fi e d t h r o u g h t h e i n c o r p o r a t i o no f sm a l l m o l e c u l e s s u c ha s s u r f a c t a n t s t o i n t r o d u c ep o r o s i t yd u r i n g s y n t h e s i s ,o rc o a t i n gw i t ho r g a n o f u n c t i o n a l s i l a n e sp o s t -s y n t h e s i s t o im p a r t d e fi n e d s u r f a c e c h em i s t r i e s [ 3 ] . I f t h e s i l a n e f u n c t i o ng r o u p i sa na l k y l -o ra r y l -m o i e t y t h e r e s u l t i n g p a r t i c l eo rg e l i s t e rm e da no r g a n i c a l l y m o d i fi e d s i l i c a (O RMO S I L ) .O r g a n i c m o d i fi c a t i o n sa r eo f t e n i n c o r p o r a t e d a s a f u n c t i o n a lb r i d g e t op r om o t e a d h e s i o nw i t h a d d i t i o n a lm o l e c u l e s .B i om a c r om o l e c u l e s b o n d i n g t os i l i c ap a r t i c l e s ,s u r f a c e s ,a n d w i t h i np o r o u sg e l sc a nb e im p r o v e d t h r o u g h O RMO S I L s e l e c t i o n [ 4 , 5 ] . P r e s e r v i n gimm o b i l i z e db i om o l e c u l ef u n c t i o n ,t h r o u g hd e fi n e do r i e n t a t i o na n d r e t e n t i o no fn a t i v ec o n fi rm a t i o n so n / i n s i l i c a s i sc r i t i c a l f o rb i om e d i c a la n dd i a g n o s t i c sa p p l i c a t i o n s [ 6 ] . I np a r t i c u l a r ,p r o t e i n -s i l i c a c om p o s i t e m a t e r i a l sa r ee x p l o r e d f o ra p p l i c a t i o n i nb i om e d i c a lfi e l d s s u c h a s imm u n o l o g y , c a n c e r r e s e a r c h ,a n dd r u gd e l i v e r y [ 7 -1 0 ] .T h e s eh y b r i d m a t e r i a l sa r ea l s o i n v e s t i g a t e d i n m a t e r i a l s s c i e n c e t e c h n o l o g i e s s u c ha s s e l f -a s s em b l e d m a t e r i a l s ,q u a n t umd o tb i o c o n j u g a t e s , s e n s o r s , a n d i n o r g a n i c m a t e r i a l s s y n t h e s i s [ 1 1 - 1 7 ] .
S i l i c a -b a s e d b i om a t e r i a l s h a v e p o t e n t i a l a p p l i c a t i o n s i n t i s s u e e n g i n e e r i n g , w h e r e b i om i n e r a l i z a t i o n i sn e c e s s a r y f o rb o n ea n d t o o t hr e p a i r , w h i l es im u l t a n e o u s l ys e r v i n ga sad r u g delivery system to stimulate surrounding tissues or prevent infection. Structural integrity can be enhanced through incorporation of fibrillar protein assemblies such as those observed for spider silk. Recombinant spider silk (SS) protein is a promising biomaterial with huge potential in the textiles, biomedical, and manufacturing industry in the form of fibers, films, hydrogels, lyogels, and adhesives [18][19][20][21][22][23][24]. SS based materials have shown potential in tissue engineering through surface modifications that promote fibroblast cell attachment and proliferation [25][26][27][28][29][30]. The extraordinary mechanical strength of SS comes from its β-sheet dominated secondary structures that form the basis for self-assembly through strong physical interactions [14]. However, SS proteins are insoluble in aqueous solution, and conventionally dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to generate spin dopes to create fibers, films, gels, and foams [31][32][33][34]. HFIP, however, is expensive and poses significant health and environmental risks. Recently, a simple microwave method has been introduced to dissolve SS in aqueous solution, providing a cost-effective and green approach to preparing SS-functionalized materials and surfaces [35].
Here, we investigate addition of simple organo-and fluoro-silanes (ORMOSILs and F-ORMOSILs) to SS as a means to tune properties of the resulting sol-gel inorganic/organic composite. Alkoxysilanes are often used as the precursor for silicone, a widely used adhesive to bridge inorganic materials with organic molecules [36]. Hydrolyzed ORMOSILs and F-ORMOSILS exhibit a strong ability to induce secondary structures of globular proteins [37]. We select hydrophobic n-propyltrimethoxy silane (nPM) and 3,3,3-trifluoropropyl trimethoxy silane (3F) as both have been shown to influence the molten globule transition and secondary structures of beta-lactoglobulin and albumin [37], which are low MW, water soluble proteins in contrast to SS. We hypothesize that hydrolyzed nPM and 3F can induce secondary structure in SS and upon condensation form bio-inorganic nanocomposites consisting of SS and silica. Using microwave-assisted dissolution of SS in the presence of hydrolyzed silanes followed by base-initiated condensation yields hybrid spider silk-silica nanocomposites under purely aqueous conditions. Spherical sub-micron particles are observed for both 3F and nPM, however, the two silanes distinctly influence the SS secondary structures as determined by FTIR analysis. The β structural content increased by 10% in the SS-nPM nanocomposites, but decreased by a net 28% in the SS-3F nanocomposites. The ability to tune protein secondary structures in the protein-ORMOSIL composite may provide a means to control subsequent growth processes, such as biomineralization with which biological function and mechanical properties of these composite organic/inorganic biomaterials can be modified.
The integration of spider silk protein with organo-and fluoro-silanes, referred to as the SS-silica nanocomposites is detailed in the Materials Section. The hybrid particles are prepared by addition of the synthetic spider silk into the acid-hydrolyzed silane solutions followed by microwave-induced solubilization of the SS protein then addition of base to induce condensation. The SS-silica nanocomposites were assessed using SEM, AFM, and infrared spectroscopy. In Figure 1, SEM images reveal nanoparticles (NPs) synthesized from the pure silanes (top row) and the SS-silica hybrid particles (bottom row). The pure fluoro (3F) and organo (nPM) silane NPs formed through the acid/base two-step method form highly regular spheres with sizes generally ranging between 350 and 550 nm. These particles resemble typical NPs prepared via Stöber sol-gel methodology; however, the 3F and nPM particles are prepared without any alcohol as co-solvent. The SS-silica nanocomposite particles, Figure 1, bottom row, are notably smaller than the pure silane controls in the top row. These images also suggest that the SS-nPM particles (Figure 1b) are more monodisperse (<50 nm) than the SS-3F particles in Figure 1d where particle sizes are multimodal, with sizes <50 nm and >300 nm clearly observed. Both SS-nPM and SS-3F particles appear highly aggregated under SEM observation. The morphological differences among the SS-silica nanocomposites may arise from templating effects of the protein on the sol-gel process, apparently favoring formation of smaller NPs. From the SEM data it, is uncertain if the larger particles in the SS-3F synthesis are also hybrid particles, or whether these reflect a competing process of forming pure silane particles. However, we believe the protein and silane to be integrated into composite particles for the SS-3F synthesis based on the following: (1) There was no evidence of protein fibrils in the SEM and AFM images of the SS-3F systems; (2) the 3F to SS molar stoichiometry was 200:1, which is selected to provide sufficient silane (~200 Da) to interact with SS protein (~100 kDa); and (3) the decrease in particle size when the sol-gel process occurred in the presence of SS protein indicated some type of templating or interaction between organic/inorganic components. Differences between nPM and 3F interactions with SS protein in their hydrolyzed states are also possible, with hydrolyzed 3F exhibiting a higher dipole moment than nPM due to the strong electronegativity of the tri-fluoro moiety pendent to the Si atom in 3F. These effects could yield the differences in the 3F-SS and nPM-SS particles observed in the SEM images.
AFM was employed as a complementary imaging modality, and in particular, is highly sensitive to analyzing pure SS fibrils, as shown in Figure 2a. No fibrillated proteins were observed upon addition of either silane, as shown in Figure 2b,c. During the microwave synthesis, the SS proteins apparently integrated with the nPM and 3F hydrolyzed monomers and condensation products, thus inhibiting silk protein self-assembly into the characteristics fibrils shown in Figure 2a. The SS-nPM silica nanocomposites and SS-3F silica nanocomposites shown in panels b and c, respectively, reveal the particles to be highly aggregated as observed in the SEM images. Both SS-nPM and SS-3F particles appear highly aggregated under SEM observation. The morphological differences among the SS-silica nanocomposites may arise from templating effects of the protein on the sol-gel process, apparently favoring formation of smaller NPs. From the SEM data it, is uncertain if the larger particles in the SS-3F synthesis are also hybrid particles, or whether these reflect a competing process of forming pure silane particles. However, we believe the protein and silane to be integrated into composite particles for the SS-3F synthesis based on the following: (1) There was no evidence of protein fibrils in the SEM and AFM images of the SS-3F systems; (2) the 3F to SS molar stoichiometry was 200:1, which is selected to provide sufficient silane (~200 Da) to interact with SS protein (~100 kDa); and (3) the decrease in particle size when the sol-gel process occurred in the presence of SS protein indicated some type of templating or interaction between organic/inorganic components. Differences between nPM and 3F interactions with SS protein in their hydrolyzed states are also possible, with hydrolyzed 3F exhibiting a higher dipole moment than nPM due to the strong electronegativity of the tri-fluoro moiety pendent to the Si atom in 3F. These effects could yield the differences in the 3F-SS and nPM-SS particles observed in the SEM images.
AFM was employed as a complementary imaging modality, and in particular, is highly sensitive to analyzing pure SS fibrils, as shown in Figure 2a. No fibrillated proteins were observed upon addition of either silane, as shown in Figure 2b,c. During the microwave synthesis, the SS proteins apparently integrated with the nPM and 3F hydrolyzed monomers and condensation products, thus inhibiting silk protein self-assembly into the characteristics fibrils shown in Figure 2a. The SS-nPM silica nanocomposites and SS-3F silica nanocomposites shown in panels b and c, respectively, reveal the particles to be highly aggregated as observed in the SEM images.
ATR-FTIR spectra (Figure 3) showed some major conformational changes in the amide I zone (1575 cm -1 to 1725 cm -1 ) of SS proteins within the SS-nPM silica nanocomposites, and SS-3F silica nanocomposites compare to the SS proteins alone. Figure 3 also includes spectra for the pure nPM and 3F silica NPs. With the amide I and amide II zones, other bending and stretching peaks are also shown in the respected spectra. Spectra representing 3F based silica NPs and SS-3F based silica nanocomposites show the C-F stretching and deformation at 840 cm -1 , 1210 cm -1 , and 1260 cm -1 [38]. Peaks at 1210 cm -1 representing the deformation vibration of Si-CH 2 -exist in both 3F and nPM, causing the increased adsorption intensity at 1210 cm -1 in 3F based silica NPs and SS-3F silica nanocomposites as it overlaps with C-F vibration [39]. Strong bands between 1210-1000 cm -1 are due to the stretching v i b r a t i o no fS i -O -S io b s e r v e d i na l l s p e c t r a e x c e p t t h eS S s p e c t r um c o n fi rm t h ep r e s e n c eo f3Dp o l ym e r i c n e tw o r ko fS iO 2 [ 3 9 , 4 0 ] . A n o t h e ra d s o r p t i o nb a n da r o u n d8 0 0cm -1 i sa l s oo b s e r v e d r e p r e s e n t i n g S i -O -S i i na l l t h e s p e c t r ao f s i l i c a -c o n t a i n i n g s am p l e s [ 4 1 h y d r o l y z e d s t a t e s a r e a l s o p o s s i b l e , w i t h h y d r o l y z e d 3 F e x h i b i t i n g a h i g h e r d i p o l e m om e n t t h a n n PM d u e t o t h e s t r o n g e l e c t r o n e g a t i v i t y o f t h e t r i -f l u o r o m o i e t y p e n d e n t t o t h e S i a t om i n 3 F . T h e s e e f f e c t s c o u l d y i e l d t h e d i f f e r e n c e s i n t h e 3 F -S S a n d n PM -S S p a r t i c l e s o b s e r v e d i n t h e S EM im a g e s .
A FM w a s em p l o y e d a s a c om p l em e n t a r y im a g i n g m o d a l i t y , a n d i n p a r t i c u l a r , i s h i g h l y s e n s i t i v e t o a n a l y z i n g p u r e S S f i b r i l s , a s s h ow n i n F i g u r e 2 a . N o f i b r i l l a t e d p r o t e i n s w e r e o b s e r v e d u p o n a d d i t i o n o f e i t h e r s i l a n e , a s s h ow n i n F i g u r e 2 b , c . D u r i n g t h e m i c r ow a v e s y n t h e s i s , t h e S S p r o t e i n s a p p a r e n t l y i n t e g r a t
] .
of SS protein with nPM and 3F during the formation of nanocomposites. The deconvoluted FTIR spectra of SS, SS-3F, and SS-nPM are shown in Figure 4a-c, respectively. Figure 4d provides a comparison of the secondary structure content in the corresponding SS proteins. In SS-3F silica nanocomposites, β-sheet was reduced by 20%, and β-turn reduced by 41% compared to the spectra reporting these secondary structures in SS proteins alone. In the SS-3F silica nanocomposites two new secondary structures corresponding to a 3 10 -helix appeared and was 25% of the total secondary structures present in the SS-3F nanocomposites.
Molecules 2018, 23, x 5 of 9 4d provides a comparison of the secondary structure content in the corresponding SS proteins. In SS-3F silica nanocomposites, β-sheet was reduced by 20%, and β-turn reduced by 41% compared to the spectra reporting these secondary structures in SS proteins alone. In the SS-3F silica nanocomposites two new secondary structures corresponding to a 310-helix appeared and was 25% of the total secondary structures present in the SS-3F nanocomposites. An opposite trend was observed in the secondary structures in the amide I zone of SS-nPM silica nanocomposites, where both of the primary β structures (sheet and turn) increased. In these nanocomposites, β-sheet increased by 14%, β-turn and α-helix decreased by 4% and 26%, respectively. These patterns of conformational changes in the secondary structures of SS proteins are consistent with conformational changes observed in BLG protein, as reported by Peng et al. [37]. BLG is a relatively low MW (~18.4 kDa), water soluble, globular protein in contrast to the 65-120 kDa hydrophobic SS proteins investigated here. [42] The solubilization of the SS proteins in the presence of the silanes through microwave energy solubilization procedure provides a facile means to synthesis hybrid silane-biomacromolecule nanoparticle-microparticle composites.
3,3,3-trifluoropropyl trimethoxy silane (3F, > 95% purity, MW = 218.3, d = 1.14 g/mL), and n-propyltrimethoxy silane (nPM, > 95% purity, MW = 164.3, d = 0.94 g/mL) were purchased from Gelest, Inc. (Morrisville, PA, USA). 50% (w/w) NH4OH was purchased from Fisher Scientific. Muscovite mica was purchased from SPI supplies for AFM study (West Chester, PA, USA). AFM tips were obtained from TED Pella Inc., Redding, CA (TAP300AL-G-50). Recombinant spider silk protein was obtained from R. Lewis, Utah State University Department of Biology. Two different An opposite trend was observed in the secondary structures in the amide I zone of SS-nPM silica nanocomposites, where both of the primary β structures (sheet and turn) increased. In these nanocomposites, β-sheet increased by 14%, β-turn and α-helix decreased by 4% and 26%, respectively. These patterns of conformational changes in the secondary structures of SS proteins are consistent with conformational changes observed in BLG protein, as reported by Peng et al. [37]. BLG is a relatively low MW (~18.4 kDa), water soluble, globular protein in contrast to the 65-120 kDa hydrophobic SS proteins investigated here. [42] The solubilization of the SS proteins in the presence of the silanes through microwave energy solubilization procedure provides a facile means to synthesis hybrid silane-biomacromolecule nanoparticle-microparticle composites.
3,3,3-trifluoropropyl trimethoxy silane (3F, > 95% purity, MW = 218.3, d = 1.14 g/mL), and n-propyltrimethoxy silane (nPM, > 95% purity, MW = 164.3, d = 0.94 g/mL) were purchased from Gelest, Inc. (Morrisville, PA, USA). 50% (w/w) NH 4 OH was purchased from Fisher Scientific. Muscovite mica was purchased from SPI supplies for AFM study (West Chester, PA, USA). AFM tips w e r eo b t a i n e d f r omT EDP e l l a I n c . ,R e d d i n g ,CA ( TA P 3 0 0A L -G -5 0 ) .R e c om b i n a n t s p i d e r s i l kp r o t e i n w a so b t a i n e d f r omR .L ew i s ,U t a hS t a t eU n i v e r s i t yD e p a r tm e n to fB i o l o g y .Tw od iff e r e n t t y p e so f S Sp r o t e i ns u c ha s m a j o ram p u l l a t es i l kp r o t e i n rM a S p 1a n d m a j o ram p u l l a t es i l kp r o t e i n rM a S p 2 w e r ep u r i fi e d f r om t h e m i l ko f t r a n s g e n i cg o a t s t h r o u g h t a n g e n t i a lfl owfi l t r a t i o n ,p r e c i p i t a t i o n ,a n d w a s h i n g ,y i e l d i n g6 5 -1 2 0kD ap r o t e i n s . [4 2 ] T h eh y b r i d s i l a n e / p r o t e i n c om p o s i t e sw e r ep r e p a r e db yfi r s tp r e p a r i n g t h e s i l a n e s o l u t i o n s .3 F ( 0 . 4 M )a n dn PM ( 0 . 4 M ) w e r eh y d r o l y z e da tpH3 . 0 i na q u e o u s m e d i a i ns e p a r a t ev i a l s . Tw oS S p r o t e i n s ( rM a S p 1 a n d rM a S p 2 )w e r e m i x e dw i t ha r a t i oo f5 0 : 5 0 [ 1%w/ vo r0 . 0 0 1 M )a n da d d e d i n5 m L3 Fa n dn PM m e d i a s e p a r a t e l ya f t e rd i l u t i n gh y d r o l y z e d3 Fa n dn PM s o l u t i o n t o0 . 0 2 M w h i c h m a k e s t o i c h i om e t r yo f3 F/ n PM : S Sa s2 0 0 : 1 .T h ep r o t e i n r em a i n e da sav i s i b l ew h i t ea g g r e g a t e i n t h e s o l u t i o n .T h e s e m i x e d s o l u t i o n sw e r e t h e n m i c r ow a v e d (H a i e r m i c r ow a v e ( 7 0 0 W ) f o r8 s , f o l l ow e d b ya3 0 sp a u s e , f o r4 t im e s i na t i g h t l yc l o s e dg l a s sv i a l s t od i s s o l v e t h eS S .F o l l ow i n g4c y c l e s , t h e t em p e r a t u r e i n t h ev i a l s r e a c h e d1 3 0 • Ca n d t h e r e s u l t i n g s o l u t i o n sw e r e c l e a r .
F o l l ow i n g m i c r ow a v e -f a c i l i t a t e dd i s s o l u t i o no f t h eS Sp r o t e i n , t h e s o l u t i o n sw e r ec o o l e d t o2 3 • C u s i n ga n i c ew a t e rb a t h . A5 m La l i q u o to f t h e s o l u t i o nw a s r em o v e d , s t i r r e da t5 0 0 r pm ,a n d5 0µ L o f NH 4 OH w a sa d d e dd r o pw i s e t oc a t a l y z e3 F/ n PMc o n d e n s a t i o n . Ac l o u d y s o l f o rm e da n da f t e r 2h t h e s o l u t i o nw a s t r a n s f e r r e d t o1 5 m L c e n t r i f u g e t u b e s , c e n t r i f u g e da t4 0 0 0 r pm t o f o rmap e l l e t . T h es u p e r n a t a n t w a sd i s c a r d e da n dp e l l e t r e s u s p e n d e d i nD I w a t e r . t y p e s o f S S p r o t e i n s u c h a s m a j o r am p u l l a t e s i l k p r o t e i n rM a S p 1 a n d m a j o r am p u l l a t e s i l k p r o t e i n rM a S p 2 w e r e p u r i f i e d f r om t h e m i l k o f t r a n s g e n i c g o a t s t h r o u g h t a n g e n t i a l f l ow f i l t r a t i o n , p r e c i p i t a t i o n , a n d w a s h i n g , y i e l d i n g 6 5 -1 2 0 kD a p r o t e i n s . a c i l i t ya tU t a hS t a t eU n i v e r s i t y .S am p l e s w e r e im a g e d w i t h2 0kVa c c e l e r a t i n gp o t e n t i a l w i t h o u t c o n d u c t i v e c o a t i n g s . A FM im a g e sw e r e t a k e nu s i n gN a n o s c o p e I I IB i o s c o p e (D i g i t a l I n s t r um e n t , I n c . , C am a r i l l o ,CA ,U SA ) i n t a p p i n g m o d e w i t hA l -c o a t e dB S -T a p3 0 0c a n t i l e v e r s f r omB u d g e tS e n s o r , Sofia, Bulgaria. 100 µL samples were drop-cast on freshly cleaved mica surfaces then gently washed with DI water and air-dried before taking AFM images.
Bonding between spider silk protein and both fluorinated and methylated silica were analyzed by FTIR using a Varian 660-IR (Agilent, Santa Clara, CA, USA) with a horizontal single reflection Pike Technologies MIRacle attenuated total reflectance (ATR) unit, fitted with a ZnSe crystal. 100 µL samples were drop cast on the ZnSe crystal of the ATR platform and air-dried before taking the reading. Readings were taken after averaging 20 scans over the range of 600 cm -1 to 1800 cm -1 with a resolution of 1 cm -1 . Prior to each reading, a background scan was acquired. The ATR-FTIR spectra were later deconvoluted using OriginPro, OriginLab Corporation (Northampton, MA, USA), to analyze the transformation in the secondary structures of spider silk protein.
An aqueous sol-gel process combined with microwave-assisted dissolution of hydrophobic synthetic spider silk is demonstrated here to yield silk-silica nanocomposite particles. Sub-micron particles were observed for both silk ORMOSIL (SS-nPM) and silk-F-ORMOSIL (SS-3F) hybrids. Incorporation of SS into the sol-gel process yielded relatively spherical 3F and nPM silk nanocomposites with a greater range of sizes and morphologies, as contrasted with the pure nPM and 3F silica NPs. In the absence of the two hydrophobic silanes, pure SS assembled into fibrillar strands exhibiting strong amide I and II peaks in the ATR-FTIR spectra. Shifts in these peaks in the nanocomposites further confirmed an intimate integration of the SS protein with the silanes in the nanocomposites. However, the influence of the organo-and fluoro-silanes on the SS secondary structures were distinct. Deconvoluted ATR-FTIR spectra showed the increased β structures in SS-nPM silica nanocomposites and decreased β structures in the SS-3F silica nanocomposites. The ability to induce defined secondary structures in the protein-silane hybrid particles may allow for bottom-up design of bioactive particles, surfaces, and monoliths where subsequent epitaxial growth and biomineralization can be tuned for user-defined applications.