Optically Active Hybrid Materials Based on Natural Spider Silk | ACS

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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 22962−22972

Optically Active Hybrid Materials Based on Natural Spider Silk Aleksandra Kiseleva,† Grigorii Kiselev,† Vadim Kessler,‡ Gulaim Seisenbaeva,‡ Dmitry Gets,† Valeriya Rumyantseva,† Tatiana Lyalina,† Anna Fakhardo,† Pavel Krivoshapkin,† and Elena Krivoshapkina*,† †

ITMO University, Lomonosova Street 9, Saint Petersburg 191002, Russia Department of Molecular Sciences, Biocenter, SLU, P.O. Box 7015, SE-75007 Uppsala, Sweden

‡

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S Supporting Information *

ABSTRACT: Spider silk is a natural material possessing unique properties such as biocompatibility, regenerative and antimicrobial activity, and biodegradability. It is broadly considered an attractive matrix for tissue regeneration applications. Optical monitoring and potential control over tissue regrowth are attractive tools for monitoring of this process. In this work, we show upconversion modification of natural spider silk fibers with inorganic nanoparticles. To achieve upconversion, metal oxide nanoparticles were doped with low concentrations of rare-earth elements, producing potentially biocompatible luminescent nanomaterials. The suggested approach to spider silk modification is efficient and easy to perform, opening up sensing and imaging possibilities of biomaterials in a noninvasive and real-time manner in bio-integration approaches. KEYWORDS: natural spider silk, hybrid material, optically active nanoparticles, zirconia, hafnia, upconversion

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in nerve tissue engineering33 and in the regeneration of peripheral nerves.34 Recombinantly produced engineered spider silk proteins can successfully be used in various forms, from films and coatings35 to particles.36 These morphologies represent original systems with adapted properties regulated by process parameters.37 Such materials have found application in bioengineering as drug carriers in clinical medicine.38 In addition, there are numerous works dedicated to the production of recombinant spider silk protein fibers with their further modification.39 However, effectively mimicking the nanofibrous structures of the extracellular matrix for fabricating biomaterials remains a challenge. The production of spider silk fiber is a very complicated process, requiring variation of the protein concentration, extensional flow, pH, and metal ion concentrations under controlled environmental conditions and following shear stress.40 Despite this, spiders in nature mastered complex silk construction processes millions of years ago. Every day, these animals create fibers with multiscale organization and a controlled fiber diameter or β-sheet crystal size.41 Exploiting such naturally available nanomaterials is a highly attractive prospect, and thus, in the present work, we demonstrate modification of natural spider silk directly obtained from curtain-web spiders (Linothele fallax). However, implementation of silk-based biomaterials for the above-mentioned biomedical applications requires understanding of the behavior of these biomaterials in vivo, which

INTRODUCTION Natural silk materials produced by spiders and silkworms1 have garnered considerable attention in the past two decades due to their extraordinary mechanical properties (high Young’s modulus, high fracture strength, and exceptional extensibility),2,3 biodegradability,4 and biocompatibility.5,6 Moreover, studies suggest that, in comparison to other biomaterials, such as collagen or polylactic acid, silk causes a significantly lower inflammatory response7 and supports proliferation of different cell types.8 In addition, depending on how it is to be used, the degradation rate of silk materials can be modulated, lasting anywhere from days to months.9,10 Furthermore, growth and adhesion factors of silk fibers as well as the genetic adaptability of proteins press for investigation of fibrous protein family for biomaterial applications.11 These properties are particularly significant when designing materials for tissue engineering. Recent results with in vitro bone and ligament formation prove the potential of such biomaterials in future applications. As the variety of silk-like fibrous proteins may be obtained from different spider and insect species, there is an array of natural or bioengineered variations available that may, in turn, be applied to a number of clinical needs. Silk-based materials have been widely utilized as biomedical sutures12 and implantable devices,13 as well as scaffolds14 for tissue regeneration,15−18 and in bone19,20 and cartilage tissue engineering.21 They have also been used in drug delivery22−24 as hydrogels for encapsulation25 and as drug carriers targeting cancer cells26,27 or, more recently, in clinical trials for the renovation of the tympanic membrane28,29 and as coatings for breast implants.30 Silk-based composites have been also used as anti-epileptic drug carriers31 and as a base for neurite growth32 © 2019 American Chemical Society

Received: March 22, 2019 Accepted: June 5, 2019 Published: June 5, 2019 22962

DOI: 10.1021/acsami.9b05131 ACS Appl. Mater. Interfaces 2019, 11, 22962−22972

Research Article

ACS Applied Materials & Interfaces

effectively spin the silk. Living in a special container, L. fallax daily covered the bottom of the container size of 10 × 20 cm with silk, which is 10 mg of spider silk from one individual spider per day. It was possible since L. fallax have long spinning glands and over a thousand microscopic spigots in their spinnerets, each producing one filament of the silk in a film-like form. Once a week, we opened the container, moved the spider into a small plastic container, removed the leftover food, and harvested the spider silk in the form of a fabric. Thus, the spider silk samples were collected without use of any forced reeling. We used dragline silk, which spiders use as a salvation and main constructing material, obtained from the major ampullate gland. Synthesis. The nanoparticles (NPs) were prepared via the sol−gel method. The hafnia and zirconia sol−gels were produced from the hydrolysis−polycondensation process of hafnium butoxide and zirconium propoxide, respectively. The water necessary for the controlled hydrolysis−condensation processes came from the acetic acid esterification.60 Trivalent rare-earth acetate hydrate salts were added to the precursor sol solution. The ratio of reagents was as follows: 1.0 mL of hafnium butoxide, 6.0 mL of isopropanol, 0.3 mL of acetic acid, and 0.01 mL of HCl were used for hafnia synthesis, while 1.0 mL of zirconium propoxide solution, 13.3 mL of isopropanol, 0.6 mL of acetic acid, and 0.01 mL of HCl were used for zirconia synthesis. First, the desired amounts of erbium acetate hydrate (1 mol %) and ytterbium acetate hydrate (1 mol %) were dissolved in propanol-2 under stirring at 60 °C. A metal alkoxide was then added to the solution under an argon atmosphere, with constant stirring for 30 min at 60 °C. Next, acids were added to the solution under stirring for 4 h at 60 °C. Subsequently, the resulting mixture was left in a closed container for gelation for 12 h. The gel was then annealed in a LOIP-LF muffle furnace at 800 °C at a heating rate of 1 °C/min before being cooled to room temperature at a cooling rate of 0.5 °C/min. Fabrication of the Upconversion Nanoparticle-Modified (Coated) Silk by Electrostatic Assembly. UCNPs were dissolved in propanol-2 (100 mg/mL, pH = 6.5 for hafnia and zirconia). Spider silk fiber (100 mg) was added to 20 mL of UCNP solution and then stirred at room temperature overnight. All colloidal systems were stable during the whole experiment; no coagulation of the nanoparticles occurred. The resulting modified samples were alternately washed three times with deionized water and propanol-2 and then dried in a LOIP-LF-25/350-VS2 drying furnace in air at 50 °C for 6 h. Repeatedly washing the samples in different solvents allows excess particles that have not interacted with the fibers to be removed. Therefore, the nanoparticles remaining on the fiber surface after washing were firmly fixed and were capable of resisting the shear stress of washing. Cell Cultures. Human embryonic lung fibroblasts (HELF) and human malignant epithelial cells (HeLa) obtained from Biolot (Saint Petersburg, Russia) were maintained in Dulbecco’s modified Eagle’s medium (DMEM-lg, Biolot) and Eagle’s medium (EMEM, Biolot), respectively, and supplemented with 10% fetal bovine serum (FBS, Gibco) and gentamicin (50 μg/mL, Biolot) at 37 °C and 5% CO2. The cells were subcultured regularly using trypsin/EDTA (Gibco). Cytotoxic Assay. The quantity of surviving cells was indirectly determined by measuring the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction by NAD(P)Hdependent cellular oxidoreductase enzyme.61 MTT is a yellow water-soluble tetrazolium dye that is reduced by live cells to a purple formazan product that is insoluble in aqueous solutions.62 When cells die, they lose the ability to convert MTT into formazan; thus, an observed color change serves as a useful and convenient marker of only the viable cells. The quantity of formazan dissolved in DMSO solution was measured at 570 nm using a Tecan Infinite 50 plate reading spectrophotometer.63 Human malignant epithelial (HeLa) cells and human embryonic lung fibroblasts (HELF) cells in their logarithmic growth phase ((5−10) × 103/well, three wells for each concentration) were plated onto 96-well plates overnight and then treated for 72 h with pristine and doped hafnium and zirconium oxide NPs dissolved directly in the culture medium (final concentrations, 31−500 μg/mL). After the completion of exposure, the medium with

remains a considerable challenge. The use of in vitro degradation tests can deliver a standard setup for characterizing properties of the silk-based biomaterials, but there are still problems in understanding their performance after implantation, both quantitatively and accurately.42 Conservative visualization techniques are often inefficient in monitoring materials in vivo.43−45 Although diagnostic methods such as magnetic resonance imaging or microcomputed tomography may be applied for in situ observations, they are limited by their low resolutions.46−48 Therefore, a novel method of providing biomaterials with noninvasive in vivo bioimaging functions is especially required. On the one hand, spider silk fibers can serve as a bonding base to merge the benefits of function constituents.49,50 On the other hand, the functionalization of the fibers with fluorescent materials provides a nice opportunity for observing the evolution of these fibers together with investigation of biochemical changes of the environment over a period of time.51,52 In this regard, modifying spider silk with selected particles may offer a budding option for bioimaging.51,53 Although the most widely used fluorescence materials are organic dyes, quantum dots, and one-photon fluorescent materials, their excitation wavelengths tend to result in relatively low penetration depths or autofluorescence of background and photo damage to surrounding tissues.54 Since the exciting near-infrared (NIR) light is located in the second biological transparency window, it can intensely penetrate into living tissues and allow less tissue scattering.55 Owing to the ability for light emission upon low-energy NIR excitation, lanthanide-doped upconversion nanoparticles (UCNPs) have attracted increasing attention in biological imaging.56,57 In contrast to quantum dots and organic dyes, UCNPs have been widely accepted as favorable advantageous alternatives. In comparison to UV excitation, application of a low-power NIR laser results in less photo damage to tissues and efficient realization of the upconversion process.58 Besides, with NIR excitation, the sensitivity of quantitative analysis and signal-to-noise ratio are notably higher due to the absence of scattering from samples and autofluorescence of the tissues.59 In this work, we tried to realize the modification with UCNPs directly on spider silk fibers while avoiding the use of costly instruments and any covering polymers. Therefore, by means of directly attaching UCNPs to the spider silk fibers, we aimed to obtain materials with a broad spectrum of optical activity using the upconversion luminescence (UCL) technique. This permitted sensing and imaging of biomaterials in a noninvasive and real-time manner approach.

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EXPERIMENTAL METHODS

Materials. Hafnium(IV) n-butoxide (99%, Aldrich), zirconium(IV) propoxide solution (70%, Aldrich), erbium(III) acetate hydrate (99.9%, Aldrich), ytterbium(III) acetate hydrate (99.95%, Aldrich), acetic acid (98%, Chimmed), hydrochloric acid (36%, Chimmed), propanol-2 (Chimmed), Nafion solution (Aldrich), thiazolyl blue tetrazolium bromide (MTT) (Aldrich), dimethyl sulfoxide (DMSO) (VWR), and PBS tablets (Gibco) were used without further purification. Spider silk was harvested from the spiders L. fallax, which were kept in a clean environment where the relative humidity was maintained between 70 and 90% and the temperature was about 25 °C. The spiders were kept in the absence of sunlight in the containers equipped with humidity and temperature sensors, lifting covers, and water bowls. The spiders were fed crickets once a week. By creating favorable conditions for keeping spiders, we were able to make them 22963


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ACS Applied Materials & Interfaces crystals was discarded, and 200 μL of MTT solution in PBS (0.5 mg/ mL) was added. This medium was left to sit for 1.5 h at 37 °C in a CO2 incubator. Then, the MTT solution was aspirated, and the formazan granules were dissolved in 200 μL of DMSO. The optical density was measured at 570 nm on a Tecan Infinite 50 spectrophotometer (Austria). Cell viability was calculated as the percentage of optical densities in wells, with each concentration of pristine and doped hafnium and zirconium oxide NPs normalized to the optical density of untreated cells (100%). Bioimaging Visualization. The UCNP-modified spider silk was disinfected with ethanol and attached to the bottom of a Petri dish using Nafion solution. After that, the Petri dish was filled with HeLa cell culture and nutrient medium and incubated for 24 h at 37 °C in a CO2 incubator. Mechanical Characterization. The individual native spider silk fibers and UCNP-modified spider silk fibers were mechanically tested as follows. The average cross-sectional diameters of the fibers were estimated using an optical microscope. The fibers were mounted on aluminum foil holders with a base length of 15 mm and fixed with tape and glue. The initial distance between the two glued ends of the fiber was measured with a caliper. A measured error was 0.1 mm. Then, the holder was secured to a frame. The upper part of the frame was affixed to the upper grip for the strain rate control. The lower part of the frame rested on a precision balance for force measuring, as described elsewhere.64 The fiber was then stretched until it broke. Tensile tests were performed at a constant speed of 1 mm/min. Single-fiber testing was performed in air at ambient conditions (22 °C and 20−30% of the relative humidity). We calculated the mechanical properties of the tested fibers using Excel.65 Characterization. The morphology of the spider silk fibers was investigated by scanning electron microscopy (SEM, Tescan Vega 3 SBH, detector of secondary electrons). The zeta potential and hydrodynamic radius were measured by dynamic light scattering using a Photocor Compact-Z analyzer. High-resolution transmission electron microscopy (HRTEM) measurements were carried out on a JEOL-2010 operating at 100 kV. Fourier transform infrared (FTIR) spectra of samples were obtained using a Prestige 21 FTIR spectrometer (Shimadzu, Japan) equipped with a DLaTGS detector; the measurements were carried out in the range from 400 to 4000 cm−1 of the electromagnetic spectrum in transmission mode. Dry samples were mixed with crystalline KBr (2 mg of sample per 700 mg of KBr) and then pressed into a disk. The spectra were collected at a resolution of 0.5 cm−1 with 20 cumulated scans, Happ-Genzel apodization, and a signal-to-noise ratio of 40000:1. The data were processed using software provided by the manufacturer (Shimadzu). The background was collected before accumulating the FTIR spectra of the samples. Spectral data treatments included three-point baseline corrections and Sm-smoothing (10 points). The phase composition of the samples was examined using X-ray diffraction (XRD) powder analysis, which was conducted on a D8 Advance Bruker powder X-ray diffractometer with Cu Kα radiation (λ = 1.5406 E) from 5° to 90° with a step of 0.01°/s and a rate of 5°/min. Photoluminescence and transmittance of samples were carried out with an Ocean Optics QE Pro spectrometer (excitation by HAL 100 and HBO 100). The 980 nm excitation light source was an infrared laser module LSI980-2000 (980 nm, 2000 mW) kit with power supply, display, and adjustment. The estimation of the fibers diameter for tensile test was performed using the optical microscope MEIJI TECHNO DK3000 with a 20× objective magnification. An Instron 4411 tensile testing machine was used to measure the force-extension characteristics of the spider silk fibers. The precision balance used for force measuring was Precisa XT220A, with the resolution of 0.1 mg.

the aggregation rate of the nanoparticles and expansion of the particle size. Thus, to provide straightforward indication for the UCNP immobilization, we investigated surface morphologies of both native and UCNP-modified spider silk fibers using scanning electron microscopy (SEM) (Figure 1). The native

Figure 1. (A) Schematic representation of UCNP modification of native spider silk fibers, (B) SEM images of unmodified spider silk fibers, (C) HfO2:Er,Yb nanoparticle-coated spider silk fibers, and (D) ZrO2:Er,Yb nanoparticle-coated spider silk fibers.

spider silk fibers have smooth surfaces with noticeable furrows aligned across the fiber axis (Figure 1B). On the other hand, stirring of spider silk in the UCNP sol causes the nanoparticles to form a thin monolayer that covers the fibers (Figure 1C,D). UCNP-coated spider silk fibers show an uneven surface. Furthermore, several specks consistently emerge on the coated fiber surfaces. This evidence suggests that hafnia and zirconia UCNPs could be successfully attached to spider silk fibers due to the electrostatic attraction of the positively charged nanoparticles to the negatively charged fibers. Remarkably, under mechanical affection, the fibers tend to form ropes. Moreover, thin fibers are fashioned into the bearing-like structure (Figure 1C,D), which may also be twisted as seen in Figure 1D. According to SEM, the fibers tend to stick together and form “bundle”-like structures after impregnation of the fibers with the nanoparticle solution. It can be supposed that its interaction with a protic medium, especially an aqueous one, results in the activation of hydrogen bonding on the surface of the fibers. The aggregation of fibers apparently leads to a decrease in the surface energy. Upconversion Properties of the Modified Spider Silk Fibers. The hafnium and zirconium oxides were doped with low concentrations of Er3+/Yb3+ pair of rare-earth elements (REE). The codoped hafnia and zirconia NPs were attached to the spider silk fibers, demonstrating a UCL visible to the naked eye (Figure 2F,G). The use of low REE concentrations (1 mol % Er3+/1 mol % Yb3+) was justified for bioapplications since they have low toxicity and high optical response. This may be confirmed by the fact that the REE were not detected by

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RESULTS AND DISCUSSION Spider Silk Fiber Modification. The size of the nanoparticles in coating is much smaller than the diameter of the fibers. Because of this, the nanoparticles could uniformly cover the spider silk fiber. However, their zeta potential appears to be not very high, which may lead to an increase in 22964


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Figure 2. Upconversion emission spectra of spider silk fibers coated with (A) HfO2:Er,Yb and (B) ZrO2:Er,Yb UCNPs under excitation with an NIR laser at 980 nm. Optical microphotographs of (C) native spider silk fibers in a photoluminescence microscope and (D) UCNP-modified spider silk fibers in a photoluminescence microscope under excitation with mercury lamp. (E) Energy transfer (ET) mechanisms showing the upconversion process between Er3+ and Yb3+ ion-codoped UCNPs. Digital photo of upconversion luminescence of spider silk modified with (F) upconversion HfO2:Er,Yb and (G) ZrO2:Er,Yb under excitation with an NIR laser at 980 nm.

Er3+ ion may be excited by the ET process from the 2F5/2 energy level of the Yb3+ ion to the 4I11/2 energy level of the Er3+ ion, the Er3+ ion may be also excited by NIR light from its ground state 4I15/2 to the 4I11/2 level directly also. This route may be summarized as 2F5/2 (Yb3+) + 4I15/2 (Er3+) → 2F7/2 (Yb3+) + 4I11/2 (Er3+) and can be achieved due to the proximity of the 2F5/2 energy level of the Yb3+ ion to the 4I11/2 energy level of the Er3+ ion.66 However, in the excitation of the Er3+ ion, the ET process from the Yb3+ ion to the Er3+ ion is dominant over the abovementioned ECA process of interactions of two Er3+ ions because the absorption cross section of the Yb3+ ion at 975 nm is superior to that of Er3+ in the 4I11/2 state.67 Therefore, the use of erbium and ytterbium combination seems to be very promising. When Er3+/Yb3+-codoped samples are excited by an NIR laser at 980 nm, part of the excitation energy of the Er3+ ion tends to relax nonradiatively from the 4I11/2 level to the 4I13/2 level following the ET process from the Yb3+ ion by multiphonon relaxation or direct Er3+ ion excitation.68 Moreover, bridging the 4I11/2 → 4I13/2 (3619 cm−1) or 4S3/2 → 4F9/2 (3217 cm−1) energy gaps usually requires more than six phonons and therefore is unlikely to happen due to the relatively low phonon energies of zirconia and hafnia oxide matrices.69,70 Besides, the emission process of six phonons is extremely unlikely; hence, the transition process in Er3+ ions takes place via a two-step ET process from Yb3+ to neighboring Er3+ ions, which follows the 2F7/2 → 2F5/2 excitation process of the Yb3+ ion, as shown in Figure 2E, conveying Er3+ ions to the

energy-dispersive X-ray spectroscopy (EDX) analysis (Figures S1 and S2) due to the low concentrations of these elements in the NPs. The proposed approach shows that it is possible to affix UCNPs directly to organic spider silk fibers without causing the destruction of the protein fibers or elimination of upconversion luminescence (Figure 2). The successful UCNP modification of spider silk is demonstrated in Figure 2C,D. The natural spider silk fibers give blue luminescence under UV excitation due to its protein nature. The attached UCNP crystals to the modified spider silk fibers produce visible green light under UV excitation. To investigate the upconversion properties of UCNPmodified spider silk fibers, room-temperature UCL spectra were obtained, as seen in Figure 2. UCL spectra and energy transitions of spider silk modified with UCNPs under NIR 980 nm excitation are shown in Figure 2A,B, from which three clear groups of peaks between 500 and 700 nm can be observed. As presented in Figure 2, the two green emissions around 523 and 546 nm are assigned to the radiative transition of 2H11/2 → 4 I15/2 and 4S3/2 → 4I15/2 of Er3+ ions, respectively. The red emission band from 650 to 673 nm is attributed to the radiative transition of 4F9/2 → 4I15/2 of Er3+ ions. The obtained data are in good agreement with the well-known model of UCL in Er3+/Yb3+-codoped NPs. A schematic energy level diagram of Er3+/Yb3+ and the energy transfer process are displayed in Figure 2E. The presence of both green and red emission bands is explained by the origin of two different processes: energy transfer (ET) and excited state absorption (ESA). While an 22965


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Figure 3. FTIR spectra of unmodified spider silk fibers (blue line), UCNP-coated spider silk fibers (pink line), and (A) powdered HfO2:Er,Yb and (B) ZrO2:Er,Yb UCNPs (purple line).

Figure 4. Representative XRD patterns of native spider silk fibers (blue), UCNP-coated spider silk fibers (purple), and powdered (A) HfO2:Er,Yb and (B) ZrO2:Er,Yb UCNPs (pink); black and gray bars indicate JCPDS card data.

F7/2 state.71 Subsequently, the populated 4F7/2 level of the Er3+ ion shortly relaxes nonradiatively to the lower 2H11/2 and 4S3/2 levels due to the smaller energy gaps of 4F7/2 → 2H11/2 (1162 cm−1) and 2H11/2 → 4S3/2 (794 cm−1). Therefore, two green emissions are observed since the above-described process causes 2H11/2 → 4I15/2 and 4S3/2 → 4I15/2 transitions centered at 523 and 546 nm, respectively. Also, the red emission band can be assigned to the 4F9/2 → 4I15/2 de-excitation process due to the population on the 4F9/2 level, which possibly comes from the 4F7/2 + 4I11/2 → 4F9/2 + 4F9/2 cross-relaxation process. The presence of the spider silk was not observed to affect the optical properties of the obtained upconversion materials. Fourier Transform Infrared Spectroscopy (FTIR). Figure 3A,B shows the FTIR spectra of UCNP-modified spider silk fibers compared with native spider silk fibers and powdered hafnia and zirconia UCNPs in the region between 350 and 4000 cm−1. On the IR spectra of annealed Er3+/Yb3+codoped hafnium and zirconium oxide NPs, a broad peak is observed in the region of 400−800 cm−1, which is a characteristic peak of metal oxides. The IR spectra of the native spider silk fibers reveal characteristic peaks of polypeptide absorbance. Two main absorption bands of amide I (1650 cm−1) and amide II (1540 cm−1) are observed due to stretching of CO bonds (amide I) and planar 4

deformation vibrations of the N−H bond (amide II), respectively. A clear peak at 1230 cm−1 appears due to mixed C−N stretching and N−H bending vibrations of amino acids of the spider silk proteins. There is also a clear peak at about 3300 cm−1 due to stretching vibrations of the N−H bond.72 The FTIR data demonstrate that the peptide backbone remains unaffected by interaction with the particles. With regard to the IR spectra of the hybrids, the position of the peaks characteristic of polypeptides is preserved, and an intense narrow band appears in the region of 2340 cm−1, corresponding to multiple/conjugated CC and CN bonds resulting supposedly from dehydration of the proteins upon action of metal oxides as catalysts.73 Spider silk contains mostly amino acids with chemically inert residues such as glycine and alanine but also glutamine, serine, leucine, valine, proline, tyrosine, and arginine with CH2−CH2 fragments that can relatively easily be transformed into CHCH ones upon contact with oxide surfaces. Phase Composition Analysis. For additional proof of spider silk fiber modification, X-ray diffraction (XRD) analysis was carried out. The crystallinity of the hafnia and zirconia UCNPs was confirmed by the XRD patterns, as presented in Figure 4A,B, respectively. As can be observed from the XRD spectra of Er3+/Yb3+-codoped HfO2 in Figure 4A (pink line), 22966


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Figure 5. Characterization of nanoparticles. (A) Hydrodynamic nanoparticle size in solution; (B) digital photos of sols of HfO2:Er,Yb (a) and ZrO2:Er,Yb (b) under UV excitation; (C, D) high-resolution TEM images of HfO2:Er,Yb and ZrO2:Er,Yb nanoparticles, respectively; photoluminescence spectra of (E) HfO2:Er,Yb and (F) ZrO2:Er,Yb nanoparticles and insets showing the transmission spectra of UCNPs.

31.84, 34.48, 35.61, 49.67, and 50.68 in the coated sample. They can be assigned respectively to the (110), (011), (−111), (111), (200), (002), (220), and (022) planes of the monoclinic HfO2:Er,Yb NPs, signifying the attachment of the UCNPs to the spider silk fibers. Similarly, after spider silk modification with ZrO2 UCNPs (Figure 4B, purple line), there appear additional peaks at 2θ = 24.46, 24.88, 28.68, and 31.94 in the coated sample. These can be respectively assigned to the (110), (011), (−111), and (111) planes of the monoclinic ZrO2:Er,Yb NPs along with peaks at 2θ = 30.64, 34.60, 35.61, 49.59, and 50.63, which can be assigned to the (111), (002), (200), (202), and (220) planes of the tetragonal ZrO2:Er,Yb NPs, respectively. This fact indicates successful attachment of the UCNPs to the spider silk fibers. Peaks ascribed to other coating materials are not seen. It should be noted that these peaks are slightly shifted to the right. Surface interactions of the particles may influence the structural strain on the nanoparticles, resulting in slight contraction of the unit cell parameters. Thus, XRD results verify the successful modification of spider silk fibers with the crystalline UCNPs. Nanoparticle Characterization. To achieve upconversion properties of the nanoparticles, the oxide matrices were doped with a pair of Er3+/Yb3+ ions known for its excellent UCL properties.56 The distribution and the local environment of lanthanide ions in a host matrix influence the upconversion efficiency. Moreover, a lower host phonon energy possesses a larger quantity of phonons connecting the emitting level with

the structure was proven to be a monoclinic phase, in accordance with the pattern of standard Joint Committee of Powder Diffraction Standards (JCPDS) card no. 34-104, as shown in Figure 4A by the black line. Additionally, a d-spacing value of 0.315 nm for the (−111) plane can be calculated with the Bragg equation based on 2θ (28.37°), which is in good agreement with high-resolution TEM data. Likewise, the XRD pattern of Er3+/Yb3+-codoped ZrO2 depicted in Figure 4B with a pink line corresponds correctly to the ZrO2 tetragonal (JCPDS card no. 17-923) and monoclinic phases (JCPDS card no. 37-1484), as shown in Figure 4B with gray and black lines, respectively. Moreover, the d-spacing value of 0.315 nm for the (−111) plane can be calculated with the Bragg equation based on 2θ (28.18°), which is in good agreement with that measured with high-resolution TEM. The XRD patterns of spider silk fibers before and after UCNP modification are shown in Figure 4A,B with blue and purple lines, respectively. For patterns of unmodified spider silk fibers, there appears one characteristic broad peak, which is located at 2θ = 10.58°, corresponding to typical XRD spider silk patterns.74 Whereas the peaks were not substantially altered after spider silk modification, the alcohol treatment and UCNPs insertion does not seem to affect and reverse the spidroin structure in the spider silk fibers. After modification of spider silk fibers with HfO2 UCNPs (Figure 4A, purple line), there appears a multitude of additional distinguishable peaks at 2θ = 24.44, 24.81, 28.51, 22967


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Figure 6. Results of MTT assays of pure HfO2 and HfO2:Er,Yb NPs after 72 h of exposure on (A) HeLa and (B) HELF cell lines. Results of MTT assays of pure ZrO2 and ZrO2:Er,Yb NPs after 72 h of exposure on (C) HeLa and (D) HELF cell lines. Shown are the average values of three measurements with standard error of the mean. Statistically significant differences are denoted as “*”.

respectively. The HRTEM data show well-defined crystalline structures of the synthesized annealed nanoparticles. To investigate the optical properties of the annealed nanoparticles, room-temperature transmittance and photoluminescence (PL) spectra were obtained. The photoluminescence spectra between 400 and 1000 nm of the Er3+/Yb3+-codoped HfO2 and ZrO2 were measured and are presented in Figure 5E,F. The PL peaks revealing the transitions from the 4I15/2 ground state to excited states of the Er3+ ions are also assigned, which confirm that Er3+ ions can absorb visible light. The green emission at around 525 nm and green emission band from 544 to 546 nm are assigned to the transition of 4I15/2 → 2H11/2 and 4 I15/2 → 4S3/2 of Er3+ ions, respectively. The red emission band from 655 to 680 nm is attributed to the transition of 4I15/2 → 4 F9/2 of Er3+ ions. The emission band from 845 to 870 nm is attributed to the transition of 4I13/2 → 4S3/2 of Er3+ ions. The wide sloping peak from 450 to 700 nm is characteristic of the oxide matrix. The corresponding UV−VIS−NIR transmittance spectra of powdered hafnia and zirconia UCNPs are also shown in the insets in Figure 5E,F. The transmission of hafnia and zirconia is about 1 and 5% in the visible region, respectively, which proves that UCNPs are highly absorbent between 400 and 1000 nm (insets in Figure 5E,F). The transmittance spectra show a clear peak located at 974 nm, which corresponds to the 2F7/2 →

the next lower level. To reduce the nonradiative relaxation probability and intensify the luminescence yield, it is essential to use a lattice with significantly lower phonon energy. The zirconia and hafnia matrices seem to be ideal for the synthesis of strongly luminescent materials due to their chemical and photochemical stability, high refractive index, and low phonon energy.75 The annealed Er3+/Yb3+-codoped oxide nanoparticles had average zeta potentials of 19.2 ± 1.0 mV for hafnia and 14.8 ± 1.3 mV for zirconia. The average hydrodynamic size of UCNPs in sols (Figure 5A,B) observed in the dynamic laser scattering test lies between 30 and 35 nm for hafnia and between 35 and 40 nm for zirconia. The obtained data correlate well with those estimated from HRTEM studies. HRTEM was carried out to explore the crystalline structure of the UCNPs. According to the HRTEM image (Figure 5C,D), the size of the nanoparticles ranges from 20 to 30 nm for HfO2:Er,Yb and from 30 to 40 nm for ZrO2:Er,Yb. Figure 5C displays clear lattice fringes of HfO2 nanoparticles with a P21/a space group value of 0.3123 nm corresponding to the (−111) planes and 0.2830 nm corresponding to the (111) planes of the monoclinic structure of HfO2:Er,Yb. ZrO2:Er,Yb nanoparticles show an identical trend (Figure 5D). P21/a space group values of 0.5034 and 0.3648 nm correspond to the (001) planes and (110) planes of the monoclinic structure of ZrO2:Er,Yb, 22968


Research Article

ACS Applied Materials & Interfaces 2

F5/2 transition of Yb3+ ions. This fact demonstrates that Yb3+ ions absorb the pump light and indicates energy transfer from Yb3+ to Er3+ ions. The transmission peaks that consist of several inhomogeneous broadened f−f transitions centered at around 489, 520, 546, 650, and 789 nm are due to the transitions from the 4I15/2 ground state to the excited 4F7/2, 2 H11/2, 4S3/2, 4F9/2, and 4I9/2 states of the Er3+ ions, respectively. The transmission peak at about 910 nm may correspond to the transitions that are associated with defects in the matrix and the specific distribution of Er3+ and Yb3+ ions. The transmission spectra peaks are in good agreement with peaks in the PL spectra. The peaks corresponding to the energy transitions in the Er3+ and Yb3+ ions are visible both in the transmission spectra and in the photoluminescence spectra. In this case, they coincide in position. Cytotoxic Assay. To evaluate the cytotoxic effects of pure and Er3+/Yb3+-codoped hafnia and zirconia nanoparticles, MTT assays were performed. The results of the MTT assay concentration-dependent viability of HELF and HeLa cell lines with pure and Er3+/Yb3+-codoped hafnia and zirconia nanoparticles are presented in Figure 6. According to the cytotoxicity study, the nanoparticles demonstrate a high degree of biocompatibility. After 72 h of exposure to the nanoparticles (31−500 μg/mL), the HeLa and HELF cells did not demonstrate any dramatic decrease in cell viability. The HELF cells were less sensitive to nanoparticle exposure than the HeLa cells. It is worth noting that the death of the studied cell lines does not exceed 35%, from which it can be concluded that the obtained nanoparticles are low in cytotoxicity. Moreover, Er3+/Yb3+-codoped nanoparticles show almost identical toxicity as pure oxide nanoparticles exposed on HELF cells, which means that the release of lanthanide ions does not occur. Nevertheless, lanthanide-doped hafnium oxide nanoparticles were much more toxic for HeLa cells than undoped nanoparticles. The obtained data show that, for the investigated concentrations, the obtained upconversion hafnia and zirconia nanoparticles in concentrations up to 125 μg/mL for zirconium oxide and 63 μg/mL for hafnium oxide are harmless to human cells while simultaneously displaying upconversion characteristics, making them a promising tool for in vivo diagnostics and therapeutics. Still, as a slight cytotoxic influence on cells of high concentrations of compounds was registered, additional in vivo studies should be conducted to make conclusions about the potential deleterious impacts on humans. Bioimaging Visualization. For additional evidence of the optical activity and simultaneous biocompatibility of the resulting hybrid materials for potential use as a bioagent, we cultivated HeLa cell culture atop the UCNP-modified spider silk, as seen from the optical microphotograph of epithelial HeLa cells on the spider silk modified with upconversion nanoparticles, which were obtained using a photoluminescence microscope in bright-field mode (Figure 7). The photograph clearly shows the presence of upconversion nanoparticles on the fibers of the modified spider silk. At the same time, we observe a large and dense cell population, which indicates the biocompatibility of the obtained upconversion hybrid materials. Bright-field mode was used since this type of imaging sufficiently supplies information about the cells outline, nuclei position, and the site of large vesicles in unstained specimens. The bright-field type of illumination depends on changes in the refractive index, light absorption, or

Figure 7. Optical microphotograph of HeLa cell line on the spider silk modified with upconversion nanoparticles in a photoluminescence microscope taken in bright-field mode.

color for producing contrast and therefore was chosen to indicate the living cells and UCNP-modified spider silk.76 Moreover, the antimicrobial activity of upconversion silkbased hybrid materials was not observed as seen in Figure S3 (Supporting Information). This suggests that the obtained materials are biocompatible. Tensile Properties. For the evidence of the stability of the UCNP-coated fibers, the mechanical properties of the fibers were determined from tensile tests at ambient conditions and are presented in Figure 8. In comparison with native spider

Figure 8. Stress−strain curves of the silk fibers tested in air. The curve of HfO2:Er,Yb-coated spider silk fibers is indicated with a pink line, the curve of ZrO2:Er,Yb-coated spider silk fibers is indicated with a purple line, and the curve of native spider silk fibers is indicated with a blue line.

silk, as seen from Figure 8, the hybrid fiber materials show superior mechanical properties. The obtained results reveal that the stress value of the native spider silk fibers is about 0.54 MPa, whereas the stress values of the modified spider silk fibers are about 0.7 MPa. Thus, stress properties of the hybrid materials are about 1.3 times higher than those of native spider silk. This fact indicates the successful UCNP modification of the native spider silk. This level of stress can be explained by the molecular weight of protein. Meanwhile, both types of materials have approximately the same strain level, which is about 10−15%. These data demonstrate that the peptide 22969


Research Article


design of NP synthesis and NP characterization. A.K., E.F., and P.K. drafted the manuscript. A.K., E.K., V.K. and G.S. completed the text.

backbone of spider silk remains unaffected by the interaction with the nanoparticles. Moreover, the elastic modulus (E) determined for hybrid fiber materials is higher than that of native spider silk fibers: E = 24.4 MPa for HfO2:Er,Yb-coated spider silk fibers and E = 23.2 MPa for ZrO2:Er,Yb-coated spider silk fibers, whereas E = 14.3 MPa for native spider silk fibers. Therefore, the tensile test results show that particles remain on the fibers after the washing procedure, proving the successful spider silk modification.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Russian Science Foundation (grant no. 18-79-00269) and the Swedish Research Council (grant 2018-03811). The X-ray powder diffraction studies were performed in the engineering center of the Saint-Petersburg State Technological Institute (Technical University).

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CONCLUSIONS Inorganic Er3+/Yb3+-codoped hafnia and zirconia nanoparticles and natural spider silk assemblies can successfully be obtained by the simple method of impregnation. NIR−visible light transition is demonstrated for the nanoparticles as well as their composites. Thus, the upconversion luminescent properties of the nanoparticles can be extended to macroscale components to achieve optically active composite silk-based materials. Such hybrid materials based on spider silk and inorganic nanoparticles may have potential bioapplications in the fields of biosensing and bioimaging due to their low phonon energy, high luminous efficiency, high transmittance of light, and stable chemical−physical properties. Besides biomedical applications, the synthesized materials can find suitable application in either ecological instrumentation (within production of upconversion optical coatings and wires) or equipment clothing as fabric with high strength and optical characteristics. Moreover, based on the hybrid material obtained, substrates for bioelectronics may be also produced. Furthermore, the obtained material may serve as a trigger for photochemical reactions in chemical technology. This will lead to the sensing of biomaterials in a noninvasive and real-time manner. In addition, the proposed material modification strategy opens up opportunities for more rational generic innovative biomaterial designs and their applications in the fields of energy and sustainability, medicine, nanobiomedical technology, and many others.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b05131. Additional characterization of upconversion nanoparticles (EDX) and microbiology test of spider silk-based upconversion hybrids (PDF)

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REFERENCES

(1) Craig, C. L. Evolution of arthropod silks. Annu. Rev. Entomol. 1997, 231. (2) Shao, Z.; Vollrath, F. Surprising Strength of Silkworm Silk. Nature 2002, 418, 741. (3) Du, N.; Yang, Z.; Liu, X. Y.; Li, Y.; Xu, H. Y. Structural Origin of the Strain-Hardening of Spider Silk. Adv. Funct. Mater. 2011, 772. (4) Vasconcelos, A.; Freddi, G.; Cavaco-Paulo, A. Biodegradable Materials Based on Silk Fibroin and Keratin. Biomacromolecules 2008, 1299. (5) Hakimi, O.; Knight, D. P.; Vollrath, F.; Vadgama, P. Spider and Mulberry Silkworm Silks as Compatible Biomaterials. Composites, Part B 2007, 38, 324−337. (6) Vepari, C.; Kaplan, D. L. Silk as a Biomaterial. Prog. Polym. Sci. 2007, 32, 991−1007. (7) Setzen, G.; Williams, E. F. Tissue Response to Suture Materials Implanted Subcutaneously in a Rabbit Model. Plast. Reconstr. Surg. 1997, 100, 1788. (8) Mandal, B. B.; Kundu, S. C. Cell Proliferation and Migration in Silk Fibroin 3D Scaffolds. Biomaterials 2009, 30, 2956. (9) Rising, A. Controlled Assembly: A Prerequisite for the Use of Recombinant Spider Silk in Regenerative Medicine? Acta Biomater. 2014, 10, 1627−1631. (10) Bai, J.; Ma, T.; Chu, W.; Wang, R.; Silva, L.; Michal, C.; Chiao, J. C.; Chiao, M. Regenerated Spider Silk as a New Biomaterial for MEMS. Biomed. Microdevices 2006, 317. (11) Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J.; Lu, H.; Richmond, J.; Kaplan, D. L. Silk-Based Biomaterials. Biomaterials 2003, 24, 401−416. (12) Kundu, B.; Kurland, N. E.; Bano, S.; Patra, C.; Engel, F. B.; Yadavalli, V. K.; Kundu, S. C. Silk Proteins for Biomedical Applications: Bioengineering Perspectives. Prog. Polym. Sci. 2014, 39, 251−267. (13) Gagner, J. E.; Kim, W.; Chaikof, E. L. Designing Protein-Based Biomaterials for Medical Applications. Acta Biomater. 2014, 10, 1542−1557. (14) Gellynck, K.; Verdonk, P. C. M.; Van Nimmen, E.; Almqvist, K. F.; Gheysens, T.; Schoukens, G.; Van Langenhove, L.; Kiekens, P.; Mertens, J.; Verbruggen, G. Silkworm and Spider Silk Scaffolds for Chondrocyte Support. J. Mater. Sci.: Mater. Med. 2008, 19, 3399− 3409. (15) Kundu, B.; Rajkhowa, R.; Kundu, S. C.; Wang, X. Silk Fibroin Biomaterials for Tissue Regenerations. Adv. Drug Delivery Rev. 2013, 65, 457−470. (16) Wang, Y.; Kim, H. J.; Vunjak-Novakovic, G.; Kaplan, D. L. Stem Cell-Based Tissue Engineering with Silk Biomaterials. Biomaterials 2006, 6064. (17) Lamboni, L.; Gauthier, M.; Yang, G.; Wang, Q. Silk Sericin: A Versatile Material for Tissue Engineering and Drug Delivery. Biotechnol. Adv. 2015, 33, 1855−1867. (18) Mandal, B. B.; Kundu, S. C. Biospinning by Silkworms: Silk Fiber Matrices for Tissue Engineering Applications. Acta Biomater. 2010, 6, 360−371.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Vadim Kessler: 0000-0001-7570-2814 Gulaim Seisenbaeva: 0000-0003-0072-6082 Elena Krivoshapkina: 0000-0001-6981-5134 Author Contributions

The manuscript was written through contributions of all authors. A.K., E.K., P.K., V.K., and G.S. designed the study. A.K., G.K., and D.G. performed the optical experiments. A.F. and A.K. made the cytotoxicity assays. V.R. and A.K. performed the microbiological tests. T.L. made the tensile examination. G.K., A.K., E.K., and P.K. contributed to the 22970


Research Article

ACS Applied Materials & Interfaces (19) Mottaghitalab, F.; Hosseinkhani, H.; Shokrgozar, M. A.; Mao, C.; Yang, M.; Farokhi, M. Silk as a Potential Candidate for Bone Tissue Engineering. J. Controlled Release 2015, 215, 112−128. (20) Melke, J.; Midha, S.; Ghosh, S.; Ito, K.; Hofmann, S. Silk Fibroin as Biomaterial for Bone Tissue Engineering. Acta Biomater. 2016, 31, 1−16. (21) Wang, Y.; Blasioli, D. J.; Kim, H. J.; Kim, H. S.; Kaplan, D. L. Cartilage Tissue Engineering with Silk Scaffolds and Human Articular Chondrocytes. Biomaterials 2006, 4434. (22) Zhao, Z.; Li, Y.; Xie, M. B. Silk Fibroin-Based Nanoparticles for Drug Delivery. Int. J. Mol. Sci. 2015, 16, 4880−4903. (23) Huang, Y.; Bailey, K.; Wang, S.; Feng, X. Silk Fibroin Films for Potential Applications in Controlled Release. React. Funct. Polym. 2017, 116, 57−68. (24) Wen, H.; Lan, X.; Zhang, Y.; Zhao, T.; Wang, Y.; Kajiura, Z.; Nakagaki, M. Transgenic Silkworms (Bombyx Mori) Produce Recombinant Spider Dragline Silk in Cocoons. Mol. Biol. Rep. 2010, 37, 1815−1821. (25) Silva, R.; Fabry, B.; Boccaccini, A. R. Fibrous Protein-Based Hydrogels for Cell Encapsulation. Biomaterials 2014, 35, 6727−6738. (26) Florczak, A.; Mackiewicz, A.; Dams-Kozlowska, H. Functionalized Spider Silk Spheres as Drug Carriers for Targeted Cancer Therapy. Biomacromolecules 2014, 2971. (27) Subia, B.; Chandra, S.; Talukdar, S.; Kundu, S. C. Folate Conjugated Silk Fibroin Nanocarriers for Targeted Drug Delivery. Integr. Biol. 2014, 6, 203−214. (28) Shen, Y.; Redmond, S. L.; Teh, B. M.; Yan, S.; Wang, Y.; Zhou, L.; Budgeon, C. A.; Eikelboom, R. H.; Atlas, M. D.; Dilley, R. J.; Zheng, M.; Marano, R. J. Scaffolds for Tympanic Membrane Regeneration in Rats. Tissue Eng. Part A 2013, 657. (29) Lee, J. H.; Kim, D. K.; Park, H. S.; Jeong, J. Y.; Yeon, Y. K.; Kumar, V.; Bae, S. H.; Lee, J. M.; Moon, B. M.; Park, C. H. A Prospective Cohort Study of the Silk Fibroin Patch in Chronic Tympanic Membrane Perforation. Laryngoscope 2016, 2798. (30) Zeplin, P. H.; Maksimovikj, N. C.; Jordan, M. C.; Nickel, J.; Lang, G.; Leimer, A. H.; Römer, L.; Scheibel, T. Spider Silk Coatings as a Bioshield to Reduce Periprosthetic Fibrous Capsule Formation. Adv. Funct. Mater. 2014, 24, 2658−2666. (31) Szybala, C.; Pritchard, E. M.; Lusardi, T. A.; Li, T.; Wilz, A.; Kaplan, D. L.; Boison, D. Antiepileptic Effects of Silk-Polymer Based Adenosine Release in Kindled Rats. Exp. Neurol. 2009, 126. (32) Roloff, F.; Strauß, S.; Vogt, P. M.; Bicker, G.; Radtke, C. Spider Silk as Guiding Biomaterial for Human Model Neurons. Biomed Res. Int. 2014, 1. (33) Allmeling, C.; Jokuszies, A.; Reimers, K.; Kall, S.; Vogt, P. M. Use of Spider Silk Fibres as an Innovative Material in a Biocompatible Artificial Nerve Conduit. J. Cell. Mol. Med. 2006, 770. (34) Yang, Y.; Yuan, X.; Ding, F.; Yao, D.; Gu, Y.; Liu, J.; Gu, X. Repair of Rat Sciatic Nerve Gap by a Silk Fibroin-Based Scaffold Added with Bone Marrow Mesenchymal Stem Cells. Tissue Eng., Part A 2011, 2231. (35) Harris, T. I.; Gaztambide, D. A.; Day, B. A.; Brock, C. L.; Ruben, A. L.; Jones, J. A.; Lewis, R. V. Sticky Situation: An Investigation of Robust Aqueous-Based Recombinant Spider Silk Protein Coatings and Adhesives. Biomacromolecules 2016, 17, 3761− 3772. (36) Spiess, K.; Lammel, A.; Scheibel, T. Recombinant Spider Silk Proteins for Applications in Biomaterials. Macromol. Biosci. 2010, 10, 998−1007. (37) Copeland, C. G.; Bell, B. E.; Christensen, C. D.; Lewis, R. V. Development of a Process for the Spinning of Synthetic Spider Silk. ACS Biomater. Sci. Eng. 2015, 1, 577−584. (38) Florczak, A.; Jastrzebska, K.; Mackiewicz, A.; Dams-Kozlowska, H. Blending Two Bioengineered Spider Silks to Develop Cancer Targeting Spheres. J. Mater. Chem. B 2017, 5, 3000−3011. (39) Menezes, G. M.; Teulé, F.; Lewis, R. V.; Silva, L. P.; Rech, E. L. Nanoscale Investigations of Synthetic Spider Silk Fibers Modified by Physical and Chemical Processes. Polym. J. 2013, 45, 997−1006.

(40) Foo, C. W. P.; Bini, E.; Hensman, J.; Knight, D. P.; Lewis, R. V.; Kaplan, D. L. Role of PH and Charge on Silk Protein Assembly in Insects and Spiders. Appl. Phys. A: Mater. Sci. Process. 2006, 82, 223− 233. (41) Buehler, M. J. Materials by Design - A Perspective from Atoms to Structures. MRS Bull. 2013, 169−176. (42) Horan, R. L.; Antle, K.; Collette, A. L.; Wang, Y.; Huang, J.; Moreau, J. E.; Volloch, V.; Kaplan, D. L.; Altman, G. H. In Vitro Degradation of Silk Fibroin. Biomaterials 2005, 3385. (43) Appel, A. A.; Anastasio, M. A.; Larson, J. C.; Brey, E. M. Imaging Challenges in Biomaterials and Tissue Engineering. Biomaterials 2013, 6615. (44) Paley, M. A.; Prescher, J. A. Bioluminescence: A Versatile Technique for Imaging Cellular and Molecular Features. MedChemComm. 2014, 255. (45) Zhang, N.; Francis, K. P.; Prakash, A.; Ansaldi, D. Enhanced Detection of Myeloperoxidase Activity in Deep Tissues through Luminescent Excitation of Near-Infrared Nanoparticles. Nat. Med. 2013, 500. (46) Schambach, S. J.; Bag, S.; Steil, V.; Isaza, C.; Schilling, L.; Groden, C.; Brockmann, M. A. Ultrafast High-Resolution in Vivo Volume-CTA of Mice Cerebral Vessels. Stroke 2009, 40, 1444−1450. (47) Mamin, H. J.; Poggio, M.; Degen, C. L.; Rugar, D. Nuclear Magnetic Resonance Imaging with 90-Nm Resolution. Nat. Nanotechnol. 2007, 2, 301−306. (48) Lin, N.; Toh, G. W.; Feng, Y.; Liu, X. Y.; Xu, H. Two-Photon Fluorescent Bombyx Mori Silk by Molecular Recognition Functionalization. J. Mater. Chem. B 2014, 2136. (49) Mayes, E. L.; Vollrath, F.; Mann, S. Fabrication of Magnetic Spider Silk and Other Silk-Fiber Composites Using Inorganic Nanoparticles. Adv. Mater. 1998, 801. (50) Rockwood, D. N.; Preda, R. C.; Yücel, T.; Wang, X.; Lovett, M. L.; Kaplan, D. L. Materials Fabrication from Bombyx Mori Silk Fibroin. Nat. Protoc. 2011, 6, 1612−1631. (51) Lin, N.; Meng, Z.; Toh, G. W.; Zhen, Y.; Diao, Y.; Xu, H.; Liu, X. Y. Engineering of Fluorescent Emission of Silk Fibroin Composite Materials by Material Assembly. Small 2015, 11, 1205−1214. (52) Kwak, J.; Bae, W. K.; Zorn, M.; Woo, H.; Yoon, H.; Lim, J.; Kang, S. W.; Weber, S.; Butt, H. J.; Zentel, R.; Lee, S.; Char, K.; Lee, C. Characterization of Quantum Dot/Conducting Polymer Hybrid Films and Their Application to Light-Emitting Diodes. Adv. Mater. 2009, 5022. (53) Tansil, N. C.; Koh, L. D.; Han, M. Y. Functional Silk: Colored and Luminescent. Adv. Mater. 2012, 1388. (54) Lee, F. S. The Optimal Design of Non-Spherical Side Mirrors and Rear-View Mirrors II. J. Technol. 2012, 27, 45−54. (55) Smith, A. M.; Mancini, M. C.; Nie, S. Bioimaging: Second Window for in Vivo Imaging. Nat. Nanotechnol. 2009, 710. (56) Auzel, F. Upconversion and Anti-Stokes Processes with f and d Ions in Solids. Chem. Rev. 2004, 139. (57) Chatterjee, D.; Rufaihah, A.; Zhang, Y. Upconversion Fluorescence Imaging of Cells and Small Animals Using Lanthanide Doped Nanocrystals. Biomaterials 2008, 937. (58) Manouras, T.; Vamvakaki, M. Field Responsive Materials: Photo-, Electro-, Magnetic- and Ultrasound-Sensitive Polymers. Polym. Chem. 2017, 8, 74−96. (59) Wang, F.; Banerjee, D.; Liu, Y.; Chen, X.; Liu, X. Upconversion Nanoparticles in Biological Labeling, Imaging, and Therapy. Analyst 2010, 1839. (60) Agoudjil, N.; Kermadi, S.; Larbot, A. Synthesis of Inorganic Membrane by Sol-Gel Process. Desalination 2008, 223, 417−424. (61) Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 1983, 55. (62) Stockert, J. C.; Blázquez-Castro, A.; Cañete, M.; Horobin, R. W.; Villanueva, Á . MTT Assay for Cell Viability: Intracellular Localization of the Formazan Product Is in Lipid Droplets. Acta Histochem. 2012, 785. 22971


Research Article

ACS Applied Materials & Interfaces (63) Riss, T. L.; Moravec, R. A.; Niles, A. L.; Duellman, S.; Benink, H. A.; Worzella, T. J.; Minor, L. Cell Viability Assays. In Assay Guidance Manual; Eli Lilly & Company and the National Center for Advancing Translational Sciences: 2013. (64) Perez-Rigueiro, J. The Effect of Spinning Forces on Spider Silk Properties. J. Exp. Biol. 2005, 2633. (65) Madurga, R.; Gañań -Calvo, A. M.; Plaza, G. R.; Guinea, G. V.; Elices, M.; Pérez-Rigueiro, J. Production of High Performance Bioinspired Silk Fibers by Straining Flow Spinning. Biomacromolecules 2017, 18, 1127−1133. (66) Auzel, F. Upconversion Processes in Coupled Ion Systems. J. Lumin. 1990, 341. (67) He, S.; Xia, H.; Zhang, J.; Zhu, Y.; Chen, B. Highly Efficient Up-Conversion Luminescence in Er3+/Yb3+ Co-Doped Na5Lu9F32 Single Crystals by Vertical Bridgman Method. Sci. Rep. 2017, 7, 8751. (68) Kumar, G. A.; De La Rosa, E.; Desirena, H. Radiative and Non Radiative Spectroscopic Properties of Er3+ Ion in Tellurite Glass. Opt. Commun. 2006, 601. (69) Patra, A.; Friend, C. S.; Kapoor, R.; Prasad, P. N. Upconversion in Er3+:ZrO2 Nanocrystals. J. Phys. Chem. B 2002, 1909. (70) Zhao, X.; Vanderbilt, D. First-Principles Study of Structural, Vibrational, and Lattice Dielectric Properties of Hafnium Oxide. Phys. Rev. B - Condens. Matter Mater. Phys. 2002, 233106. (71) Boyer, J. C.; Van Veggel, F. C. J. M. Absolute Quantum Yield Measurements of Colloidal NaYF4: Er3+, Yb3+upconverting Nanoparticles. Nanoscale 2010, 1417. (72) Stock, B. Bio-Rad Laboratories. Anal. Chem. 1983, 237A. (73) Parejas, A.; Montes, V.; Hidalgo-Carrillo, J.; Sánchez-López, E.; Marinas, A.; Urbano, F. Microemulsion and Sol-Gel Synthesized ZrO2-MgO Catalysts for the Liquid-Phase Dehydration of Xylose to Furfural. Molecules 2017, 2257. (74) Karthikeyani, R.; Divya, A.; Mathavan, T.; Asath, R. M.; Benial, A. M. F.; Muthuchelian, K. Structural and Optical Studies on Selected Web Spinning Spider Silks. Spectrochim. Acta, Part A 2017, 170, 111− 116. (75) Smits, K.; Jankovica, D.; Sarakovskis, A.; Millers, D. UpConversion Luminescence Dependence on Structure in Zirconia Nanocrystals. Opt. Mater. 2013, 35, 462−466. (76) Rottenfusser, R. Proper Alignment of the Microscope. Methods in Cell Biology 2013, 43.

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