Hard and Transparent Films Formed by Nanocellulose–TiO2 Nanoparticle Hybrids

The formation of hybrids of nanofibrillated cellulose and titania nanoparticles in aqueous media has been studied. Their transparency and mechanical behavior have been assessed by spectrophotometry and nanoindentation. The results show that limiting the titania nanoparticle concentration below 16 vol% yields homogeneous hybrids with a very high Young’s modulus and hardness, of up to 44 GPa and 3.4 GPa, respectively, and an optical transmittance above 80%. Electron microscopy shows that higher nanoparticle contents result in agglomeration and an inhomogeneous hybrid nanostructure with a concomitant reduction of hardness and optical transmittance. Infrared spectroscopy suggests that the nanostructure of the hybrids is controlled by electrostatic adsorption of the titania nanoparticles on the negatively charged nanocellulose surfaces.


Introduction
Organic-inorganic nanocomposites or hybrids have attracted much interest due to their current and potential applications as they can combine useful chemical, optical and mechanical characteristics. [1,2] Traditionally, organic-inorganic nanocomposites have had a focus on the polymeric matrix, being e.g., formed from vinyl polymers, condensation polymers or polyolefins filled with relatively passive inorganic components such as layered silicates, i.e., montmorillonite or hectorite. [1,2] With the strong movement towards biodegradable, renewable, sustainable, and carbon-neutral polymeric materials, it is also of importance to develop viable and facile production routes for nanocomposites using such biopolymers. In this respect, nanocellulose [3] is emerging as a cheap and sustainable polymeric material with useful functional properties such as tailored hydro/oleophilicity, optical transparency and remarkable mechanical performance both as films and aerogels. [4][5][6][7][8][9] The exploration of nanocellulosenanoparticle hybrids is still relatively sparse but has increased pronouncedly since the pioneering report on multifunctional magnetic nanocellulose hybrids. [10] Recent studies have suggested various applications for different nanocellulose-inorganic hybrids: nanocrystalline cellulose-amorphous calcium carbonate hybrid films resemble biogenic materials such as dentin, [11] nanocellulose-clay nanopaper has shown good fire retardancy and gas barrier functions, [12] nanocellulose aerogels coated with titania using a CVD approach display a photoswitchable hydrophobicity [13] and oil adsorption, [14] and nanocellulose-silver hybrids were evaluated as potential antibacterial agents. [15] Moreover, it should be noted that other biopolymers such as silk, [16,17] chitin, [18,19] or collagen [20] also can be utilized in the production of organic-inorganic hybrids.
In this work, we demonstrate the facile fabrication of nanocellulose-titania nanoparticles hybrids with high inorganic content by the adsorption of TiO2 (anatase) nanoparticles on wood-derived nanofibrillated cellulose. The nanostructure of the hybrids was characterized mainly by electron microscopy and the optical transparency and mechanical performance of the hybrids were evaluated using spectrophotometry and nanoindentation tests, respectively. We show that the effective Young's modulus, hardness and transparency of the hybrids are determined by their nanostructure, in particular, by the homogeneity of the inorganic and organic components. The optimum range of inorganic content, where the modulus and hardness of the hybrids exceed that of pure nanocellulose and the transparency is high, is identified and the mechanisms for the nanocellulose-titania interactions and agglomeration are discussed.

Materials
Commercial TiO 2 (anatase) nanoparticles were dispersed in a 0.1 M HCl aqueous solution with a stock concentration c TiO2~3 0 mg=cm 3 . Nanofibrillated cellulose (NFC) was prepared by TEMPO oxidation of wood fibers according to a previously reported procedure which resulted in surface-functionalized fibrils with carboxylic groups with a total charge of 1.84 mmol/g. [6].
Aqueous dispersion of hybrids were prepared by adsorbing TiO 2 nanoparticles onto NFC in an aqueous media. TiO 2 nanoparticles, NFC (stock concentration c NFC~0 :75 mg=cm 3 ) and water (Millipore, resistivity §18 MV=cm) were mixed in different ratios, see Table 1, and their composition was also assessed using thermal analysis (see Supplementary Information, Figure S2). The dispersions were shaken for two hours and then the pH was adjusted to 8 with aqueous solutions of diluted NH3 and HCl.
Films were prepared by depositing 0:2 cm 3 of an aqueous dispersion of the hybrids on circular glass slides (diameter ~1:2 cm) and placed in a Binder atmospheric chamber at 30uC and 50% relative humidity. The thickness of the obtained films was approximately 20 mm. Alternatively, the aqueous dispersions of hybrids were centrifuged for 30 min at 3800 g with a Hettich EBA 21 centrifuge, the supernatant was discarded and the remaining portion was freeze-dried at 240uC and a pressure of 10 {4 mbar using a SRK GT2 freeze-drier.

Morphological Characterization
Transmission Electron Microscopy (TEM). Transmission electron microscopy (TEM) images of the titania nanocrystals were obtained using a JEOL JEM-2000 FX microscope equipped with a LaB6 filament operated at 200 kV (Cs = 3.4 mm, point resolution = 0.31 nm). The specimens were prepared by depositing a drop of a diluted dispersion of nanoparticles onto carbon-coated copper grids and allowing the solvent to evaporate. The images were recorded with a CCD camera (Keen View, SIS analysis, 1376|1032 px 2 , pixel size 23:5|23:5 mm 2 ). The particle length and width, x i were manually measured on 200 nanoparticles from the TEM micrographs. The mean length or width, x x, and its standard deviation, s, were determined by fitting the corresponding histogram (see supplementary Figure S1) with a Gaussian distribution function.
Scanning Electron Microscopy (SEM). NFC and hybrid films were deposited on silicon chips (1|1 cm 2 ) and mounted on aluminium stubs using carbon ink and coated with a thin carbon layer (v20 nm). Alternatively, hybrid aerogels were glued on aluminum stubs using a double-sided carbon tape and also coated with a thin carbon layer (v20 nm). Scanning electron microscopy (SEM) images of the hybrids were acquired using a JEOL JSM-7401F field-emission gun microscope in secondary electron imaging mode at an accelerating voltage of 2 kV and probe current of 10 mA and working distance of 3 mm. The imaging of the films was carried out under the 'GB High' setting.
Atomic Force Microscopy (AFM). Atomic force microscopy (AFM) images were recorded in tapping mode with the aid of a MultiMode instrument with Veeco NanoScope V controller using Veeco MPP-11100-10 silicon probes with nominal spring constants of 40 N/m. The force was kept minimal during scanning by routinely decreasing it until the tip left the surface and subsequently increasing it slightly to just regain contact. The scan rate was 0.5 to 2 lines per second. All images with 512|512 px 2 were analyzed with non-commercial software WSxM. [40].

Spectroscopic Characterization
UV-Visible spectrophotometry. In-line transmittance spectra of the NFC and hybrids in the form of films on glass slides and as aqueous dispersions in the visible region (400-800 nm) were obtained with a Perkin-Elmer Lambda 19 UV-Vis-NIR spectrophotometer using a clean glass slide as a background. The aqueous dispersions were filled in a 1:4 cm 3 semi-micro rectangular quartz cuvette using Millipore water as background.
Infrared spectroscopy. Infrared (IR) spectra of NFC, TiO 2 and NFC-TiO 2 hybrids materials were measured on a Varian 670-IR FTIR spectrometer, equipped with an attenuated total reflection (ATR) detection device (Goldengate by Specac) with a single reflection diamond ATR element. 32 scans were accumulated in the spectral region of 390{4000 cm {1 with a spectral resolution of 4 cm {1 . To maximize the signal of the carboxylic band the pH of aqueous dispersions of NFC and was adjusted to pH = 3 with few microliters of diluted HCl and NH3 prior to contacting them with the aqueous dispersions of TiO 2 . The pH of the pristine aqueous dispersions of TiO 2 was also adjusted to pH = 3 before the measurements. The materials were then freeze-dried before the measurements using the protocol previously described.

Mechanical Characterization
The mechanical properties of the hybrids were evaluated using a Fischer-Cripps Laboratories Ultra-Micro-Indenter system (UMIS) equipped with a Berkovich pyramidal-shaped diamond tip. The value of maximum applied force was chosen to be 500 mN to ensure that the maximum penetration depth was kept well below one tenth of the overall film thickness (a necessary condition to avoid having an influence of the substrate on the measured mechanical properties of the film). [41] The thermal drift during nanoindentation was kept below 0.05 nm/s. Proper corrections for the contact area (calibrated with a fused quartz specimen), instrument compliance, and initial penetration depth were applied. The hardness, H, and effective reduced Young's modulus, E r , values were derived from the load-displacement curves at the beginning of the unloading segment using the method of Oliver and Pharr. [42] From the initial unloading slope, the contact stiffness, S, was determined as S~d P dh where P and h denote, respectively, the applied load and the penetration depth during nanoindentation. The effective reduced Young's modulus was evaluated based on its relationship with the contact area, A, and contact stiffness S~b 2 ffiffi Here, b is a constant that depends on the geometry of the indenter (b~1:034 for a Berkovich indenter), and E r is defined as 1 Er~1 Ei . [43] The reduced modulus takes into account the elastic displacements that occur in both the specimen, with effective Young's modulus E and Poisson's ratio n, and the diamond indenter, with elastic constants E i and n i . Note that for diamond, E i~1 140 GPa and n i~0 :07. Remarkably, for most materials, including NFC or TiO 2 , where n i *0:26 [44] and 0.27, [45,46] respectively, the contribution of the tip to E r is almost negligible, i.e., E r &E (5% overestimation). The hardness was calculated as H~P max A where P max is the maximum load applied during nanoindentation. Finally, the elastic recovery was evaluated as the ratio between the elastic and the total (plastic + elastic) energies during nanoindentation, W el =W tot . These energies were calculated from the nanoindentation experiments as the areas between the unloading curve and the x-axis (W el ) and between the loading curve and x-axis (W tot ). [43] The results presented here represent the statistical average of a set of 50 indentations for each sample, whereas up to 200 indentations were carried out on the samples with low inorganic content (v16vol%).

Results and Discussion
Figures 1a and 1b show electron microscopy images of the hybrid constituents, i.e., TiO 2 nanoparticles and NFC fibers, respectively. Analysis of the TEM images showed that the TiO 2 particles had a length l~26+3 nm and a width w~16+2 nm, i.e., an aspect ratio AR~1:6+0:3 (see Supplementary Information, Figure S1). The cellulose nanofibrils had a width distribution between 3-5 nm and length of 0:3{1 mm. However, during the adsorption of TiO 2 the fibrils tend to agglomerate somewhat and form bundles with thickness between 10-20 nm, as can be see in Figure 1c which shows a SEM micrographs of the hybrid aerogel with 9 vol% of inorganic content. Hybrids of NFC and TiO 2 nanoparticles were prepared also as films. Representative SEM images of hybrid films with low (4 vol% TiO 2 ) and medium (16 vol% TiO 2 ) inorganic content are shown in Figures 1d and 1e, respectively. The images show that the amount of fibers on the surface decreases with an increase of the fraction of nanoparticles. Films with a large amount of TiO 2 nanoparticles (w vol w16 vol%) display an even granular surface. AFM images (Figure 1f) show that the inorganic nanoparticles are distributed on the surface of the films and also in between the fibrils. Figure 2a shows a photograph of the films deposited on glass. The films are transparent at low inorganic content but tend to become milky as the concentration of nanoparticles increases, suggesting that light scattering becomes increasingly important. The optical transmittance, T, in the visible region of the different film samples with different inorganic content is shown in Figure 2b. The figure shows that the NFC film has a high optical transmittance over the visible range, as expected from its low absorption coefficient [47] and smoothness of the films. The hybrids with relatively low concentration of inorganic nanoparticles have a high transmittance in the visible area which decreases toward the ultraviolet region, when the bandgap of anatase is approached. In the case of hybrids, in the absence of significant absorption, the transmitted light across a hybrid film can be described using the Rayleigh formalism for scattering, as indicated by Eq. (1). [ where T is the transmittance, l the wavelength, n TiO 2 &2:49 and n NFC &1:58 the average refractive indices of anatase [49] and cellulose [47], respectively, d the diameter of the particles, and x&20 mm the thickness of the film. Note that the model assumes that the NFC matrix is dense and nonporous and that the particles are point scatterers much smaller than the wavelength, i.e., dv0:1l. The equation shows that the transmittance decreases with an increase of: the concentration of nanoparticles, the particle size, the difference of refractive indices, and the thickness of the films, and with a decrease of the wavelength. Indeed, as anatase and cellulose have a very low absorption in the visible region it is possible to use Eq. (1) to model the response of the hybrids using the experimental data. Figure 2c shows the optical transmittance of the films at l~550 nm. Plotted along the experimental points are the calculated transmittance for three particle sizes: (i) particle diameter with an equivalent particle volume as the TiO 2 nanoparticles used for the fabrication of the hybrids (d TiO2~1 9 nm); (ii) particles with a diameter equal to the upper limit of the experimental particle size (d up TiO2~3 0 nm); and (iii) particles with a diameter similar to the observed agglomerates d agg TiO2~5 0 nm. The experimentally observed transmittance of the hybrid films with an inorganic content w TiO2 ƒ16% can be well described within the boundaries described by (i) and (ii) (hatched region). Alternatively, the transmittance of the hybrids with w TiO2 w16% is between the boundaries defined by (ii) and (iii), suggesting that the number of agglomerates becomes increasingly important. Notice that both the reflectivity [39] and the surface roughness of the films [50] also contribute to a slightly lowered transmittance.
Previous work on the fabrication of poly(vinyl alcohol)-TiO 2 (rutile) nanoparticle (PVAL-TiO 2 ) nanocomposites have also demonstrated high transparency in hybrid films. [51] The hybrids, with a thickness x&100 mm, were formed in a similar fashion as the ones described in the present work, i.e., by the ex-situ nucleation of nanoparticles (d&2:5 nm) and their subsequent mixing and drying with the polymer. As a comparison, PVAL-TiO2 hybrids with a w vol~4 vol% showed a T l~400nm &90%. Sasaki et al. prepared poly(diallyldimethylammonium chloride)-Ti 1{d O 4d{ 2 (d~0:0875) nanoplatelet nanocomposite films using the layer-by-layer technique (LbLTiO2). [39] The hybrids were composed of alternating layers of polymer and Ti 1{d O 4d{ 2 nanoplatelets (thickness ca. 1.2 nm, lateral dimensions in the sub-mm regime). However, the LbLTiO2 hybrids showed a substantial reflectivity which decreased the optical transmittance. For instance, a 10-repeat multilayer with a thickness of x&20 nm showed a reduced T l~550nm &90%. Regarding the current work, it is interesting to note that despite the relatively large size of the anatase nanoparticles and the fibrillated structure of the nanocellulose, the transparency of the hybrids is very high and comparable to those systems prepared from smaller particles.
The mechanical behavior of the films was tested using nanoindentation measurements where the typical load-displacement nanoindentation curves and AFM images of the indents are shown in the supplementary information ( Figures S4 and S5). Figure 3 shows the effective reduced Young's modulus, E r , and hardness, H, of the films as a function of inorganic content, w vol . The E r value corresponding to NFC is in close agreement with the reported values of the transversal Young's modulus of native cellulose. [52] The addition of a small amount of TiO 2 nanoparticles resulted in a slight increase of E r . This initial increase of E r as w vol increases can be described with a simple linear rule of mixtures, i.e., E r,hyb &w vol E TiO2 z(1{w vol )E NFC . Figure 3a includes the estimates for hybrids with low w vol (hatched area) using the experimentally reported values for the elastic modulus of anatase thin films (nanoindentation), E film TiO2 &170 GPa, [53,54] and nanoparticles (high pressure X-ray), E NP TiO2 &330 GPa. [45] The experimental value obtained for sample S1 is assigned to pure NFC, i.e., E NFC &38 GPa. However, as the concentration of nanoparticles increased further the E r value of the hybrid films decayed abruptly. Indeed, at higher concentration of nanoparticles, w vol §30%, the films became looser and compliant, leading to a decrease of E r . This behavior suggests that the bonding and microstructure of the hybrids change significantly with increasing anatase content. Remarkably, the NFC and hybrid films with TiO 2 concentrations up to 24 vol% had extraordinarily high effective Young's modulus when compared to organic-inorganic hybrids previously reported (see [41] and references therein) and some high-performance lightweight materials, such as magnesium [55] or concrete. [56,57] The linear increase in E r at low TiO 2 additions strongly indicates that the inorganic nanoparticles are homogeneously distributed and bonded to the NFC network, thus increasing the modulus (and hardness) with increasing amount of the stiff and hard constituent. At a critical concentration, the homogeneity of the hybrid and the anatase nanoparticle distribution decreases. Recent reports have indeed shown that nonsintered films composed of anatase TiO 2 nanoparticles have Young's moduli as low as 22.5 GPa. [58,59] This strong dependence of the mechanical behavior on the TiO 2 nanoparticle content is much more evident in Figure 3b where the hardness, H, of the NFC and hybrid films is depicted. The hardness of the NFC and hybrids with w vol ƒ9% is roughly constant about 3.4 GPa (first hatched area). As the concentration of nanoparticles further increases there is a sharp decrease of the hardness values at volume fractions w vol §16% (second hatched area), with H falling below 1 GPa. Note that AFM analysis of the indentations shows little pile-up and sink-in at high TiO 2 contents (30 vol%) thus having a reduced influence on the overall trends shown in Figure 3 see supplementary information Figure S5).
We have used IR spectroscopy to obtain more information on the bonding and interaction between the inorganic nanoparticles and the nanocellulose. The IR spectra are shown in Figure 4a for NFC, TiO 2 nanoparticles, and the hybrids with different inorganic content. The broad band at the low frequency end of the spectra (also partially related to the librational mode of adsorbed water) is assigned to the Ti-O band and increases with the TiO 2 content. To facilitate the analysis of the interactions between the nanoparticles and the NFC, the nanocellulose and the hydroxyl groups on the surface of the titania nanoparticles were protonated prior to the formation of the hybrid. Hence, by observing the C = O stretching region of the (protonated) carboxylic groups on the surface of the fibrils it is possible to correlate the reaction between the fibrils and the positively charged TiO 2 nanoparticles. Figure 4a shows that a decrease in the intensity of carboxyl band corresponding to the acidic C = O (1725 cm {1 ) decreases with an increase of the concentration of TiO 2 . The formation of an ester between the carboxylic group of the nanocellulose and the hydroxyl groups on the surface of the nanoparticles was excluded as no C = O band was detected at higher frequencies than that corresponding to the acidic C = O. Using a difference spectra, the bands that take part in the NFC-TiO 2 interactions are readily observed. Figure 4b shows the spectrum of freeze-dried TiO 2 nanoparticles and the difference spectra of a hybrid with 16 vol% TiO 2 from which the spectra of NFC was subtracted. The negative band at 1725 cm {1 shows the decrease in the amount of carboxyl groups whereas the positive band at 1595 cm {1 (antisymmetric stretching) suggests an increase in the amount of carboxylate group. Note that the band at 1440 cm {1 is due to ammonium ions, [60] whereas the bands at 1405 and 1260 cm {1 in the spectrum of TiO 2 are likely to correspond to nitrate and nitrite ions [60] arising from the photooxidation of ammonia [61] used during pH adjustment as these bands do not appear in the IR spectrum of pristine TiO 2 (see supplementary information Figure  S3). The bands at 1460 and 1410 cm 21 could arise from the symmetric stretching of carboxylate ions, although the presence of artifacts during spectral subtraction cannot be ruled out.
Analysis of the spectra suggests that the nanocellulose and the nanoparticles interact through electrostatic interactions between the dissociated carboxylic group and the positively charged groups on the nanoparticles, i.e., {COO 7 Á Á Á + TiO{. Other groups have prepared polymer-titania hybrids using a silane groups as grafting agent, resulting in a covalent modification of titania nanoparticles(e.g., a Ti-O-Si bond) formed in-situ, [23,33,35] whereas the the formation of polyaniline-titania probably also proceeds via electrostatic interactions. [21,22] The formation of hybrids using ex-situ synthesized nanoparticles allow for a larger range of inorganic content that is accessible. However, the results also suggest that a careful balance of the electrostatic interactions between the nanocellulose and titania nanoparticles and their dispersability in aqueous media is key to ensure optimized optical and mechanical properties.

Conclusions
Hybrids composed of nanofibrillated cellulose and anatase nanoparticles with variable inorganic content were fabricated through the adsorption of ex-situ prepared nanoparticles. Electron  Table 1), as a function of the wavelength, l. (C) Transmittance of the NFC and hybrid films at l~550 nm as a function of the TiO 2 content, w vol . The symbols represent the experimental data whereas the three lines correspond to the calculated transmittance of films composed of particles with three different sizes, i.e., d~19, 30, and 50 nm according to Eq. (1), see text for details. The hatched area shows the region where the hybrids display high transparency (w80%). doi:10.1371/journal.pone.0045828.g002 microscopy shows that the homogeneity of the hybrids decreases towards high concentration of nanoparticles. The reduction in homogeneity resulted in a reduced hardness and reduced optical transparency. Infrared spectroscopy demonstrated that the nanocellulose and nanoparticles are bound through electrostatic interactions and not through the formation of covalent bonds. The hybrids with an optimized inorganic content presented in the current work showed extraordinary optical and mechanical properties, with high transmittances in the visible region and high effective Young's modulus and hardness superior to previously reported materials. These properties suggest a potential use of nanocellulose-based hybrids as transparent coatings where high wear resistance and UV activity are required.