Noncovalent Dispersion and Functionalization of Cellulose

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Noncovalent Dispersion and Functionalization of Cellulose Nanocrystals with Proteins and Polysaccharides Wenwen Fang, Suvi Arola, Jani-Markus Malho, Eero Kontturi, Markus B Linder, and Päivi Laaksonen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00067 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 24, 2016

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Noncovalent Dispersion and Functionalization of Cellulose

Nanocrystals

with

Proteins

and

Polysaccharides Wenwen Fanga, Suvi Arolab,c, Jani-Markus Malhod,e, Eero Kontturif, Markus B. Linderb, Päivi Laaksonen*,a a

Aalto University, Department of Materials Science, P.O. Box 16200, FI-00076 Aalto, Finland

b

Aalto University, Department of Biotechnology and Chemical Technology, P.O. Box 16100, FI-00076 AALTO, Finland c

VTT Technical Research Centre of Finland, Tietotie 2, P.O. Box 1000, FI-02044, Espoo, Finland d

Aalto University, Department of Applied Physics, P.O. Box 15100, FI-00076 Aalto, Finland

e

Université de Bordeaux/CNRS, Laboratoire de Chimie des Polymères Organiques, UMR5629, /CNRS/Bordeaux-INP ENSCBP 16, avenue Pey Berland, 33607 Pessac Cedex, France

f

Aalto University, Department of Forest Products Technology, P.O. Box 16300, FI-00076 Aalto, Finland

E-mail: [email protected]

Abstract

ACS Paragon Plus Environment

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Native cellulose nanocrystals (CNCs), are a valuable high quality materials with potential for many applications including manufacture of high performance materials. In this work, a relatively effortless procedure was introduced for the production of CNCs, which gives a nearly 100% yield of crystalline cellulose. However, the processing of the native CNCs is hindered by the difficulty in dispersing them in water due to the absence of surface charges. To overcome these difficulties, we have developed a onestep procedure for dispersion and functionalization of CNCs with tailored cellulose binding proteins. The process is also applicable for polysaccharides. The tailored cellulose binding proteins are very efficient for the dispersion of CNCs due to the selective interaction with cellulose, and only small fraction of proteins (5 – 10 wt-%, corresponds to about 3 µmol g-1) could stabilize the CNC suspension. Xyloglucan (XG) enhanced the CNC dispersion above a fraction of 10 wt-%. For CNC suspension dispersed with carboxylmethyl cellulose (CMC) we observed the most long-lasting stability, up to 1 month. The cellulose binding proteins could not only enhance the dispersion of the CNCs, but also functionalize the surface. This we demonstrated by attaching gold nanoparticles (GNPs) to the proteins, thus forming monolayer of GNPs on the CNC surface. Cryo transmission electron microscopy (Cryo-TEM) imaging confirmed the attachment of the GNPs to CNC solution conditions.

Introduction Cellulose nanocrystals (CNCs), pure crystalline cellulose with nanoscale dimensions, are attractive materials because of their high elastic modulus (∼130 GPa), low density (∼1.6 g/cm3), low cost, nontoxic character and high accessible surface area.1 Due to these properties and interaction with other materials, cellulose exists in plants and other life forms as a component in robust support structures. The key to the great properties of these materials is in hierarchy and order of the different components, which turns the interest in CNC research towards controlled alignment. In dispersions, CNCs can self-


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organize into a liquid crystal phase at sufficiently high concentration and the chiral nematic order can be largely preserved in films after drying of the dispersions.2 The alignment happens through intrinsic properties of the cellulose crystals. Being able to precisely control and manipulate the location and alignment of individual CNCs beyond their intrinsic abilities would open up new possibilities to understand and build new materials with enhanced mechanical properties.

In laboratory scale, CNCs as a green and low-cost material have been applied in diverse fields3, 4, 5, including composites, specific enzyme immobilization, antimicrobial and medical materials, green catalysts, emulsion stabilizers, biosensors and drug delivery. Production and processing of the native cellulose nanocrystals for applications, have certain limitations that need to be further developed before the material can be effectively utilized on large scale and its fine properties fully benefited in technological solutions. Extraction of the cellulose nanocrystals with the method presently common, using concentrated sulfuric acid, has large water consumption and the yield far away from ideal. In this process, only 30-60 % yield is achieved depending on the parameters used in the hydrolysis process.6, 7 Recently, alternative methods for CNC production have been developed, including enzymatic8, mechanochemical99, oxidation10, 11and esterification12 routes. These include changes to the CNC through chemical modification and some lead to reduction of crystallinity of the CNCs. In this work, we prepare native CNCs using a very recent innovation introducing hydrolysis of cotton filter paper by hydrochloric acid (HCl) vapor.13 This is a rather effortless procedure to achieve high quality nanocrystals with yield higher than 98 %. After hydrolysis, the CNCs are still trapped in the confines of the hierarchical fiber cell wall and have been dispersed only by treatment with concentrated formic acid and prolonged sonication times.13 We have developed this method further and show how the CNCs can be dispersed in mild aqueous conditions assisted by wet-milling and biomolecules.


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Different methods have been employed to enhance the dispersion of CNCs to overcome the low stability of unmodified CNC dispersions.10, 14 Introducing electrostatic repulsion between the crystals by increasing the surface charges, is an efficient method to enhance the dispersion. (2,2,6,6tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidized CNC has been shown to form homogeneous water suspension due to the presence of newly introduced carboxyl groups adding negative charges on the surface of the nanocrystals.15, 16Positive charges have been introduced by cationic surface functionalization of the CNCs.17 Another possibility is to increase the colloidal stability by surface charges and steric repulsion by grafting a polymer, such as poly(ethylene glycol) (PEG), on the surface of the CNC.18, 19Besides the chemical routes, colloidal stability can also be enhanced by non-covalent modification of the CNCs employing molecules or polymers, such as carboxylmethyl cellulose (CMC), xyloglucan (XG) or biomolecules like DNA that can adsorb onto cellulose.20, 21 XG is widely used as a sizing agent for textiles and effective dispersion agent for pulp fibers.22, 23The adsorption of CMC on fibers is driven by the similar conformation between CMC and cellulose24 and is known to depend on the degree of substitution of CMC, pH and ionic strength of the medium and the fabrication level of the fibers.25 CMC has also been applied to bacterial cellulose nanocrystals to improve their colloidal stability.20

Cellulose binding modules (CBMs) are structural motifs found in cellulose-degrading enzymes, which have a specific affinity to cellulose surfaces.26 A recombinant double CBM, denoted as dCBM, can be constructed by genetic engineering techniques by fusing the individual CBMs via a polypeptide linker.27 Our hypothesis is that as the dCBM binds to the surfaces of CNC, it shields the cellulose-cellulose interactions between the nanocrystals and thus enhances their colloidal stability by preventing aggregation. Hydrophobins, in turn, are amphiphilic surfactant-like proteins produced by filamentous fungi.28 The hydrophobin HFBI has a size of approximately 70 amino acids and is globular with a cross-


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section of about 2 nm. HFBI has been linked to dCBM through genetic engineering29 to form a recombinant fusion protein (HFBI-dCBM) that has found potential applications, in self-assembly of nanofibrillated cellulose, in stabilizing emulsions30, in dispersing drug nanoparticles31 and as a building block in biomimetic nanocomposites.29, 32 Here, the fusion protein was anchored to the surface of the CNCs to stabilize their dispersion and also to functionalize them. Attachment of the protein layer was visualized by labelling the protein molecules by gold nanoparticles.

We present a comparison of dispersion of the CNCs hydrolyzed by HCl vapor from filter paper through two different types of non-covalently bound molecules, the first type being the cellulose binding proteins and the second type being polysaccharides. The aim was to demonstrate how the hydrolyzed paper can be processed into dispersion of CNCs simultaneously functionalized by self-assembling adhesive molecules. The results convince that the presented method has potential for production of CNCs having new capabilities, for instance controlled binding or alignment, through attached genetically engineered molecules. Materials and Methods The Whatman filter paper was hydrolyzed in a desiccator saturated with hydrochloric acid vapor for 4 hours and then rinsed with deionized water to remove the remained acid. The CNC suspension was prepared using a wet-milling technique performed in a planetary ball mill (Pulverisette 7 Premium, Fritsch Co., Idar-Oberstein, Germany). 1 g of coarsely ground CNC was wetted with 6 ml water in the milling bowl containing 30 g of milling pearls (zirconium oxide, diameter 1mm). The wet paste of CNC was milled at 1100 rpm for 8 ×3 min with a pause of 15 min in between. After milling, the CNC paste was separated from the grinding pearls by sieving and washing with deionized water.

The morphology of the ball milled CNC was imaged using a field emission scanning electron microscope (Zeiss Sigma VP FE-SEM) under the acceleration voltages of 2 keV. The samples were sputtered with a


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thin layer of gold/platinum (Emitech K950X/K350) to prevent the charging of the samples. Transmission electron microscopy (TEM) imaging of CNC/GNP-dCBM sample was performed with high-resolution Tecnai F20 200 kV FEG microscope.

Ball milled CNCs were imaged by atomic force microscopy (AFM) in ambient conditions. The samples were prepared by drop casting of the CNC suspensions on silicon wafer, which was treated with ozone to make the surface more hydrophilic. The Nanoscope IIIa multimode scanning probe microscope (Digital Instruments Inc., Santa Barbara, CA) operating in tapping mode was employed. Silicon cantilevers (NSC15/AIBS, MicroMasch, Tallinn Estonia) with a driving frequency around 300−360 kHz were used. The radius of the tip according to the manufacturer was less than 10 nm. Flattening was the only image processing applied.

3 mL of CNC suspension with concentration ratios of CMC (Sigma-Aldrich, DS 0.7) or XG (Megazyme, >95 %) from 0 to 1 were tip sonicated (Branson S-450D sonifier, 1/8” stepped microtip, USA) for 10 min using 20 % of the full output power (about 1800 J) and the concentration of CNC was kept at 2.5g L-1. The salts in CMC were removed by dialysis before use. For CNC with HFBI-dCBM or dCBM, 1.2 mL sample was tip sonicated in ice bath for 5 min with 15 % output power (about 300 J) and the concentration of CNC was 1 g L-1.

The CNC suspensions dispersed with additives were centrifuged at 5000 rpm for 10 min. The supernatant (0.3 mL) was dried in an oven at 40 °C for overnight and subjected to a two-step hydrolysis according to the standard NREL/TP-510-42618. The recovered neutral sugar monomers were analyzed by a Dionex ICS 3000 high-performance anion exchange chromatograph with pulsed amperometric detection (HPAEC-PAD) equipped with a CarboPacPA20 column (Dionex, Sunnyvale, CA, USA).


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CNC, CMC and XG all contain glucose, so the CNC glucose and glucose stemming from CMC or XG have to be distinguished. It was found that the 6-O-carboxymethyl glucose peak correlated linearly with the glucose peak in CMC and the xylose, galactose and arabinose correlated linearly with glucose in XG.20 Therefore, different concentrations of CMC and XG standards were hydrolyzed and analyzed and this proportionality was used in the case of CNC/CMC and CNC/XG (Figure S1-S3 in supporting information). The amount of 6-O-carboxylmethyl glucose and xylose was used to calculate the amount of glucose derived from CMC and XG respectively. The amount of glucose derived from CNC was calculated by subtracting the glucose derived from CMC or XG from the total glucose.

The mean particle size of dispersed CNC was determined by photon correlation spectroscopy (PCS) on a Malvern Zetasizer 3000 (Malvern Instrument, Malvern, UK). Because PCS is a light-scattering method, the measured CNC particle size values quoted are the z-average (intensity mean) hydrodynamic diameters of equivalent spheres and do not represent actual physical dimensions of the needle-like CNC particles. However, they are valid for comparison purposes. The measurements were performed three times for each sample.

5 nm N-hydroxysulfosuccinimide (NHS) -activated gold nanoparticles were purchased from Cytodiagnostics Inc. and conjugated with proteins according to the supplier’s instructions. CdTe quantum dots (QD) terminated with –COOH group (Mw ca. 25000 g mol-1, 540 emission maximum, 2.3 nm) were purchased from PlasmaChem GmbH (Berlin, Germany). 10 mM of 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 20 mM of (NHS) were reacted with 1 g L-1 of QD in aqueous solution, pH ∼ 5, for 20 min at 20°C. Activated QD were then conjugated with HFBIdCBM (1 g L-1) in 25 mM phosphate buffer (pH 7.4) for 3h at 20°C. The QD-labeled HFBI-dCBM were mixed with CNC and incubated at 4°C for at least 1 h before TEM imaging. A negative control was made by leaving out EDC and NHS and performing reactions otherwise, as described above.


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Results and Discussions

CNC preparation

After the HCl vapor treatment, the cotton filter paper became brittle. The apparent loss of toughness indicated that the degree of polymerization of the cellulose microfibrils had been severely reduced due to hydrolysis of fibrils into shorter crystalline segments. There is also recent evidence of recrystallization of cellulose due to such treatment.13 The integrity of the fibers and the paper itself was retained upon hydrolysis because the HCl vapor leaves the morphology intact meaning that the crystals are still aligned as they were in the initial fibers.

Different methods were tested for the separation of the packed cellulose nanocrystals including Wiley mill and tip sonication, but none of them were efficient enough to fully disintegrate the macroscopic fiber-like particles into individual nanocrystals (Figure 1a). Wet milling is a technique, which has been extensively used for nano-sizing drug suspensions.33 The morphology of the CNC suspensions obtained by Wiley milling and wet milling were compared with SEM. Macro-fibers were obtained for the sample ground with Wiley mill and followed by tip sonication of the suspension (Figure 1 a). Wet milling proved to be efficient enough to separate the cellulose nanocrystals as shown in Figure 1 b: nano-sized crystalline cellulose was obtained. The morphology of the CNCs was also characterized by cryo-TEM and AFM (Figure 1 c, d), and most of the CNCs were small bundles instead of single crystals. The reported diameter of an individual CNC is around 7 nm,13 whereas the CNCs after wet milling mostly had a diameter between 10-50 nm. There was also a small fraction of micron-sized fibers present. From the cryo-TEM and AFM images in Figure 1, most of the CNCs clearly consist of parallel aggregates of individual CNCs. Same kind of aggregates were also reported by Elazzouzi-Hafraoui et al.,34 who found aggregates of parallel CNCs within even the sulfuric acid hydrolyzed CNCs, which have typically length


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from 100 to 300 nm and the thickness between 5 and 15 nm. It was speculated that the association originates from biosynthesis and not from the preparation technique.

Figure 1. The morphology and properties of obtained crystalline cellulose. (a) SEM image of hydrolyzed filter paper treated by grinding and (b) ball milling. (c) Cryo-TEM image of cellulose nanocrystals suspended in water. (d) AFM image of cellulose nanocrystals.

CNC dispersion

According to conductometric titration, the crystalline cellulose prepared by HCl vapor hydrolysis had almost no charges on the surface (Figure S4, supporting information). Thus, there were merely attractive interactions between the nanocrystals and no electrostatic repulsion to stabilize the colloid leading to aggregation of the CNCs.35, 36In this work, cellulose-binding molecules of two different types were investigated for dispersing the CNCs: 1) tailored cellulose binding proteins (dCBM and HFBI-dCBM) and 2) polysaccharides (CMC and XG). The interaction of the cellulose binding modules is very selective and is directed by attachment of three aromatic amino acids of each well-defined CBM unit on the cellulose


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crystal.37 Binding of the polysaccharides is more random and is mostly due to multiple weak interactions between the polysaccharides and cellulose.20

A colloidal stability test, based on centrifugation, was developed. Less particle aggregation and sediment indicate higher colloidal stability, so a higher concentration of the CNCs in the supernatant after centrifugation means higher colloidal stability. Plain CNCs without any additives were used as a reference and a total sedimentation during centrifugation was evaluated based on the quantification of the total sugar contents (Figure 2). The CNCs were dispersed with all different additives in a concentration-dependent manner either showing a saturation of the maximal dispersed fraction (XG, HDBI-dCBM and dCBM) or a linear increase of the dispersed fraction as a function of the additive concentration (CMC).

For dCBM/CNC, 13 wt-% of the CNCs were detected in the supernatant at the mass ratio of 0.015 due to the higher colloidal stability provided by the protein. At 0.05 mass ratio, about 28 wt-% of the CNCs remained in the supernatant. From 0.05 onwards, the CNC concentration in the supernatant was near wt-29 % and the dispersion stability did not show more significant increase. The amount of dispersed nanocrystals is limited due to the saturation of the bound proteins at the cellulose interface reported in an earlier study.36 There are possibly also some limitations due to tight bundling of the CNCs, which were not mechanically separated by the wet milling and a certain fraction of the CNCs remained in the bundles.

The fusion protein HFBI-dCBM was also used for the dispersion of CNCs. The HFBI was linked to the cellulose binding module via a flexible linker, meaning that the protein has larger dimensions and may therefore provide more steric repulsions between cellulose crystals as shown in the illustration in Figure 2b. It has been observed earlier, that in a hydrogel of nanofibrillated cellulose (NFC), the same molecule may increase the cohesion between the NFC fibrils as higher values for the viscoelastic moduli were


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recorded when HFBI-dCBM was introduced to NFC.38 Here, the nanocellulose has remarkably smaller aspect ratio and does not form a hydrogel but a colloidal suspension, where the particles move more freely due to diffusion and are not stagnant as in a hydrogel. Thus, the particles are less likely to find position next to each other and we do not expect strong adhesion and aggregation due to the attachment of the proteins to the CNCs. A higher concentration of the CNC in the supernatant (35 % max) indicated a slightly better colloidal stability for HFBI-dCBM/CNC. Only the addition of a very small amount of dCBM and HFBI-dCBM, wt-5% and wt-10% respectively (corresponds to about 3 µmol g-1 CNC) could reach the maximum dispersion of the CNCs. When comparing to dCBM, this may be explained by the difference in their molar masses. The presented results enable us to propose that cellulose binding proteins could stabilize CNCs efficiently by coating the surfaces to inhibit the attraction between the CNCs. However, the cellulose biding modules were not able to break down large CNC bundles, but they were present even after the protein treatment of the samples.

Xyloglucan could stabilize the CNCs up to a saturation level comparable to the proteins, but the stabilization at low concentration (less than 10 wt-%) was less efficient. Based on previous reports, it is known that XG prefers to adhere to cellulose as a monolayer rather than multilayers in dilute aqueous solutions and that the adhesion appears to be irreversible.39, 40 Saturation of the cellulose surface by XG is a reasonable explanation for leveling off of the dispersed fraction in the supernatant. Before saturation, the dispersed amount of the CNCs remained very low until the XG/CNC ratio was 0.1, which indicates that XG is not effectively stabilizing the CNCs until a full surface layer has been formed.

The effect of CMC on the dispersion stability was studied by mixing different concentrations of CMC with CNC by tip sonication. When the mass ratio of CMC/CNC was less than 10 %, the CNCs almost completely sedimented during centrifugation. Previous work on the dispersion of bacterial CNCs demonstrated that only a full coverage of cellulose surface with CMC yields stable suspensions.19 In the


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present work, the concentration of the CNC in the supernatant kept increasing with an increasing amount of CMC showing no saturation (Figure 2). At mass ratio 1, there was about 60 % of the CNCs in the supernatant, which is a much higher fraction than what was observed for the proteins and XG. The continuous increase of stabilized CNCs means that binding of CMC is not limited to concentration ratio as it is for the proteins and XG that can only occupy certain surface sites. The high electrostatic repulsion of CMC, combined with its rather large molecular mass also indicates that it can probably stabilize larger CNC bundles than the other stabilizers.

Figure 2. a) The percentage of CNCs dispersed and remained in the supernatant after centrifugation plotted against the ratio of the dispersant/CNC. The samples dispersed by CMC are denoted with black squares, xyloglucan by red circles, HFBI-dCBM by green upward triangles and dCBM by blue downward triangles. b) An illustration showing the possible mechanism of the dispersion of CNC with different additive molecules.

The size measurement of the CNCs dispersed by different stabilizers by dynamic light scattering gave insight into the changes of CNC bundle size with addition of the additives. The storage times of the


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different dispersions were also studied by recording the size of the particles as a function of time. The measured CNC particle size values are hydrodynamic diameters which are not equal to the actual physical particle dimensions. The measured size corresponds to the effective particle movement within a liquid, and it strongly correlates with particle length. Therefore, the size shown in Figure 3 is an estimated value of the particle size and only valid for comparative study.41 Note that here the centrifugation was not carried out, but the size is representative for the whole sample.

The particle size decreased with increasing concentration of the cellulose binding proteins until the mass ratio reached value 0.1 and the mean size of the CNC particles had decreased to around 350 nm as presented in Figure 3a.

The stability of the CNC suspensions at room temperature was investigated by size measurement as a function of time. During the first few hours, all the samples went through an increase of particle size. After that, the increase of the size slowed down. The CNCs dispersed with CMC were the most stable and the particle size remained close to 600 nm even after storage for 12 days. The negative charges introduced by the CMC adsorption is one of the reasons for the stability. For dCBM/CNC, the size increased from 370 nm to 530 nm in the first 5 h and then slowly increased with time. The HFBIdCBM/CNC sample had a similar trend at the beginning, but the particle size rapidly increased from the fifth day. In solution, the HFBI forms dimers and tetramers due to the hydrophobic interactions,42 which may cause the aggregation of the CNCs during the storage. The CNC/HFBI-dCBM could be well redispersed under the treatment of ultrasonic waves. The mean size of XG/CNC kept increasing along with storage time, which indicates the XG can only provide weak stabilization of the dispersions.

AFM was used to determine the morphology and dimensions of the CNCs (Figure 4). Note, that the whole sample without centrifugation was taken for AFM imaging. Therefore, there is a small fraction of large CNC bundles in all samples which are not disintegrated by wet milling, and these large bundles


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were excluded in the diameter analysis. Because the sample was prepared by drop casting, the CNCs may have formed network during the drying process. The average diameter of the original CNCs, determined as the thickness of the CNC, was 22.2 nm and the distribution was rather wide. From the diameter histogram, almost no individual CNC exists without any dispersing additives (Figure 4a). The average diameter of CNCs dispersed with dCBM decreases to 16.4 nm and a fraction of individual crystals appears, which suggested that dCBM could stabilize and inhibit aggregation of CNCs. CBM has a very well-defined structure and a diameter of about 3 nm30 and it becomes very tightly associated with the CNC surface after binding. However, the repulsive interactions between CNCs decorated with the dCBM appeared not to be strong enough to further disintegrate the CNC bundles, therefore there were still bundles in the suspension. For both of the samples CNC/CMC and CNC/XG, not very high resolution was obtained with AFM and the average diameters were close to 20 nm, practically similar to the dispersion without any stabilizer. This might be due to the large size of CMC and XG, which prevents them from adsorbing to the small cavities between the individual CNCs and results in a randomly adsorbed CMC and XG layers on the surface of the CNC bundles.


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Figure 3. a) The size of the CNC particles as a function of the protein/CNC ratio measured immediately after dispersion. The black squares denote the samples dispersed dCBM and red circles are the samples dispersed by HFBI-dCBM. Note that the protein/CNC ratio is presented based on concentration due to the significant difference in the molar masses of the proteins. b) The size of the CNC particles measured as a function of time. The concentration ratios of CMC/CNC (black squares), XG/CNC (red circles), HFBIdCBM/CNC (green upward triangles) and dCBM/CNC (blue downward triangles) were 1, 1, 0.1 and 0.05 respectively.


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Figure 4. The morphology and diameter histogram of CNC characterized with AFM (5umx5um). (a) Plain CNC. (b) CNC with dCBM. (c) CNC with CMC. (d) CNC with XG. The diameter of CNC was analyzed with section function in NanoScope Analysis software.

To further increase the fraction of individual CNCs, we combined wet milling with the addition of the stabilizing agent. Our hypothesis was that the dispersion would be more efficient if the additive molecules could adsorb to the CNC surfaces during the mechanical treatment. Therefore, we milled the crystalline cellulose together with the CMC solution. During the wet milling, the CNCs were separated and the CMC adsorbed on its surface, enhancing the disintegration process. As shown in Figure 5, there were small bundles of CNC after wet milling (a, c), but single nanocrystals were obtained if milled with CMC (b, d). Addition of CMC during the wet milling led to more homogeneous CNC suspensions where the thickness of the CNCs was around 10 nm whereas the thickness of the milled plain CNC was in the range of 10-50 nm.


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Figure 5. The morphology of the CNCs dispersed by wet milling with and without CMC. AFM topography images of a) CNC dispersed by wet milling in water and b) CNC dispersed by wet milling together with CMC. The graphs in c) and d) are typical cross sections taken from the corresponding AFM images. CryoTEM images of the CNC dispersions wet milled in water e) and with CMC f).

To visualize how the cellulose binding proteins functionalize the surface of the nanocrystals, we carried out experiments with proteins labeled with GNPs and imaged them by TEM. The NHS-activated GNPs can react to the primary amine groups of the proteins as shown in the insert schematics of Figure 6. There is only one primary amine group in the single cellulose binding module, which can react with a


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GNP.30 This is the N-terminal amine of the protein and it is not in close proximity of the binding face. The cellulose binding face has a length of about 3 nm and the GNPs used here have a diameter of around 5 nm, which means the separation of the GNP and the cellulose surface is at least 6 nm in extended conformation. TEM images of nanocrystals dispersed with GNP labeled proteins are presented in Figure 6. The nanoparticles are associated very selectively to the cellulose nanocrystals to form a monolayer for both HFBI-dCBM and dCBM, but the binding of the nanoparticles varied from nanocrystal to nanocrystal. The alignment of the GNPs attached via HFBI-dCBM, was less regular compared with the dCBM sample. The HFBI has some primary amine groups38 which may bind GNP and cause the less orderly distribution of nanoparticles. In the reference sample which only contained CNCs mixed with GNPs, the GNPs were not associated with the CNC but were assembled into patterns all over the sample.

In Figure 6, the surfaces of the CNCs seems to be densely covered by a labelled protein monolayer. This was quite surprising, because in the conditions where samples were prepared, the surface coverage was not expected to be this high. There are reasons in the sample preparation method that may explain this. The TEM images were taken from drop-cast samples after drying, which may have caused an artefact in the binding density of the proteins due to the change of concentration during the drying process. As the solvent evaporates, the protein concentration near the CNCs increases and may increase the amount of bound proteins near the surface saturation, even though the original protein concentration should have lead to binding much below the saturation. The binding of the dCBM on cellulose is an equilibrium event and there are always free proteins which are not bound to cellulose. The amount of bound dCBM on cellulose increases with increasing dBCM concentrations until the binding reaches a maximum value.38 The CNC/GNP-dCBM sample for TEM imaging was very dilute, so only a small amount of the dCBM was originally bound to CNC. With the drying of the sample, the concentration of dCBM was increased which drove the dCBM bind to cellulose and therefore, formed the monolayer of GNPs. To avoid the drying effects, imaging of the CNC/GNP-dCBM sample was also carried out by cryo-TEM (Figure 7). The cryo-


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TEM micrograph rather clearly shows that the GNP labeled dCBM were bound to the CNCs even in the actual solution state. The distances between GNPs to the nearest CNCs were measured and are summarized in the histogram in Figure 7. Most GNPs were located on the surface of CNCs with apparent zero distance and a small fraction of the GNPs were bound very close to the CNCs (a normal distribution around 6 nm). The distance corresponds well with the length of the PEG molecules capping the GNPs together with the size of the CBM. The large number of zero distance particles may be due to the fact that the image is a 2 dimensional projection of a 3 dimensional structure being surrounded by the attached NPs. In addition to these, there was also a population of particles further away from the CNCs, which probably consists of the free GNPs in the solution.

Figure 6. TEM images of assembled gold nanoparticles on the surface of CNCs in the presence of HFBIdCBM (a), dCBM (b) and reference (c). HFBI-dCBM and dCBM were decorated with gold nanoparticles through EDC-mediated esterification. The inserts are the schematic presentation of the covalent conjugation of GNPs to HFBI-dCBM and dCBM.


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Figure 7. Cryo-TEM image of the assembled gold nanoparticles on the surface of CNCs in the presence of dCBM and the histogram of distances between GNP and the surface of CNCs (insert). The column of 50 nm stands for those free unbounded GNPs.

Conclusions

We report how HCl hydrolysed paper can be turned into dispersions of CNC, coated with cellulose binding proteins or polysaccharides, in a one-step procedure with high yield. The hydrolysis of paper was carried out by HCl vapor, which provides an efficient and unique process for the preparation of uncharged CNCs with high crystallinity. Even though the low colloidal stability of the native CNCs introduces a challenge for production of the CNCs, the resulting cellulose nanocrystals had very high quality and were available on reasonable quantities. Only 5-10 wt-% of the cellulose binding proteins could significantly improve the colloidal stability of the CNC suspension, which demonstrated the high


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efficiency of the biomolecules. The protein provided the surface with new features, demonstrated by labeling the proteins with gold nanoparticles that were then visualized by TEM and Cryo-TEM near the surface of the CNCs. This shows how precisely designed and genetically engineering molecules can be efficiently employed in preparation and functionalization of nanomaterials.

Acknowledgement

We thank Riitta Suihkonen and Michael Bayley for purifying and fermenting proteins, Yanling Ge for the help with HR-TEM, Timo Laaksonen for the support with DLS and Herbert Sixta for the support of HPAEC. The work was supported by the Academy of Finland (Centres of Excellence Program 2014-2019). EK acknowledges the Academy of Finland for his funding (Project 259500). Supporting Information Description and graphs related to sugar analysis and conductometric titration are provided in the Supporting Information.

References

1. Dufresne, A., Nanocellulose: a new ageless bionanomaterial, Mater. Today 2013, 16 (6), 220-227.

2. Revol, J. F.; Bradford, H.; Giasson, J.; Marchessault, R. H.; Gray, D. G., Helicoidal self-ordering of cellulose microfibrils in aqueous suspension, Int. J. Biol. Macromol. 1992, 14 (3), 170-172.

3. Moon, R. J.; Martini, A.; Nairn, J.; Simonsen, J.; Youngblood, J., Cellulose nanomaterials review: structure, properties and nanocomposites, Chem. Soc. Rev. 2011, 40 (7), 3941-3994.


21

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 35

4. Klemm, D.; Kramer, F.; Moritz, S.; Lindström, T.; Ankerfors, M.; Gray, D.; Dorris, A., Nanocelluloses: A New Family of Nature-Based Materials, Angew. Chem. Int. Ed. 2011, 50 (24), 5438-5466.

5. Lam, E.; Male, K. B.; Chong, J. H.; Leung, A. C. W.; Luong, J. H. T., Applications of functionalized and nanoparticle-modified nanocrystalline cellulose, Trends in Biotechnology 2012, 30 (5), 283-290.

6. Bondeson, D.; Mathew, A.; Oksman, K., Optimization of the isolation of nanocrystals from microcrystalline celluloseby acid hydrolysis, Cellulose 2006, 13 (2), 171-180.

7. Fan, J.-s.; Li, Y.-h., Maximizing the yield of nanocrystalline cellulose from cotton pulp fiber, Carbohydr. Polym. 2012, 88 (4), 1184-1188.

8. Satyamurthy, P.; Jain, P.; Balasubramanya, R. H.; Vigneshwaran, N., Preparation and characterization of cellulose nanowhiskers from cotton fibres by controlled microbial hydrolysis, Carbohydr. Polym. 2011, 83 (1), 122-129.

9. Tang, L.; Huang, B.; Yang, N.; Li, T.; Lu, Q.; Lin, W.; Chen, X., Organic solvent-free and efficient manufacture of functionalized cellulose nanocrystals via one-pot tandem reactions, Green Chem. 2013, 15 (9), 2369-2373.

10. Leung, A. C. W.; Hrapovic, S.; Lam, E.; Liu, Y.; Male, K. B.; Mahmoud, K. A.; Luong, J. H. T., Characteristics and Properties of Carboxylated Cellulose Nanocrystals Prepared from a Novel One-Step Procedure, Small 2011, 7 (3), 302-305.


22

Page 23 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

11. Yang , H.; Alam, M. N.; Ven, T. G. M., Highly charged nanocrystalline cellulose and dicarboxylated cellulose from periodate and chlorite oxidized cellulose fibers, Cellulose 2013, 20 (4), 1865-1875.

12. Braun, B.; Dorgan, J. R., Single-Step Method for the Isolation and Surface Functionalization of Cellulosic Nanowhiskers, Biomacromolecules 2009, 10 (2), 334-341.

13. Kontturi, E.; Meriluoto, A.; Penttilä, P. A.; Baccile, N.; Malho, J.-M.; Potthast, A.; Rosenau, T.; Ruokolainen, J.; Serimaa, R.; Laine, J.; Sixta, H., in preparation.

14. Rusli, R.; Shanmuganathan, K.; Rowan, S. J.; Weder, C.; Eichhorn, S. J., Stress Transfer in Cellulose Nanowhisker Composites—Influence of Whisker Aspect Ratio and Surface Charge, Biomacromolecules 2011, 12 (4), 1363-1369.

15. Montanari, S.; Roumani, M.; Heux, L.; Vignon, M. R., Topochemistry of Carboxylated Cellulose Nanocrystals Resulting from TEMPO-Mediated Oxidation, Macromolecules 2005, 38 (5), 1665-1671.

16. Habibi, Y.; Chanzy, H.; Vignon, M. R., TEMPO-mediated surface oxidation of cellulose whiskers, Cellulose 2006, 13 (6), 679-687.

17. Hasani, M.; Cranston, E. D.; Westman, G.; Gray, D. G., Cationic surface functionalization of cellulose nanocrystals, Soft Matter 2008, 4 (11), 2238-2244.

18. Araki, J.; Wada, M.; Kuga, S., Steric Stabilization of a Cellulose Microcrystal Suspension by Poly(ethylene glycol) Grafting, Langmuir 2001, 17 (1), 21-27.


23

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 35

19. Araki, J.; Mishima, S., Steric Stabilization of “Charge-Free” Cellulose Nanowhiskers by Grafting of Poly(ethylene glycol), Molecules 2015, 20 (1), 169.

20. Winter, H. T.; Cerclier, C.; Delorme, N.; Bizot, H.; Quemener, B.; Cathala, B., Improved Colloidal Stability of Bacterial Cellulose Nanocrystal Suspensions for the Elaboration of Spin-Coated CelluloseBased Model Surfaces, Biomacromolecules 2010, 11 (11), 3144-3151.

21. Mangalam, A. P.; Simonsen, J.; Benight, A. S., Cellulose/DNA Hybrid Nanomaterials, Biomacromolecules 2009, 10 (3), 497-504.

22. Zhou, Q.; Rutland, M. W.; Teeri, T. T.; Brumer, H., Xyloglucan in cellulose modification, Cellulose (Dordrecht, Neth.) 2007, 14 (6), 625-641.

23. Yan, H.; Lindstroem, T.; Christiernin, M., Some ways to decrease fibre suspension flocculation and improve sheet formation, Nord. Pulp Pap. Res. J. 2006, 21 (1), 36-43.

24. Blomstedt, M.; Kontturi, E.; Vuorinen, T., Optimising CMC sorption in order to improve tensile stiffness of hardwood pulp sheets, Nord. Pulp Pap. Res. J. 2007, 22 (3), 336-342.

25. Blomstedt, M.; Vuorinen, T., Modification of softwood kraft pulp with carboxymethyl cellulose and cationic surfactants, J. Wood Sci. 2007, 53 (3), 223-228.

26. Linder, M.; Teeri, T. T., The roles and function of cellulose-binding domains, J. Biotechnol. 1997, 57 (1-3), 15-28.


24

Page 25 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

27. Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T. T., Characterization of a double cellulose-binding domain. Synergistic high affinity binding to crystalline cellulose, J. Biol. Chem. 1996, 271 (35), 2126821272.

28. Linder, M. B., Hydrophobins: Proteins that self assemble at interfaces, Curr. Opin. Colloid Interface Sci. 2009, 14 (5), 356-363.

29. Laaksonen, P.; Walther, A.; Malho, J.-M.; Kainlauri, M.; Ikkala, O.; Linder, M. B., Genetic Engineering of Biomimetic Nanocomposites: Diblock Proteins, Graphene, and Nanofibrillated Cellulose, Angew. Chem. Int. Ed. 2011, 50 (37), 8688-8691.

30. Varjonen, S.; Laaksonen, P.; Paananen, A.; Valo, H.; Hähl, H.; Laaksonen, T.; Linder, M. B., Selfassembly of cellulose nanofibrils by genetically engineered fusion proteins, Soft Matter 2011, 7 (6), 2402-2411.

31. Valo, H.; Kovalainen, M.; Laaksonen, P.; Haekkinen, M.; Auriola, S.; Peltonen, L.; Linder, M.; Jaervinen, K.; Hirvonen, J.; Laaksonen, T., Immobilization of protein-coated drug nanoparticles in nanofibrillar cellulose matrices-Enhanced stability and release, J. Controlled Release 2011, 156 (3), 390397.

32. Linder, M. B.; Qiao, M.; Laumen, F.; Selber, K.; Hyytiae, T.; Nakari-Setaelae, T.; Penttilae, M. E., Efficient Purification of Recombinant Proteins Using Hydrophobins as Tags in Surfactant-Based TwoPhase Systems, Biochemistry 2004, 43 (37), 11873-11882.


25

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 35

33. Liu, P.; Rong, X.; Laru, J.; van Veen, B.; Kiesvaara, J.; Hirvonen, J.; Laaksonen, T.; Peltonen, L., Nanosuspensions of poorly soluble drugs: Preparation and development by wet milling, Int. J. Pharm. 2011, 411 (1-2), 215-222.

34. Elazzouzi-Hafraoui, S.; Nishiyama, Y.; Putaux, J.-L.; Heux, L.; Dubreuil, F.; Rochas, C., The Shape and Size Distribution of Crystalline Nanoparticles Prepared by Acid Hydrolysis of Native Cellulose, Biomacromolecules 2008, 9 (1), 57-65.

35. Viet, D.; Beck-Candanedo, S.; Gray, D. G., Dispersion of cellulose nanocrystals in polar organic solvents, Cellulose (Dordrecht, Neth.) 2007, 14 (2), 109-113.

36. Ponni, R.; Kontturi, E.; Vuorinen, T., Accessibility of cellulose: Structural changes and their reversibility in aqueous media, Carbohydr. Polym. 2013, 93 (2), 424-429.

37. Linder, M.; Mattinen, M. L.; Kontteli, M.; Lindeberg, G.; Stahlberg, J.; Drakenberg, T.; Reinikainen, T.; Pettersson, G.; Annila, A., Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I, Protein Sci. 1995, 4 (6), 1056-64.

38. Malho, J.-M.; Arola, S.; Laaksonen, P.; Szilvay, G. R.; Ikkala, O.; Linder, M. B., Modular Architecture of Protein Binding Units for Designing Properties of Cellulose Nanomaterials, Angewandte Chemie (International ed. in English) 2015, 54 (41), 12025-8.

39. Chambat, G.; Karmous, M.; Costes, M.; Picard, M.; Joseleau, J.-P., Variation of xyloglucan substitution pattern affects the sorption on celluloses with different degrees of crystallinity, Cellulose (Dordrecht, Neth.) 2005, 12 (2), 117-125.


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Page 27 of 35

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Biomacromolecules

40. Park, Y. B.; Cosgrove, D. J., Xyloglucan and its interactions with other components of the growing cell wall, Plant Cell Physiol. 2015, 56 (2), 180-94.

41. Boluk, Y.; Danumah, C., Analysis of cellulose nanocrystal rod lengths by dynamic light scattering and electron microscopy, J. Nanopart. Res. 2014, 16 (1), 2174/1-2174/7, 7 pp.

42. Szilvay, G. R.; Nakari-Setaelae, T.; Linder, M. B., Behavior of Trichoderma reesei Hydrophobins in Solution: Interactions, Dynamics, and Multimer Formation, Biochemistry 2006, 45 (28), 8590-8598.


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For Table of Contents Use Only. Noncovalent Dispersion and Functionalization of Cellulose Nanocrystals with Proteins and Polysaccharides Authors: Wenwen Fang, Suvi Arola, Jani-Markus Malho, Eero Kontturi, Markus B. Linder, Päivi Laaksonen 88x34mm (300 x 300 DPI)


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The morphology and properties of obtained crystalline cellulose. (a) SEM image of hydrolyzed filter paper treated by grinding and (b) ball milling. (c) Cryo-TEM image of cellulose nanocrystals suspended in water. (d) AFM image of cellulose nanocrystals. 82x82mm (150 x 150 DPI)


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a) The percentage of CNCs dispersed and remained in the supernatant after centrifugation plotted against the ratio of the dispersant/CNC. The samples dispersed by CMC are denoted with black squares, xyloglucan by red circles, HFBI-dCBM by green upward triangles and dCBM by blue downward triangles. b) An illustration showing the possible mechanism of the dispersion of CNC with different additive molecules. 170x77mm (150 x 150 DPI)


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a) The size of the CNC particles as a function of the protein/CNC ratio measured immediately after dispersion. The black squares denote the samples dispersed dCBM and red circles are the samples dispersed by HFBI-dCBM. Note that the protein/CNC ratio is presented based on concentration due to the significant difference in the molar masses of the proteins. b) The size of the CNC particles measured as a function of time. The concentration ratios of CMC/CNC (black squares), XG/CNC (red circles), HFBI-dCBM/CNC (green upward triangles) and dCBM/CNC (blue downward triangles) were 1, 1, 0.1 and 0.05 respectively. 170x73mm (150 x 150 DPI)


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The morphology and diameter histogram of CNC characterized with AFM (5umx5um). (a) Plain CNC. (b) CNC with dCBM. (c) CNC with CMC. (d) CNC with XG. The diameter of CNC was analyzed with section function in NanoScope Analysis software. 170x153mm (150 x 150 DPI)


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The morphology of the CNCs dispersed by wet milling with and without CMC. AFM topography images of a) CNC dispersed by wet milling in water and b) CNC dispersed by wet milling together with CMC. The graphs in c) and d) are typical cross sections taken from the corresponding AFM images. Cryo-TEM images of the CNC dispersions wet milled in water e) and with CMC f). 82x121mm (150 x 150 DPI)


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TEM images of assembled gold nanoparticles on the surface of CNCs in the presence of HFBI-dCBM (a), dCBM (b) and reference (c). HFBI-dCBM and dCBM were decorated with gold nanoparticles through EDCmediated esterification. The inserts are the schematic presentation of the covalent conjugation of GNPs to HFBI-dCBM and dCBM. 170x57mm (150 x 150 DPI)


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Cryo-TEM image of the assembled gold nanoparticles on the surface of CNCs in the presence of dCBM and the histogram of distances between GNP and the surface of CNCs (insert). The column of 50 nm stands for those free unbounded GNPs. 82x82mm (150 x 150 DPI)


Noncovalent Dispersion and Functionalization of Cellulose

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