Green Templating of Ultra-Porous Cross-Linked Cellulose Nanocrystal

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Green Templating of Ultra-Porous CrossLinked Cellulose Nanocrystal Microparticles Daniel Levin, Sokunthearath Saem, Daniel A. Osorio, Aline Cerf, Emily D. Cranston, and Jose M. Moran-Mirabal Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b03858 • Publication Date (Web): 12 Oct 2018 Downloaded from http://pubs.acs.org on October 14, 2018

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Chemistry of Materials

Green Templating of Ultra-Porous Cross-Linked Cellulose Nanocrystal Microparticles Daniel Levin,† Sokunthearath Saem,† Daniel A. Osorio,§ Aline Cerf,‡ Emily D. Cranston,§ Jose M. Moran-Mirabal†,* †Department

of Chemistry and Chemical Biology, McMaster University, 1280 Main St. W., Hamilton, Ontario L8S 4M1 Canada. §Department of Chemical Engineering, McMaster University, Hamilton, ON, L8S 4M1, Canada. ‡LAAS-CNRS,

Université de Toulouse, CNRS, INSA, UPS, 7 Avenue du Colonel Roche, 31400 Toulouse, France

ABSTRACT: Cellulose nanocrystals (CNCs) are rigid rod-like nanoparticles derived from natural cellulose. Their high surface area, mechanical strength and non-cytotoxicity have elicited interest in their use for various applications, including composite and construction materials, cosmetic, food, and biomedical products. However, few methods exist to control the morphology and dimensions of assembled CNC structures in the micrometer range. Here, we use water-in-oil droplet microfluidics to template uniform spherical CNC droplets in a non-toxic and sustainable manner. Subsequent evaporation of the water within the droplets promotes the chemical cross-linking of surface-modified CNCs, resulting in ultra-porous and flexible micrometer-sized particles. Changing the size of the microfluidic channel or the concentration of the CNC suspension results in microparticles with tunable sizes. The microparticles swell in polar solvents, with larger swelling observed for microparticles fabricated from less concentrated CNC suspensions. While swelling is pH independent, it is impacted by ionic strength for microparticles with low cross-link densities. Scanning electron microscopy reveals that the microparticles have macro- and mesopores, supporting a large specific surface area. These porous microparticles have potential for a range of applications, such as drug delivery or sorption agents, or as biodegradable beads for use in cosmetic and food applications.

INTRODUCTION High surface area, porous and uniform microparticles are attractive for many applications, including enzyme immobilization,1 size exclusion chromatography,2 and drug delivery.3 Various methods have been reported for creating microparticles, including spray-drying,4 bulk emulsification,5 and droplet microfluidics.6 The latter approach has become popular due to its ability to work under tuneable fabrication conditions to rapidly create uniform microparticles.6 Droplet microfluidics involves the use of two immiscible phases flowed perpendicularly to one another in sub-millimeter channels, typically made of glass or poly(dimethylsiloxane) (PDMS), to generate uniform droplets.7 The size of these droplets depends on a variety of factors, including channel diameter, flow rates, viscosities of the two phases and wettability of the channel walls. In addition, the channel design plays a pivotal role in the fabrication of droplets.8 Some examples of designs include the T-junction9 and flow-focusing,10 where either one or two streams of the continuous phase (e.g., oil) are flowed perpendicularly to a stream of the droplet or discontinuous phase (e.g., water). While the flow-focusing design is amenable to parallelization scale-up, the T-junction is simpler to use and permits the creation of uniform droplets over a wider range of flow rates.11 Solute-containing droplets produced in such devices can be turned into microparticles through a variety of methods, including drying,3,12 ion13 and solvent-induced gelation,14 chemical cross-linking,15 and thermal16 or light6,17,18 activated polymerization. Thus, droplet microfluidics allows microparticle production that can be optimized to the physical properties of the fabrication materials involved.

An advantage of droplet microfluidics is its high throughput, where a single device can be used to produce 105-106 particles/hour, and the devices can be parallelized to increase the particle yield.16 Due to the ability to scale up production, droplet microfluidics have been tested for the fabrication of a wide range of shape-templated microparticles. For example, Tjunction microfluidic devices have been used to create uniform poly(1,6-hexanediol diacrylate) microparticles in the 30-120 m range by photo-induced polymerization.6 Similarly, flowfocusing designs have been used to fabricate solid carboxylated copolymer particles.17 Using flow-focusing designs and controlled photo-polymerization, porous microparticles with controllable pore size, shape and size have also been created from solutions containing tripropyleneglycol diacrylate16,19 or copolymers.20,21 While the above methods yielded porous microparticles, they relied on traditional fossil fuel-derived materials and used harsh solvents during fabrication. Given the growing societal push to replace fossil fuel-derived materials in manufacturing processes, environmentally friendly materials are being considered for microparticle production. A variety of biodegradable materials have been incorporated into droplet microfluidic fabrication. For example, T-junction devices have been used to fabricate porous alginate microparticles for use as 3D cellular microenvironments.22 Similarly, the flow-focusing geometry has been used to fabricate uniform, porous poly(lactic-co-glycolic acid) (PLGA) microparticles for effective drug release,3 as well as chitosan microparticles with a high BSA loading efficiency for pharmaceutical applications.23 Furthermore, calcium alginate has been used with soybean oil as the continuous phase to

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generate shape-controlled microparticles in shapes that included spheres, disks and threads.13 The successful integration of these bio-based materials into porous microparticles has motivated a quest to incorporate cellulose, which is renewable, biodegradable, highly abundant, and could provide additional advantages, including high surface area and tunable mechanical properties.24 To date, several types of cellulose-based microparticles have been fabricated using sol-gel ion induced gelation1,2,25 and solution regeneration.14,26,27 In this way, uniform, porous, and magnetic Fe3O4/cellulose microparticles have been demonstrated for enzyme immobilization and protein delivery.25 Regenerated cellulose microparticles have also been fabricated via sol-gel processing for use in size exclusion chromatography, with nanoscale pores and high adsorption capacities.2 In another study, hollow micron-sized cellulose capsules were produced by saturating a cellulose solution with CO2, resulting in the precipitation of cellulose around spherical gas bubbles giving capsules that were lightweight and compressible.26,27 Similar cellulose capsules, intended for drug delivery, were fabricated using a flow-focusing microfluidic device with solvent-induced gelation as the driving force for capsule formation.14 In these applications, cellulose was used for its advantageous hydrophilic, non-toxic and biodegradable nature. Cellulose nanocrystals (CNCs) are commercially available rodlike nanoparticles typically produced by strong-acid hydrolysis of cellulose.28,29 They are attractive nanomaterials for the production of microparticles due to their high surface area, biocompatibility/degradability, renewability, and impressive mechanical strength (axial Young’s modulus of ~150 GPa).30,31 The use of sulfuric acid during hydrolysis introduces sulfate half-ester groups onto the CNC surface that are beneficial for keeping them colloidally-stable in aqueous media.28 However, these anionic groups make creating microparticles from aqueous suspensions challenging due to the electrostatic repulsion that keeps CNCs from interacting or forming networked structures. For this reason, to date CNCs have been primarily mixed in biopolymer solutions to generate composite hydrogels for drug delivery, with the CNCs serving as reinforcement to improve the mechanical properties of, for example, alginate microparticles.32 Two recent studies investigated CNC self-assembly within droplets templated using flow-focusing microfluidics and studied the internal structure of microdroplets of different sizes.12,33One study also explored the distribution of nanocolloids within the microdroplets,33 while the other demonstrated that spherical chiral nematic “all-CNC” solid microparticles could be templated if the aqueous droplets were allowed to dry.12 These microparticles had reduced porosity and thus lost the high specific surface area that makes CNCs attractive for many applications. In both studies only unmodified CNCs were used, which resulted in CNC assemblies held together by physical interactions that would dissolve upon rehydration. Furthermore, both studies used hexadecane or fluorinated oils for templating the microdroplets, which are not ideal from a sustainability standpoint. As all-CNC microparticles may provide an effective means of drug delivery or molecule absorption/capture due to the nanomaterial’s high surface area, a more sustainable and non-toxic fabrication method would be preferred for biomedical applications.

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In this work, our aim was to produce porous cross-linked allCNC microparticles that retain the high surface area of individual CNCs but are easier to process and are no longer classified as nanomaterials. Since an additional goal was to employ sustainable materials, our droplet microfluidic fabrication method only used water, food-grade soybean oil, CNCs, and polyglycerol polyricinoleate (PGPR), a green surfactant used in chocolate as an emulsifier.34 To enable crosslinking, the CNCs were surface grafted with reactive aldehyde or hydrazide functionalities as previously reported.35,36 Hydrazone cross-linking chemistry with CNCs is promising as it does not require added stimuli such as heat or light36 and has been used to create hydrogels35 and aerogels37 that display low cytotoxicity.38 Microparticle formation was promoted through drying of the microfluidic templated droplets in a soybean oil bath, which allowed the reactive CNCs to come into close proximity, overcoming electrostatic repulsion, to form hydrazone bonds. The resulting cross-linked solid microparticles were uniform, with sizes and mechanical properties tunable through the fabrication conditions and were stable in a variety of solvents. The ability to control microparticle shape and size using droplet microfluidics and then set the structure through cross-linking offers advantages over other micrometer-sized formats of cellulose. Unlike microcrystalline cellulose (e.g., Avicel, CF11) found in cosmetics, pharmaceutical products and foodstuff,39,40 the allCNC microparticles presented here have much higher porosity, are spherical, compressible, and easily tunable. The simplicity of the fabrication method and low toxicity of the materials makes these microparticles particularly attractive for applications in the personal care, food, and pharmaceutical industries. EXPERIMENTAL SECTION Materials. Whatman cotton ashless filter air was purchased from GE Healthcare (Mississauga, Canada) and sulfuric acid (95-98%) was purchased from VWR (Mississauga, Canada). Adipic acid dihydrazide (ADH, 98%), ethylene glycol (99.8%), N’-ethyl-N-(3-(dimethylamino)propyl)-carbodiimide (EDC, commercial grade), N-hydrosuccinimide (NHS, 97%), silver (I) oxide (99.99% trace metals basis), sodium periodate (NaIO4, >99.8%), sodium carbonate (>99.5%), Sodium phosphate monobasic (99%), (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO, 99%, purified by sublimation) and 14 kDa dialysis membranes were all purchased from Sigma-Aldrich (Oakville, Canada), and used without further purification. Hydrogen chloride was purchase from LabChem Inc. (Toronto, Canada), while sodium hydroxide, sodium bicarbonate (>99.7%) and sodium phosphate dibasic (99%, anhydrous) were purchased from EMD Millipore (Etobicoke, Canada). Ethyl alcohol (EtOH, 95% and anhydrous) were purchased from Commercial Alcohols Inc (Brampton, Canada). Sodium chloride (99%), sodium acetate (99%) dimethyl sulfoxide (DMSO, reagent grade), acetic acid (99.7%, glacial) and hexane (HPLC grade) were purchased from Caledon Laboratory Chemicals (Georgetown, Canada). Soybean oil was purchased from Loblaw’s Companies (retail food stores, Brampton, Canada) and polyglycerol polyricinoleate (PGPR) was a donation from Sakamoto Yakuhin Kogyo Co., Ltd (Chuo-Ku, Japan). All water used was purified Type 1 water with a resistivity of 18.2 M cm (Milli-Q Advantage A10 Water Purification system, Millipore Sigma, Etobicoke, Canada).


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Preparation of Cellulose Nanocrystals (sCNCs). Conventional sulfated cellulose nanocrystals were produced inhouse through the sulfuric acid (64 wt%) hydrolysis of Whatman cotton ashless filter aid (40 g) at 45C for 45 minutes, as described previously.47 Briefly, 40 g of filter aid was blended into a wet pulp and dried at 80C overnight. This pulp was added to 700 mL of 64 wt% sulfuric acid at 45C for 45 minutes at constant stirring. The CNC precipitate was quenched with 4C water (10:1), with the resulting suspension repeatedly pelleted by centrifugation (HeraeusTM MultifugeTM X1 Centrifuge Series, Thermo Scientific) at 14600 g for 10 minutes, decanting and washing with fresh water to remove excess sulfuric acid. The concentrated CNC suspension was then dialyzed with a dialysis membrane (MWCO = 14 kDa) for 2 weeks with successive water exchange until the dialysis water pH remained steady between 5-6. CNC suspensions were then sonicated (Sonifier 450, Branson Ultrasonics, Danbury, CT) in an ice bath for three, 15 minute cycles before being passed through a Whatman glass filter paper. These CNCs are very similar to CNCs available commercially.29 The CNC concentration in the final suspension was measured by gravimetric analysis and determined to be 0.6 wt%. The suspension was used in its acid form (pH = 5.5) stored at 4C and concentrated to the appropriate concentration for further experiments by evaporation at ambient conditions. Sulfuric acid-hydrolyzed CNCs were used to prepare the surfacemodified CNCs as described below. The resulting CNCs had apparent “size” by dynamic light scattering (DLS), electrophoretic mobility and sulfate half-ester content of 98.4  0.7 nm, -54  3 mV and 0.45  0.08 mmol of S/g of CNCs, respectively. AFM dimensions of sCNCs were 120  80 nm in length and 4  2 nm in height (N = 200). Aldehyde-Modified CNC Preparation (aCNCs). Sodium periodate (NaIO4, 12 g) was dissolved in 90 mL of 3% CNC aqueous suspension (3 g of CNCs), with the pH adjusted to 3.5  0.2 using 0.1 M HCl and NaOH. The mixture was allowed to stir for 4 hours at 45C while covered with aluminum foil to prevent photo-induced decomposition of NaIO4. Ethylene glycol (5.45 mL) was added to the suspension to stop the reaction. The suspension was then dialyzed for two weeks, sonicated and filtered as described above and stored in acid form at 4C. The resulting CNCs had apparent “size” by DLS, electrophoretic mobility and aldehyde content of 86  2 nm, 17  3 mV and 0.9  0.1 mmol/g of CNCs, respectively. TEMPO-oxidation of CNCs. (2,2,6,6-Tetramethylpiperidin1-yl)oxyl (TEMPO) (0.0888 g) and NaBr (0.972 g) were dissolved in 55 mL of water by stirring at ambient conditions overnight. Sodium hypochlorite (NaClO, 18 g, 12.5%) was then added dropwise as an aqueous solution and allowed to react for 3 hours, with the pH monitored to remain at 10  0.2 with 1 M NaOH. After 3 hours, 8.68 g of anhydrous ethanol were added to stop the reaction, with the pH of the suspension adjusted to 2.0  0.1 with 1.0 M HCl. This was then centrifuged for one round at 24,300 g at 10C for 10 minutes. The suspension was then dialyzed for two weeks, sonicated and filtered as described above and stored in sodium form at 4C. The resulting CNCs had apparent “size” by DLS, electrophoretic mobility and carboxylic acid content of 75.6  0.6 nm, -40  6 mV and 0.76  0.02 mmol/g of CNCs, respectively. Hydrazide-Modified CNC Preparation (hCNCs). ADH (0.6 g) was dissolved in 0.435 wt% aqueous suspension (200 mL) of

TEMPO-oxidized CNCs. NHS (0.014 g) and EDC (0.06 g) were respectively suspended in 1:1 DMSO:H2O solutions (4 mL) each. These solutions were then sequentially added dropwise to the flask, with a resulting, stable suspension pH of 6.8 by adjusting with 0.1 M NaOH and HCl. This reaction was carried out for one hour under ambient conditions. The suspension then dialyzed for two weeks before being stored in acid form at 4C. The resulting CNCs had apparent “size” by DLS, electrophoretic mobility and hydrazide content of 71.1  0.3 nm, -40  6 mV and 0.7  0.1 mmol/g of CNCs, respectively. Degree of Functionalization. Sulfate half-ester,54 aldehyde,55 carboxylic acid and hydrazide37 content on CNCs was quantified by conductometric titrations as previously described. CNCs (12-15 mg dry) were added to a 1 mM NaCl solution to measure conductivity. Sulfate half-ester content was titrated with 0.250 mL aliquots of 2 mM NaOH. Aldehyde, hydrazide and carboxylic acid content was titrated using 0.05 mL aliquots of 10 mM NaOH, with 0.3 mL of 0.1 M HCl added to the CNC suspension for any carboxylic acid titrations. The carboxylic content remaining on NH2NH-CNCs was subtracted from the carboxylic content on tCNCs to quantify the number of hydrazide groups on the NH2NH-CNC surface. To measure aldehyde content, these groups were selectively oxidized into carboxylic acid groups through the Ag2O oxidation reaction55 by adding CHO-CNCs (0.1006 g), NaOH (0.05432 g) and silver(I) oxide (0.3883 g) into 20 mL of water. After stirring overnight, the suspension was diluted with water to 60 mL, set to a pH of 3.3 with 1 M HCl and titrated with 10 mM NaOH. A comparison of DLS apparent size, zeta potential and degree of functionalization data between unmodified vs. modified CNCs is available in Supporting Information Table S1. Dynamic Light Scattering (DLS). DLS was used to determine the apparent particle size for CNCs within a suspension. As CNCs are rod-shaped particles and DLS assumes spherical particles, the term “apparent” is used in recognition that sizes are relative and used to monitor aggregation. The values themselves should not be taken as absolute and do not accurately represent the length or cross-section of the CNCs. Measurements were made on CNC suspensions diluted to 0.025 wt% using a Malvern Zetasizer Nano particle analyzer (Malvern Instruments Ltd, Malvern, UK). Particle size readings were taken in triplicate with the temperature maintained at 23C. Standard deviation was calculated from three individual sample measurements. Zeta Potential/Electrophoretic Mobility. The zeta potential of CNC samples was measured using a ZetaPlus electrophoretic mobility analyzer (Brookhaven Instruments Corp, Holtsville, NY). Samples were prepared by first diluting CNC suspensions to 0.25 wt% and adding 10 mM NaCl to help with accuracy. Zeta potential values were taken from an average of 10 measurements of 15 cycles per sample. Standard deviations were calculated from three individual sample measurements, with the exception of hydrazide samples that had two. Atomic Force Microscopy (AFM). Suspensions of 0.1 wt% sulfate half-ester CNCs were spin-coated onto silicon wafers at 4000 RPM for 30 seconds. A Nanoscope IIIa MultiMode Scanning Probe Microscope with an E scanner (Bruker AXS, Santa Barbara, CA) was used in tapping mode to image the samples, using probes from Nano World (FMR type 2.8 N/m spring constant and 75 kHz resonance frequency).



Measurements and images are available in Supporting Information Fig. S4. Scanning Electron Microscopy (SEM) Imaging. Microparticles fabricated from 3 wt% CNC suspensions were swollen in water and allowed to incubate for 24 hours to better remove oil/PGPR from the surface and within the microparticles. After 5 rinses with water, microparticles were submerged into liquid nitrogen and allowed to freeze dry over 24 hours. Samples were mounted on a 1” diameter stainless steel stub on carbon tape. Nickel paint was placed around the sample for Fig. 5B-C to help create a better connection to the sample stage and avoid charge build-up. Aerogels were sputtered with 5 nm platinum and imaged in a JEOL 7000SF SEM (JOEL, Tokyo, Japan). All images were taken at a working distance between 4 – 6 mm and at an acceleration voltage of 2 kV. Polarized Optical Microscopy Imaging. Microparticles were imaged with an Infinity 1 colour camera (Lumenera, Ottawa, Canada) equipped with 4 and 10x objectives and the images were captured and saved using the Infinity Capture software (Lumenera, Ottawa, Canada). Transmitted images were taken between cross-polarizers, with a 530 nm retardation waveplate used to impart colour into the sample. Confocal Imaging. Microparticles fabricated from 0.3 and 3 wt% suspensions (MP-03 and MP-3) were stained with Calcofluor White for 1 minute and placed on a glass slide. They were then imaged with a 4x objective on a Nikon A1 Confocal Eclipse Ti microscope with Nikon A1plus camera and Nikon Elements software. Microfluidic Device Fabrication. The moulds used in the fabrication of the droplet microfluidic devices were generated using two approaches, through a benchtop process using xurography and through conventional photolithography. The benchtop fabrication of microfluidic devices (Fig. S3) relied on cutting of the microfluidic device shape into an adhesive film to generate an adhesive mould that could be used to cast PDMS over, as previously reported.56,57 Briefly, a T-junction layout with a channel width of 250 m and triangular inlets (0.5 cm) was designed in Adobe Illustrator (Adobe Systems, San Jose, CA) and cut into a ByTac® PTFE surface protection laminate (Sigma-Aldrich, St. Louis, MO) using a blade cutter (ROBOPro CE5000-40-CRP, Graphtec America Inc., Irvine, CA). The ByTac cut-out, with a characteristic height of 150 m, was then adhered onto a plastic dish, where it could be used as a mould. The moulds for the 100 m-wide channel droplet microfluidic devices were fabricated in silicon wafers using standard photolithography and reactive ion etching processes, which resulted in moulds of 100 m depth but otherwise the same Tjunction layout as those made through the benchtop process. The silicon moulds were then passivated through vapor deposition of a monolayer of perfluoroalkyl silane to prevent the irreversible bonding of PDMS to the mould. All-PDMS T-junction droplet microfluidic devices were fabricated as previously reported.57 Briefly, Sylgard 184 (Dow Corning, Midland, MI) elastomer and hardener were mixed at 9:1 (v/v) ratio and degassed. The pre-cured mixture was cast over the mould in a plastic dish where small pieces of silicone tubing (Masterflex, Gelsenkirchen, Germany) were placed over the inlets, and allowed to partially cure at 60C for 2 hours. Similarly, flat PDMS slabs were made by casting the pre-cured mixture and partially curing them. Immediately before bonding the microfluidic layer with the flat slab, they were removed

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from the petri dish in which they were pre-cured and flipped to expose the bonding surfaces to oxygen plasma treatment for 30 s (30 sccm air flow, 600 mTorr) at high power setting (30W) in a PDC expanded plasma cleaner (Harrick, Ithaca, NY). The two PDMS layers were then aligned, pressed on top of one another and allowed to bond at 60C for 24 hours. After the two layers were bonded, a blade was used to cut through the exiting portion of the microfluidic channel to be able to expose it directly to a surrounding oil bath, removing the need for outlet tubing and minimizing the disturbance to the templated droplets. Microparticle Fabrication through Droplet Templating. To fabricate CNC microparticles via droplet templating, the discontinuous aqueous phase was prepared at varying CNC wt% (sCNCs or aCNC-hCNC mixtures) and flowed perpendicularly to the soybean oil-PGPR continuous phase with the use of a syringe pump (Harvard Apparatus, Holliston, MA). sCNCs microparticles were produced from 3 wt% aqueous suspensions, while chemically cross-linked microparticles were fabricated with aqueous suspensions containing 0.1 – 3 wt% CNCs. In both cases, the discontinuous (aqueous) phase was flowed at controlled rates of 4.0 and 1.6 L/min for the 250 and 100 m-wide microfluidic devices respectively, while the continuous phase (7% PGPR dissolved in soybean oil at 50C for 30 minutes) was flowed at 5.0 and 2.0 L/min for the 250 and 100 m-wide microfluidic devices respectively. Once monodisperse droplets were produced, they were allowed to flow out of the microfluidic device and collected in a 7% PGPR/soybean oil bath which surrounded the microfluidic device. The CNC-containing droplets suspended in the oil were then dehydrated at 40C over a period of 72 hours to produce solid CNC microparticles. Swelling Experiments. To assess the swelling of CNC microparticles, solvent exchange was performed from oil to water, ethanol or hexane. This was performed by pipetting 20 40 L of the intended solvent into the area surrounding the microparticles and video recording the microparticle swelling behaviour for 30 seconds under both transmitted illumination polarization and bright field microscopy using a Nikon Eclipse LV100N POL epifluorescence microscope (Nikon Instruments, Mississauga, ON) equipped with an Infinity 1 color camera. The polarizer was set 90 degrees with respect to the analyzer, and a 530 nm retardation plate was inserted between the polarizer and analyzer. Microparticles were also tested for swelling in 2 mM acetate (pH 3.95), phosphate (pH 7.20) and carbonate (pH 9.70) buffers, and in water containing various concentrations of NaCl in 2 mM phosphate buffer. The video recorded data was analyzed using ImageJ software, with the diameter recorded over time as a measure of swelling ability. With the diameters of both dry and swollen microparticles known, the volumes were calculated. The swollen microparticle volume was then divided by the dry volume, resulting in a normalized, swelling ratio. Dry Microparticle Size Measurements. To determine the size distribution of dry microparticles, a Cytation 5 Imaging Reader (BioTek, Winooski, VT) was used to acquire images of sample populations and measure their diameters. Due to optical resolution limitations, microparticles from 250 and 100 m devices were observed with 4x and 10x objectives, respectively. Density Measurements. The density of MP-1, MP-2 and MP3 microparticles fabricated in 100 × 100 m channels was measured through gravimetric analysis. A droplet containing


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swollen microparticles was placed on a pre-weighed glass slide and then imaged to determine the number of microparticles (N) within the droplet. The average diameter of these microparticles was measured with ImageJ software, and used to calculate average microparticle volume (V). The sample was then weighed after drying in an oven at 60C until the mass of the sample no longer changed. The mass of the pre-weighted slide was then subtracted, yielding the mass of the CNC material (m). With the density of cellulose nanocrystals (cell) taken from previous works as 1.5 g/cm3,49 the following equation could be used to determine the CNC microparticle porosity (P%).

[

𝑚

𝑃% = 1 ― 𝑁 × 𝑉 × 𝜌

𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒

] × 100%

(1)

Mechanical Properties of Microparticles. To measure the difference in the mechanical properties of swollen MP-0.3 and MP-3 chemically cross-linked microparticles in response to a constant compressive force, a CellScale MicroSquisher apparatus was used (Waterloo, Canada) with a cantilever diameter of 0.5488 mm. To provide a contact area between cantilevers and microparticles, a 2 × 2 mm platen was glued to the cantilevers before being mounted to the device. The microparticle samples were then pipetted out of a soybean/PGPR oil bath into a water bath within the MicroSquisher. A constant force of 100 N was applied, with images being taken throughout the compression experiments.

Figure 1. Schematic of the production of CNC microparticles via droplet microfluidic templating. (A) All-CNC microparticles were fabricated by flowing suspensions of either sCNCs or mixed aldehyde and hydrazide CNCs (aCNCs + hCNCs) through a droplet microfluidic device. (B) The aqueous CNC suspension (blue arrow) was flowed perpendicular to the continuous phase (yellow arrow, 7 w/v% polyglycerol polyricinoleate – PGPR in soybean oil), which resulted in the production of uniform aqueous microdroplets. (C) The microdroplets were collected in a surrounding bath of the continuous phase. (D) The bath containing the microdroplets was placed in an oven to evaporate the water. (E) When aldehyde (red functional groups) and hydrazide (blue functional groups) groups are in close proximity, they form hydrazone bonds (purple) that chemically cross-link the CNCs into porous microparticles. RESULTS AND DISCUSSION Droplet Microfluidic Templating of CNC Microparticles. To produce porous and uniform all-CNC microparticles, a sustainable fabrication method was used which made use of Tjunction droplet microfluidics. Aqueous suspensions of sulfated cellulose nanocrystals (sCNCs), or aldehyde + hydrazide cellulose nanocrystals (aCNCs + hCNCs) were prepared as described in the Experimental Section and Supporting Information Table S1. Suspensions of either sCNCs or mixtures of cross-linkable aCNC + hCNCs were flowed into a droplet microfluidic device through a channel perpendicular to the flow of the continuous phase solution of 7 w/v% PGPR in soybean oil. Soybean oil and PGPR, a common chocolate emulsifier, were selected as the continuous phase and surfactant, respectively, because they are sustainable, non-toxic and edible,

making these materials attractive for the development of green and safe porous microparticles. Varying concentrations of PGPR were tested, and it was found that less than 7 w/v% resulted in microdroplet coalescence and more than 7 w/v% led to no noticeable improvement in the microdroplet stability. This optimization resulted in a continuous stream of monodisperse CNC-containing droplets that were collected in a soybean oil bath (Fig. 1). Upon exiting the microfluidic device, the microdroplets sank to the bottom of the surrounding oil due to their higher density, and after 72 hours at 40C, dry microparticles could be easily collected. The slow sedimentation of the microdroplets during drying was a key step, as it ensured that microparticles were templated as spheres due to the reduction of surface tension. During the evaporation of water from the microdroplets, the



chemically-modified CNCs came close enough to cross-link with each other, which constrains the possible geometric configurations, and due to the rigidity of CNCs, porous microparticles were produced. PGPR played a key role in stabilizing the microdroplets during drying, since the absence of PGPR during drying would lead to increased coalescence and reduced microparticle uniformity. Furthermore, the higher density of the dry microparticles compared to soybean oil allowed for easier separation and recovery. After production, the microparticles retained a yellow hue, which disappeared during the swelling of microparticles in water over several hours. Thus, the yellow hue was attributed to the entrapment of soybean oil or PGPR, which are both yellow in colour and are removed as the particles are swollen in water. In this way, the use of droplet microfluidics led to the creation of spherical, porous, all-CNC microparticles with uniform shape and tunable size. Microparticle Size and Uniformity. The size of the microparticles could be tuned as a function of the microfluidic channel width used to fabricate them. The devices used for microparticle templating were made using moulds fabricated through either xurography (250 x 150 m channel crosssection) or photolithography (100 x 100 m cross-section). Dry microparticles made from 3 wt% aCNC + hCNC suspensions using the xurographic and photolithographic microfluidic channels had average diameters of 110  50 m and 30  10 m, respectively (Fig. 2). We further observed that the crosssectional area of the dry microparticles scaled proportionally to the cross-sectional area of the microfluidic channel, which highlights the ability to control the microparticle size through the device design. The obtained diameter values fall within the range of previously reported microparticles containing cellulose, such as alginate-CNC (927  14 m),32 PLGA (11, 28, 41 m),23 regenerated cellulose (5.5, 315, 631, 1096 m)2 and precipitated hollow cellulose capsules (32 – 62 m).14 While microfluidic devices made through both fabrication approaches generated well-defined populations of dry microparticles, occasionally “satellite” microparticles < 5 m in diameter were also observed. This is consistent with previous reports for particles generated in other T-junction devices,6 and suggests that further downstream processing might be needed to remove this smaller microparticle population if highly uniform microparticle sizes are required.

Figure 2. Size distribution of dry CNC microparticles (from 3 wt% CNC suspensions). The diameter of dry cross-linked microparticles fabricated from T-junction droplet microfluidic devices with channel cross-sections of 250 × 150 m and 100 ×

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100 m was characterized through optical microscopy. Microparticles fabricated from the 100 m device were more uniform than those fabricated from the larger device. Microparticles from the 250 m device were measured with a 4x objective and those from the 100 m device were measured with a 10x objective, with a lower diameter threshold of 3 pixels used. For this reason, microparticles below this threshold were not included in the histogram. Histograms correspond to n = 7933 and m = 2130 particles for the 250 and 100 m devices, respectively. Since xurography makes use of benchtop cutting equipment to fabricate adhesive vinyl channel moulds,41 whereas photolithography creates moulds through patterning and etching using microfabrication equipment,42 the comparison between these devices also allowed us to assess the impact of the fabrication technique on microparticle size dispersity. It was observed that the dispersity in microparticle diameter was consistently better for photolithographic devices, with the 100 x 100 m channels producing more uniform microparticles (Fig. 2B). Given the similarity in Reynolds numbers for the two devices at the corresponding operating conditions (330 and 270 for xurographic and photolithographic devices, respectively), this difference was attributed to the precision provided by the microfluidic mould fabrication technique. The larger dispersity in the microparticles produced from the xurography devices appears to be due to a bimodal distribution, which could arise due to instabilities during the droplet generation, leading to two populations of microparticles with slightly different sizes (of ~60 and ~100 m diameter). The xurography process relies on the direct cutting of an adhesive film using a blade, which results in microfluidic channel mould edges that are rougher than those produced through photolithography. Thus, it is reasonable to expect that smoother channels translated into greater uniformity in the final microparticle diameters. However, the xurography process is simple, rapid, and inexpensive, which enables the fabrication of microfluidic devices without resorting to photolithography masks or cleanroom procedures. Irrespective of this, both microfluidic device fabrication methods enabled the reproducible production of microparticle populations with tunable diameters. Furthermore, microfluidic devices fabricated through both methods could be re-used (with soybean oil/water cleanings between runs) for the fabrication of multiple microparticle batches, highlighting the robustness of the microfluidic devices and the microparticle fabrication technique. Microparticle Structure and Swelling. Polarized optical microscopy was used to follow CNC self-assembly during the drying step and subsequently to follow microparticle behavior during the rehydration of sCNC (non-cross-linked) and aCNC + hCNC (chemically cross-linked) microparticles (Fig. 3). CNCs are birefringent, a property that can be conveyed to the materials they are incorporated into.43 During the drying process for sCNC microparticles, a temporal evolution of increasing chiral nematic ordering was observed under polarization with a retardation plate, which imparts colour into the images facilitating the visualization of CNC alignment within the droplet. Following the first two hours of drying, sCNC microdroplets showed tactoid formation and the characteristic “fingerprint texture” of chiral nematic liquid crystals (Supporting Information Fig. S1).44 This was expected, as evaporation of surrounding solvent induced the isotropic to anisotropic phase transition.45 The tactoids are created by small


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anisotropic droplets of higher local concentration, which will ultimately coalesce with other tactoids as evaporation continues. By 24 hours, the sCNCs in the microparticles had likely reached kinetic arrest,46 as there were no observable differences between 24 and 72 hours of drying. At the end of the drying process, the dry sCNC microparticles exhibited varying degrees of order under polarization (Fig. 3A, t = 0 s), which implies that the individual particles may have arrested at different points during the self-organization process. This supports previous reports of chiral nematic ordering in droplets containing unmodified CNCs.12,33 On the other hand, droplets containing mixtures of aCNCs and hCNCs did not exhibit chiral nematic ordering during the drying process (Fig. 3B, t = 0 s). The reactive CNCs likely cross-linked before concentrating to a point where liquid crystal ordering would be expected (~ 5 wt%).47 This difference in the ordering of the CNCs was the first indication that cross-linking had occurred within the microdroplets. Overall, both microparticles maintained a spherical shape by the end of the drying process, despite exhibiting starkly different degrees of self-organization.

Figure 3. Swelling of microfluidic droplet-templated CNC microparticles (from 3 wt% CNC suspensions). When observed under polarized optical microscopy, (A) Dry sCNC microparticles displayed variable chiral nematic texturing due to the occurrence of kinetic arrest during drying, while (B) chemically-modified (and thus cross-linked) microparticles exhibited uniform radial ordering. (C) sCNC (non-cross-linked) microparticles were swollen through the addition of excess water over a period of 60 seconds. The microparticle undergoes progressive disordering from its initial state before ultimate microparticle disintegration. (D) Cross-linked aCNC + hCNC microparticles swollen in water showed minimal changes in internal ordering and excellent shape stability. The dried CNC microparticles were subsequently rehydrated to test their stability and evaluate the structural changes during swelling. For the swelling experiments, the dry microparticles were pipetted out of the oil bath and transferred onto glass slides, where they were swollen by the addition of excess water. It was observed that immediately after the addition of water, the microparticles underwent internal rehydration and swelling, as observed under polarized optical microscopy by the formation of a dark ring corresponding to the advancing water front (Fig. 3C, t = 2 s). The symmetry of this ring suggests that the microparticles have consistent porosity throughout as the water was able to penetrate the particle at a uniform rate. During the swelling process, sCNC microparticles transitioned from a textured appearance, reflecting a collapsed structure containing

birefringent domains, to a radially ordered structure with a Maltese cross pattern, but lacking apparent chiral nematic texture. The colour changes observed in the cross pattern (Fig. 3C, t = 10-25 s) are attributed to an increase in the radial alignment of CNCs as the porous microparticles continue to rehydrate and swell over time leading to decreased local CNC concentrations and suppressed self-assembly (i.e., liquid crystal) tendencies. The Maltese cross pattern observed during the swelling step suggests a microparticle “memory” effect, where the droplet templating process induces the planar alignment of the CNC local director to the droplet surface causing a radial alignment that is recovered from the dried microparticle during rehydration. In the final stages of microparticle swelling, after sufficient rehydration and expansion, the microparticles made from sCNCs (non-cross-linked) lost all internal order and disintegrated into small CNC aggregates that could no longer be resolved by optical microscopy (Fig. 3C, t = 60 s). The observed disintegration of the sCNC microparticles mirrors the process reported by Parker et. al.12 Their study also followed the formation of droplet templated sCNC microparticles, but with the important difference that microdroplet drying was done on a flat surface, which led to the collapse of three-dimensional particles to circular CNC thin films. In contrast, our study retained the spherical microparticle structure through drying as a suspension in an oil bath. Despite the variability in the initial state of self-organization of the sCNC microparticles, they all eventually disintegrated. The ultimate breakup of the sCNC microparticles indicated that the physical interactions between CNCs promoted during a short drying step (72 hours) was not enough to produce a structure that was stable against rehydration. Interestingly, particles stored in an oil bath over several months became more strongly associated and resisted complete particle disintegration in water over a 3-month period, suggesting that provided enough time and sufficient drying to induce significant hydrogen bonding, the physical interactions between CNCs are favoured over the interactions with the entrapped oil leading to more structurally stable microparticles. On the other hand, cross-linked microparticles made from aCNC+hCNC suspensions could be rehydrated and swollen without disintegrating. Prior to rehydration, the dry microparticles appeared to have radially-oriented CNCs and to be nearly identical to each other based on observation with polarized optical microscopy (Fig. 3B-D, t = 0 s). The rehydration process was tracked over time and it was seen that once water was added, the oil entrapped inside the dry microparticles was quickly displaced, as evidenced by the formation of a ring boundary that advanced over time towards the microparticle core (Fig. 3D, t = 2-10 s). The influx of water resulted in swelling of the cross-linked microparticles, but no significant change in order or structural stability was observed (Fig. 3D, t = 60 s). Confocal imaging of cross-linked microparticles labeled with Calcofluor white, a fluorescent dye that adsorbs onto cellulose, confirmed that the internal structure of the particles remained uniform after rehydration (Supporting Information Fig. S2). Additionally, the confocal images showed no significant fluorescence gradient along the radius of the rehydrated microparticle. Further swelling experiments performed on microparticles from different fabrication runs showed consistent swelling behavior across microparticle batches (Supporting Information Fig. S3). These results demonstrated that in contrast to the droplets containing native CNCs, those made from aCNC+hCNC suspensions produced



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uniform cross-linked microparticles that withstood rehydration and swelled in aqueous environments.

Figure 4. Behavior of chemically cross-linked microparticles swollen in aqueous media. (A) Brightfield micrographs of the temporal evolution of the microparticles exposed to water show that changing the starting concentration of aCNC+hCNC suspensions results in changes in the swelling behavior. Most notably, 0.1 wt% suspensions result in insufficient cross-linking between CNCs such that the microparticles cannot withstand the pressures of swelling, resulting in particle disintegration. (B) The effect of starting CNC concentration on the diameter of dry microparticles. (C) The temporal evolution of the microparticle swelling shows that changing the starting concentration affects the microparticle swelling ratio, but not the swelling rate.

Effect of CNC Concentration on Microparticle Swelling. To study the effect of the starting CNC concentration on swelling, microparticles were fabricated from suspensions containing 0.1 – 3 wt% of aCNC + hCNC mixtures (labeled as MP-0.1 to MP3 with the number corresponding to the total CNC wt%). Swelling was tracked using brightfield microscopy because the contrast between the particle and the background allowed a more accurate quantification of the changes in the microparticle diameter (Fig. 4). It was observed that all the suspensions produced uniform dry spherical microparticles, and that their diameters increased with increasing concentration of the

starting suspensions (Fig. 4A, t = 0 s, and 4B). Upon addition of water, the penetrating water front was seen as a dark ring (Fig. 4A), similar to the observation with polarization microscopy (Fig. 3C-D), and all the microparticles swelled rapidly. This behaviour was anticipated, since it has been reported that cryo-templated aerogels made from aCNC + hCNC mixtures present a combination of meso- (1 m) that take up water quickly.37 During swelling of the microparticles, water displaced the PGPR and soybean oil entrapped within these pores and had a plasticizing effect due to the exchange of CNC-CNC hydrogen bonds with CNC-water hydrogen bonds.48 For particles produced from


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suspensions with CNC concentrations > 0.2 wt%, swelling occurred within the first 10 seconds and the diameter then leveled-off to a final value that remained constant thereafter (Fig. 4C). On the other hand, MP-0.2 particles reached their final size at 30 s and MP-0.1 particles swelled continuously until the microparticle broke apart. Thus, it was determined that the threshold CNC concentration for obtaining stable, swollen microparticles was 0.2 wt%, and that any lower concentration did not present enough cross-links between the individual CNCs to withstand the capillary forces arising from rehydration of the microparticle. Finally, changing the initial particle size for a given CNC concentration could be used to further tune the microparticle swelling properties (Supporting Information Fig. S3). It was observed that MP-3 particles templated in 250 mwide channels (larger particles) had a lower swelling ratio (defined as swollen volume over dry volume, Vf/V0) than those templated in 100 m-wide channels (smaller particles). This suggests that the starting CNC mass and the surface to volume ratio of the dry microparticles is a major determinant in their ultimate swelling ability. Overall, these results show that our droplet templating and drying method is able to produce structurally stable cross-linked microparticles, where their size and swellability could be tuned by varying the starting CNC concentration and microfluidic channel dimensions.

Figure 5. Scanning electron microscopy images of porous, cross-linked MP-3 particles. (A) Low magnification images of freeze dried MP-3 samples showed spherical particles. (B) On the surface of the microparticle, distinctive porosity was

observed, with (C) closer observation revealing both macroand mesoporosity. Microparticle Porosity. The porosity of cross-linked microparticles was assessed through scanning electron microscopy (SEM) and gravimetric analysis. Dry MP-3 particles were rehydrated and washed extensively in water prior to freeze drying and transfer to carbon tape for SEM imaging. At low magnifications, SEM images showed spherical particles with smooth surfaces occasionally exhibiting cracks (Fig. 5A). Closer inspection of the microparticle surface revealed a porous structure composed of macro- and mesopores (Fig. 5B-C), similar to those observed in freeze-dried CNC aerogels.37 Although some of the internal structure might have collapsed during the sample preparation for SEM, these images suggested that after swelling in aqueous solutions the microparticles were highly porous. To further assess the effect of starting CNC concentration on microparticle porosity, the average microparticle density was measured. Samples containing microparticles suspended in water were weighed before and after extensive drying. The microparticle mass obtained was then divided by the known density of cellulose,49 the number of microparticles in the sample, and their average hydrated volumes. The obtained value could then be subtracted from 100% to determine the porosity of the microparticles.37 It was found that MP-0.3, MP-1, MP-2 and MP-3 particles fabricated using the 100 x 100 m microfluidic channels had porosities of 96  0.4, 94  0.5, 90  2 and 70  4%, respectively. These results indicate that lower concentrations of reactive CNCs led to particles with higher porosities. These porosities, on the order of 70 – 96%, are lower than the porosity of all-CNC aerogels fabricated via cryo-templating (98.6 – 99.6%),37 but within the porosity range seen in regenerated cellulose microspheres (95%)2 and magnetite/cellulose microspheres (83 – 95%),25 and much higher than those seen in noncrystalline cellulose capsules (24%).27 These results demonstrate that our droplettemplated cross-linked CNC microparticles are highly porous and consequently retain the high specific surface area that makes CNCs attractive for a wide range of applications. Impact of Solution Composition on Microparticle Swelling. The swelling behaviour of chemically cross-linked microparticles was also studied in different media. When swollen in water, it was observed that the swelling ratio of the microparticles varied as a function of the reactive CNC concentration, with a smaller concentration leading to significantly more swelling (Fig. 6A). This indicates that at increased concentrations, more aCNC-hCNC hydrazone crosslinks form, which give the particle increased structural stability and resistance to swelling. While the swelling ratio in water was inversely proportional to CNC concentration, microparticles exposed to ethanol or hexane did not exhibit any appreciable swelling (Fig. 6A). This finding was similar to cross-linked CNC aerogels, which have previously been shown to take up ethanol, DMSO and dodecane without noticeable changes in volume.37 As ethanol and hexane do not disrupt CNC-CNC interactions (H-bonding, van der Waals etc.) to the same extent as water,50 cross-linked networks retain their initial structure. This finding also suggests that in addition to weak interactions, the swelling in aqueous environments is promoted by the electrostatic repulsion between charged groups on the CNC surfaces (i.e., sulfate half-ester groups with an apparent pKa of ~ 2.5).51



To assess the role of electrostatic repulsion on swelling, MP-0.3 and MP-3 microparticles were swollen in aqueous solutions with increasing ionic strength. It was observed that MP-0.3 exhibited reduced swelling as the ionic strength was increased, but that this was not the case for MP-3 (Fig. 6B). Swelling of MP-0.3 decreased with increasing ionic strength which correlates with increased electrostatic screening of the CNC surface charges and minimized electrostatic repulsion. On the other hand, the observation that MP-3 microparticles were unaffected by ionic strength implied that a CNC network with more CNCs and a larger number of hydrazone cross-links was stronger and thus less susceptible to swelling due to electrostatic repulsion (and less affected by charge screening). Finally, the impact of pH on MP-0.3 swelling was studied in acetate (pH = 3.95), phosphate (pH = 7.20) and carbonate (pH = 9.70) buffers at different NaCl concentrations (Fig. 6C). In these pH environments, the sulfate half-ester groups on CNCs are expected to be fully charged, and the small ionic strength contribution from the buffer (2 mM) is assumed to be insignificant compared to the added NaCl (10 – 100 mM). The results indicated that ionic strength once again affected swelling, while pH did not. This was not surprising because although hydrazone linkages are known to hydrolyze more rapidly in acidic environments,52,53 the relatively short time over which the swelling experiments were conducted resulted in minor bond cleavage. We anticipate that prolonged exposure to acidic environments or solutions with lower pH would lead to the partial disintegration (limited by any physical cross-links present in the particle) of the microparticles due to hydrazone bond cleavage. The ability to store and process microparticles in oil or organic solvents, like ethanol and hexane, without expansion, and then tune the degree of microparticle swelling in aqueous solutions according to cross-link density or ionic strength, regardless of pH, makes this a very versatile system with interesting potential for loading and release of bioactive/functional materials for in vivo applications. Mechanical Properties of CNC Microparticles. The compressibility of MP-0.3 and MP-3 microparticles was tested under a fixed applied force to determine if the starting CNC concentration significantly changed the mechanical properties of the microparticles. These measurements were performed using a flat plate attached to a cantilever on a CellScale Microquisher apparatus, which was able to trap and compress individual microparticles immersed in a water bath. MP-0.3 and MP-3 microparticles were subjected to a compressive force of 100 N and it was observed that MP-0.3 microparticles were significantly weaker. Fig. 7 shows typical data, where a MP-0.3 microparticle was drastically deformed (and in this case broken, at t = 1.2 s) in less than two seconds due to the compressive force. On the other hand, MP-3 microparticles remained unchanged when subjected to the same force over a period of 3 s. The difference in mechanical strength offers another tunable quality of these microparticles, which is important for applications that require microparticles to withstand different compressive forces. It is also worth noting that all of the microparticle types fabricated were flexible and robust and generally easy to handle with tweezers; however, the microparticles produced from low starting concentrations of CNCs were slightly more susceptible to damage during handling.

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Figure 6. Swelling of chemically cross-linked microparticles as a function of solvent composition. (A) The swelling ratio of chemically cross-linked microparticles made from 0.2 – 3 wt% CNC suspensions was quantified in water (pH ~5.5), anhydrous ethanol, and hexane. (B) Microparticle swelling was also tested in phosphate buffer solutions (pH 7.2, 2 mM buffer) with varying NaCl concentrations. (C) The effect of pH on swelling, MP-0.3 microparticles were tested in 2 mM acetate (pH 3.95), phosphate (pH 7.20) and carbonate (pH 9.70) buffer systems at varying NaCl concentrations.


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Chemistry of Materials Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS

Figure 7. Compression of cross-linked MP-0.3 and MP-3 microparticles. MP-0.3 and MP-3 microparticles fabricated in channels with 250 × 150 m-cross-sections were compressed by a flat plate under a force of 100 N. The red lines in the MP0.3 sample panels indicate the change in the plate position over time and the yellow dashed lines highlight the particle contour. After 1.2 seconds the MP-0.3 microparticle broke and 1.8 seconds it collapsed, whereas the MP-3 microparticle did not significantly deform even after 3s. CONCLUSIONS We have presented a method to produce stable spherical cellulose microparticles through the chemical cross-linking of aldehyde and hydrazide-modified CNCs. The strategy is based on droplet templating with microfluidics and employs solely sustainable and non-toxic materials, which is attractive for use in downstream applications. The resulting microparticles are highly porous and have tunable swelling properties. The ability of the CNC microparticles to selectively swell in water can be exploited in storage/loading applications and the dimensional tunability offered by ionic strength can be optimized for specific environments. Furthermore, the microparticle’s ability to withstand a variety of pH environments, solvents and compressive strains without loss of structural integrity demonstrates their robustness. Finally, the production of microparticles with high porosity maintains the attractive high specific surface area of individualized CNC nanoparticles and could make these microparticles attractive for uses in food, cosmetic, drug delivery, and contaminant ad/absorption applications where larger particles are favored from a processing, handling and safety perspective. Our current efforts focus on conferring functionality to the microparticles to enable the adsorption and/or release of small molecules or ions for environmental monitoring and drug delivery. ASSOCIATED CONTENT Supporting Information Supporting information includes: Polarized optical microscopy images of sCNC microparticle formation; epifluorescence and confocal images of MP-0.3 and MP-3 microparticles; Quantification of batch-to-batch reproducibility of microparticle swelling; physicochemical properties of sulfuric acid hydrolyzed and chemically modified (cross-linkable) CNCs; representative atomic force microscopy height images of sCNCs This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author

The authors thank Prof. T Hoare, Prof. H Stover, and Prof. R. Wylie for access to equipment, and X. Ding and A.H. Jesmer for useful conversations and SEM help. The authors acknowledge Sakamoto Yakuhin Kogyo Co. Ltd for the generous donation of the PGPR used for experimentation. DL was partially supported through an NSERC USRA and SS was supported through an OGS award. JMM and EDC are recipients of Early Researcher awards from the Ontario Ministry of Research and Innovation. JMM holds the Tier 2 Canada Research Chair in Micro- and Nanostructured Materials, and EDC holds the Tier 2 Canada Research Chair in Bio-Based Nanomaterials. Funding from the Natural Sciences and Engineering Research Council of Canada through Discovery Grants to JMM and EDC is gratefully acknowledged. The authors acknowledge the French RENATECH network for technological support. This research made use of instrumentation from the Canadian Centre for Electron Microscopy and Biointerfaces Institute at McMaster University.

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