Article Cite This: Langmuir 2017, 33, 12344-12350
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Injectable Shear-Thinning Fluorescent Hydrogel Formed by Cellulose Nanocrystals and Graphene Quantum Dots Amir Khabibullin,† Moien Alizadehgiashi,† Nancy Khuu,† Elisabeth Prince,† Moritz Tebbe,† and Eugenia Kumacheva*,†,‡,§ †
Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto, M5S 3H6 Ontario, Canada Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, Toronto, Ontario M5S 3G9, Canada § Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada ‡
S Supporting Information *
ABSTRACT: In the search for new building blocks of nanofibrillar hydrogels, cellulose nanocrystals (CNCs) have attracted great interest because of their sustainability, biocompatibility, ease of surface functionalization, and mechanical strength. Making these hydrogels fluorescent extends the range of their applications in tissue engineering, bioimaging, and biosensing. We report the preparation and properties of a multifunctional hydrogel formed by CNCs and graphene quantum dots (GQDs). We show that although CNCs and GQDs are both negatively charged, hydrogen bonding and hydrophobic interactions overcome the electrostatic repulsion between these nanoparticles and yield a physically cross-linked hydrogel with tunable mechanical properties. Owing to their shearthinning behavior, the CNC-GQD hydrogels were used as an injectable material in 3D printing. The hydrogels were fluorescent and had an anisotropic nanofibrillar structure. The combination of these advantageous properties makes this hybrid hydrogel a promising material and fosters the development of new manufacturing methods such as 3D printing.
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INTRODUCTION Physical hydrogels are water-swollen networks formed by noncovalent cross-linking of small molecules,1,2 polymers,3,4 colloidal particles,1,5,6 or a combination of the colloidal building blocks and molecular cross-linkers.7−9 Physical interactions are reversible and are responsive to the changes in the ambient environment, thereby making physical hydrogels function as adaptive stimuli-responsive materials. For example, temperature-responsive hydrogels formed by polymer-functionalized cellulose nanocrystals can be liquefied upon heating, which makes such systems useful for growth and the subsequent release of cells10 or cancer spheroids.11 Another example is a hydrogel formed by wormlike block copolymer micelles, which undergoes dissolution on cooling due to a worm-to-sphere micelle transition.12 Cross-linking of physical hydrogels occurs by hydrogen bonding,13,14 host−guest chemistry,15 coordination,16 forces of electrostatic origin,17 hydrophobic interactions,18 and the entanglement of molecules or supramolecular species.19 Competing attraction and repulsion forces between the building blocks of these hydrogels are of particular interest, owing to the capability to fine tune the balance between them and thus control hydrogel properties. The variation in temperature, pH, or ionic strength of the system can be used as an external trigger to make a particular force dominant11,20 and thus trigger gel formation or dissolution. © 2017 American Chemical Society
An important property of physically cross-linked hydrogels is their shear-thinning behavior, that is, a substantial reduction in viscosity under applied shear stress with the subsequent recovery of gel properties after stress removal.21 The shearthinning property enables the use of physically cross-linked hydrogels as injectable materials,5,21,22 with applications in tissue engineering4 and in extrusion-based fabrication methods (e.g., 3D printing8). A special class of hydrogels includes nanofibrillar gels that are formed by high-aspect-ratio colloidal building blocks, each composed of many molecules. In comparison with hydrogels formed by molecules, nanofibrillar hydrogels have a larger pore size (and thus enhanced transport properties) and straindependent mechanical properties that originate from preferential nanofiber alignment.23,24 These properties of protein and polysaccharide hydrogels10,23,25 have motivated the synthesis and assembly of biomimetic manmade nanofibrillar gels.12,23 In the search for new building blocks of physical nanofibrillar hydrogels, cellulose nanocrystals (CNCs) have attracted considerable attention because of their sustainability, biocompatibility,26 mechanical strength,27,28 and ease of surface functionalization.28−30 Physically cross-linked nanofibrillar Received: August 18, 2017 Revised: September 14, 2017 Published: September 27, 2017 12344
DOI: 10.1021/acs.langmuir.7b02906 Langmuir 2017, 33, 12344−12350
Article
Langmuir
ester groups was purchased from the University of Maine Process Development Center and diluted using deionized water to a final CNC concentration of 7 wt %. Synthesis of GQDs. Graphene quantum dots were synthesized from graphite following a procedure reported elsewhere.44 Briefly, 3.75 g of graphite was mixed with 180 mL of concentrated sulfuric acid and 60 mL of concentrated nitric acid. The mixture was sonicated for 3 h and placed in an oil bath preheated to 120 °C for 24 h. Subsequently, the mixture was cooled to room temperature, diluted with deionized water, and neutralized with sodium carbonate. The resulting solution was filtered using filter paper with 1 μm pores. The solution of GQDs was dialyzed against deionized water (MWCO of dialysis membrane 14 000 Da) for 7 days. The dimensions of the GQDs were characterized using atomic force microscopy (AFM). The electrokinetic potential (ζ-potential) of the GQDs was measured using a Malvern Zetasizer Nano ZS-ZEN3600 instrument. The elemental composition of GQDs was characterized by X-ray photoelectron spectroscopy (XPS) using a ThermoFisher Scientific K-Alpha instrument. Modification of CNCs with Amino Groups. The CNCs were modified with amino groups following a procedure reported elsewhere.45 First, the CNCs were functionalized with epoxy groups via the reaction with epichlorohydrin (6 mmol/g cellulose) in 1 M sodium hydroxide (132 mL/g cellulose). After 2 h at 60 °C, the reaction mixture was dialyzed against deionized water (MWCO of dialysis membrane 14 000 Da) until the pH was below 12. Next, the epoxy groups reacted with ammonium hydroxide to introduce primary amino groups onto the CNC surface. After adjusting the value of pH to 12 with 50% (w/v) sodium hydroxide, ammonium hydroxide (29.4 wt %, 5 mL/g cellulose) was added and the reaction mixture was held at 60 °C for 2 h. The product was purified by dialysis until the solution pH was 7. Preparation of Hydrogels. The concentration of CNCs in suspension was in the range from 10 to 50 mg/mL, controlled by the addition of deionized water to the CNC suspension with a solid content of 7 wt %. The hydrogels were prepared by dissolving a different amount of a dry powder of GQDs in aqueous suspensions of CNCs in 20 mL scintillation vials. The final concentration of GQDs varied from 1 to 10 mg/mL. The capped vials were subsequently vortex mixed for 15 s and left to rest. The time of hydrogel formation varied from 5 min to several hours, depending on the concentrations of CNCs and GQDs. The photographs of the hydrogels in glass vials were taken using a Nikon D7200 camera. Rheology. The mechanical properties of CNC-GQD hydrogels were characterized using an AR-1000 TA Instruments 40-mmdiameter cone-and-plate rheometer. An integrated Peltier plate was used to control the hydrogel temperature. Solvent evaporation was minimized by using a solvent trap. Strain and frequency sweep experiments were conducted to determine the linear viscoelastic response and the shear-thinning behavior of the hydrogel at 22 °C. Strain sweep experiments were performed at strain varying from 0.1 to 50% and a frequency of 1 Hz. Frequency sweep experiments were performed at a frequency varying from 1 to 100 Hz and a strain of 1%. At a 1% strain and a frequency of 1 Hz, the hydrogels exhibited a linear viscoelastic response. These conditions were used in the rheological characterization of the hydrogel. The time sweep experiments were performed at 22 °C to determine the storage modulus, G′, and the loss modulus, G″, of the hydrogels. Prior to all experiments, the hydrogels were equilibrated for 50 min at 22 °C. AFM Imaging. A GQD solution (0.001 mg/mL, 0.25 mL) was spin-coated at 2000 rpm for 1 min on a freshly cleaved mica substrate. The AFM images were acquired using a Dimension Icon (Bruker Corporation). High-resolution probes from MikroMasch (Hi’ResC19/Cr-Au-5) with a nominal spring constant of 0.5 N/m, a resonance frequency of 65 kHz, and a tip radius of 1 nm were used. Imaging was carried out in intermittent contact mode (ac) under soft tapping conditions within the repulsive interaction force regime. Obtained AFM images were analyzed using the NanoScope Analysis software by Bruker and open source software Gwyddion. Images were leveled by third-order polynomial planefit and image flattening
hydrogels have been formed by CNC cross-linking in the presence of salts or surfactants17,31−33 or by the association of polymer molecules grafted to the CNC surface.10,11 Using cross-linkers that in addition to CNC binding can tailor an additional functionality to the CNC hydrogel is an attractive approach that is yet to be explored. In the present work, we used as such a cross-linkergraphene quantum dots (GQDs), which are two-dimensional biocompatible34 and photoluminescent35−37 nanoparticles with excitation-dependent emission.38,39 Recently, GQDs have attracted strong interest in the materials science community. Their applications have been explored in theranostics,34 catalysis,40 photovoltaics, and the fabrication of organic light-emitting diodes.36,41 As an additive, GQDs have been introduced into a nanofibrillar gel formed by low-molecular-weight molecules such as 9-anthracenemethyloxycarbonyl-protected L-phenylalanine or L-tyrosine. Although these gels formed without GQD addition, the GQDs added in a small amount that served as a secondary cross-linker due to the π−π stacking with amino acids. Notably, the same interactions led to electron-transfer-induced quenching of fluorescence of the anthracene moieties in the gel.37 In another system, S- and N-doped GQDs were embedded in a preformed CNC hydrogel,18 thus making it fluorescent. However, they did not serve as cross-linkers or building blocks of the gel and thus did not affect its mechanical properties. In the present work, we report the CNC-GQD hydrogel in which GQDs are used as a cross-linking agent for CNCs. Both types of nanoparticles have amphiphilic properties. In cellulose, the equatorial direction of a glucopyranose ring has hydrophilic character because all three hydroxyl groups are located at equatorial positions on the ring, whereas the hydrogen atoms of C\H bonds are located at axial positions on the ring, thus making the axial direction of the ring hydrophobic.42,43 In addition, GQDs are hydrophobic in their basal plane and hydrophilic on the edges as a result of the presence of carboxylic acid groups.44 Thus, CNCs and GQDs can exhibit hydrophobic interactions and form hydrogen bonds between the hydroxyl groups of CNCs and the carboxylic acid groups of GQDs. In addition, CNCs carry a negative charge due to the presence of surface sulfate half-ester groups or carboxylic groups. Therefore, CNCs and GQDs exhibit electrostatic repulsion. We show that because of the dominant role of hydrogen bonding and hydrophobic interactions between CNCs and GQDs, nanofibrillar hydrogels were formed and exhibited shear-thinning behavior, fluorescence, and birefringence. The storage modulus of the hydrogel was tuned from ∼50 to ∼260 Pa by varying the hydrogel composition. Using 3D printing, we show the application of this hydrogel as an injectable material. The use of abundant and nontoxic building blocks of this hydrogel, its ease of preparation, the tunability of the mechanical properties, the fluorescence, the CNC alignment, and the capability of fabrication using 3D printing makes the CNC-GQD hydrogel a promising material in tissue engineering, in chemical and biological sensing, and in the development of new manufacturing methods in 3D printing.
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EXPERIMENTAL SECTION
Materials. Graphite flakes, dialysis tubing (MWCO 14 000 Da), urea, epichlorohydrin (99%), and magnesium chloride hexahydrate (99%) were purchased from Sigma-Aldrich Canada. Ammonium hydroxide (29.4% assay NH3), nitric acid (68%), and sulfuric acid (98%) were purchased from Caledon Laboratory Chemicals. An aqueous 12 wt % suspension of CNCs carrying surface sulfate half12345
DOI: 10.1021/acs.langmuir.7b02906 Langmuir 2017, 33, 12344−12350
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Langmuir (excluding large features by selective masking), followed by applying a low-pass filter to suppress high spatial frequency components. Data analysis was performed by applying a linear cross-section to individual GQDs. The number of analyzed GQDs was 102. Scanning Electron Microscopy Imaging. The gels were freezedried using liquid propane in order to suppress the crystallization of water.33 This method of sample preparation was selected as an alternative to supercritical point drying of the hydrogel46 because GQDs are not colloidally stable in methanol or ethanol used in the supercritical point drying method. After sample freezing in liquid propane, the water was removed by freeze drying for 1 day. The resulting CNC-GQD aerogels were coated with gold using an SC7640 high-resolution sputter coater (Quorum Technologies) for 60 s at 2.0 kV. Scanning electron microscopy (SEM) imaging was carried out on the Quanta FEI scanning electron microscope. The thickness of the nanofibrils was measured using ImageJ software. Three-Dimensional Printing of the Hydrogel. The extrusion of the CNC-GQD hydrogel was carried out using a PRUSA i3 3D printer. Prior to extrusion, the heating extruder and the heating bed were removed from the printer. The 3D printing pattern was designed using the AutoCad 2016 student version (Autodesk, CA, USA). The G-code generated by Slic3r software (http://slic3r.org) was transferred to the 3D printer using open source Pronterface software (http://www.pronterface.com). A hydrogel sample (1 mL) at CNC and GQD concentrations of 50 and 10 mg/mL, respectively, was extruded onto a horizontally placed polystyrene surface from a 5 mL syringe. The extrusion was carried out at a constant pressure of 2.4 psi using a digital pressure regulator (Marsh Bellofram) connected to a nitrogen cylinder (Grade 4.8). The digital pressure regulator was controlled using a custom-based program (software: NI LabVIEW 2013). Photographs of the extruded gel were were recorded using a camera (Nikon D7200) connected to the binocular port of a polarized optical microscope (Olympus BX 51).
repulsion between the CNCs and GQDs, thus leading to physical cross-linking of CNCs and the formation of a nanofibrillar hydrogel (Figure 1B). Characterization of GQDs and CNCs. Figure 2A shows transmission electron microscopy (TEM) images of the rod-
Figure 2. (A) TEM image of dried CNCs. The scale bar is 100 nm. Inset: Schematic of an individual CNC with an average length, L, and diameter, D, of 183 and 23 nm, respectively, and a ζ-potential of −60 mV (measured at CCNC = 0.1 mg/mL at pH 7.0). (B) AFM image of GQDs. The scale bar is 200 nm. The height scale is 400 pm. Inset: Schematic of an individual GQD with an average diameter, D, and height, h, of 12.8 ± 0.9 and 0.250 ± 0.021 nm, respectively, and a ζpotential of −15 mV (measured at CGQD = 5 mg/mL and pH 7.0) The functional groups on the CNC and GQD surfaces in the insets are omitted.
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RESULTS AND DISCUSSION Figure 1 shows a schematic of the formation of the CNC-GQD network. We envisioned that interactions between the CNCs
shaped CNCs and a drawing of an individual CNC (inset). On the basis of the image analysis, the CNCs had an average length and diameter of 183 and 23 nm, respectively.11 Because of the presence of surface half-ester sulfate groups, the CNCs were negatively charged and had an electrokinetic potential (ζpotential) of −60 mV at pH 7. Figure 2B shows the AFM image of disk-like-shaped GQDs deposited onto a freshly cleaved mica substrate from an aqueous GQD solution. The height and the diameter of GQDs were 0.250 ± 0.021 and 12.8 ± 0.9 nm, respectively. Although the height could be accurately determined by the AFM measurements, the measured value of the diameter was influenced by tip convolution (Supporting Information Figure S1). The GQDs have a basal graphene plane and mainly oxygen-rich carboxyl groups on the edges (Figure S2).44 The XPS analysis of the GQDs revealed that they contained mainly C and O atoms, with traces of Na, Si, S, and N, which remained after GQD synthesis and purification (Figures S5 and Table S1). At pH 7, the GQDs had a ζ-potential of −15 mV. The negative surface charge of GQDs was due to the deprotonation of the carboxyl groups localized mostly at the GQD edges.44 The aqueous solutions of GQDs exhibited photoluminescence in the spectral range of 400−680 nm when excited at 365 nm. Because GQDs are prone to aggregation 48 at a concentration >1 mg/mL, their photoluminescence varied nonmonotonically with GQD concentration (Figure S3). Hydrogel Formation. A mixed suspension of GQDs and CNCs had a color change from yellow to brown with increasing GQD concentration, which was caused by GQD absorption in the wavelength range from 230 and 700 nm.36,48 The formation of the hydrogel upon mixing aqueous solutions of CNCs and
Figure 1. Schematic of the formation of the CNC-GQD hydrogel. (A) Building blocks of the hydrogel: a CNC carrying surface hydroxyl and half-ester sulfate groups (left) and a GQD carrying surface edge carboxylic groups (right). (B) Hydrogel formation upon mixing of the suspension of CNCs and GQDs. The inset shows the formation of the hydrogen bond between the carboxyl groups of GQDs and the hydroxyl groups of CNCs.
and GQDs would be governed by three nanoscale forces. The attraction force originated from the hydrogen bonding between the surface hydroxyl groups of the CNCs and oxygen-rich carboxyl groups on the GQD edges (Figure 1A) and the hydrophobic interactions between the CNCs hydrophobic faces and the GQDs basal planes.42,47 The repulsion forces were due to electrostatic interactions between the negatively charged surface sulfate half-ester groups of the CNCs and the carboxylic groups of the GQDs at pH 7. We hypothesized that attractive hydrogen bonding and hydrophobic forces would overcome the 12346
DOI: 10.1021/acs.langmuir.7b02906 Langmuir 2017, 33, 12344−12350
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Langmuir GQDs was first examined qualitatively by using a flip test (Figure 3A).
gelled within 1 h. Notably, in the presence of urea no precipitate was formed. In another experiment, the CNCs were modified with amino groups at an average grafting density of ∼0.2 NH2 group/nm2. Because the hydrogen bond between the hydroxyl and amino groups is weaker than that formed between carboxyl and hydroxyl groups,51 a mixture of amino-groupfunctionalized CNCs and GQDs formed a weaker gel (Figure 4B) than the gel prepared at the same CNC and GQD concentrations of CCNC = 50 mg/mL and CGQD = 5 mg/mL (illustrated in Figure 3A). Finally, we suppressed electrostatic interactions between the GQDs and CNCs by increasing the ionic strength of the medium. A mixed suspension was prepared with CCNC = 50 mg/mL and CGQD = 2.5 mg/mL. Under these conditions, gelation did not occur (state diagram in Figure 3B). Subsequently, MgCl2 was added to the mixed suspension to a concentration of 3 mM MgCl2. On the basis of our earlier work,17 this concentration of MgCl2 was not sufficient to cause the gelation of an aqueous CNC suspension as a result of the increased ionic strength and suppressed electrostatic repulsion of CNCs. In the present work, 30 min after the addition of MgCl2 to the mixed CNC-GQD suspension, a strong gel was formed (Figure 4C). Notably, without the addition of MgCl2, the suspension remained in a sol state for at least 24 h. The results of these three control experiments validated our hypothesis that a hydrogel formed as a result of the hydrogen bonding and hydrophobic interactions between the CNCs and GQDs, which overcame electrostatic repulsion between these nanoparticles. We note that reduction of electrostatic repulsion between CNCs due to the addition of charged GQDs (with traces of Na+ ions present after GQD synthesis) could also contribute to hydrogel formation.17,32 Gel Characterization. The mechanical properties of the CNC-GQD hydrogels were characterized in oscillatory shear experiments (Figure 5). Three hydrogels prepared at CCNC = 50 mg/mL and CGQD = 10 mg/mL (a strong gel), CCNC = 20 mg/ mL and CGQD = 10 mg/mL, and CCNC = 50 mg/mL and CGQD = 5 mg/mL (both corresponding to the weak gels) were characterized. Using these hydrogel compositions, we separately examined the effect CCNC or CGQD in the mixed suspension. At 10% strain, the values of G′ of the hydrogels decreased with applied strain, thus exhibiting nonlinear viscoelastic behavior. This effect was attributed to the disruption of the physically cross-linked network.11 At CCNC = 50 mg/mL, the value of G′ of 266 Pa for the gel prepared at CGQD = 10 mg/mL was approximately 5-fold greater than that of the gel formed at CGQD = 5 mg/mL (G′ = 54 Pa). This change in G′ was consistent with the GQDs acting as physical cross-linkers of the CNCs: a larger number of crosslinking points resulted in the formation of a stronger hydrogel. At CGQD = 10 mg/mL, reducing CCNC from 50 to 20 mg/mL resulted in a 2.5-fold decrease in the G value, which was also in agreement with the state diagram (Figure 3), indicating that increasing the concentration of CNC building blocks resulted in the formation of a stronger gel with a higher cross-linking density. Thus, the mechanical properties of the gel could be controlled by varying either CCNC or CGQD. The shear-thinning behavior of the hydrogels was evaluated by varying strain values from 1 to 50% (Figure 5B). At 1%
Figure 3. (A) Photographs of an aqueous CNC-GQD suspension at CCNC = 50 mg/mL and CGQD = 5 mg/mL taken immediately after mixing (left) and 1 h later (right). (B) State diagram outlining the CNC and GQD concentration regimes of the formation of a hydrogel. The diagram shows the state of the aqueous CNC-GQD mixture 4 h after mixing at pH 7.
Figure 3B shows the state diagram for the aqueous CNCGQD mixture, plotted for varying CNC and GQD concentrations, CCNC and CGQD, respectively. The hydrogels formed at CCNC ≥ 20 mg/mL and CGQD ≥ 5 mg/mL. At CCNC = 50 mg/ mL and CGQD > 7 mg/mL, the suspensions gelled within ∼30 min (these gels were termed as strong). At lower CCNC and CGQD, gelation occurred within 1−4 h (the gels were termed as weak). The mixtures with CCNC < 20 mg/mL or CGQD < 5 mg/ mL remained in a liquid state within at least 24 h. Both strong and weak hydrogels exhibited shear-thinning behavior: the gels liquefied when subjected to shaking or sonication for 1−2 s. After resting for ∼15−30 min, the mixed suspension gelled again. Notably, the pH value of all CNC-GQD suspensions remained in the range of 6.9−7. To examine the factors that favor or suppress hydrogel formation, we carried out several control experiments. In the first series of experiments, the CNCs and GQDs were mixed in the presence of urea, a chaotropic agent that disrupts hydrogen bonds.49 In addition, urea weakens hydrophobic interactions because it increases the free energy of cavity formation in water.50 Moreover, urea interacts with nonpolar species via van der Waals forces, which improves their solubility in water and weakens their hydrophobic interactions.50 Upon addition of urea to the CNC-GQD mixture (CCNC = 50 mg/mL, CGQD = 5 mg/mL) to a concentration of 1 M, gelation did not occur within 24 h (Figure 4A), whereas without urea the same system
Figure 4. Exploring the mechanism of hydrogel formation from the aqueous CNC and GQD mixed suspension at (A) CCNC = 50 mg/mL and CGQD = 5 mg/mL in the presence of 1 M urea and (B) CCNC = 50 mg/mL and CGQD = 5 mg/mL for NH2-functionalized CNCs. (C) CCNC = 50 mg/mL and CGQD = 2.5 mg/mL in a 3 mM solution of MgCl2.The photographs were taken immediately after CNC and GQD mixing (1) and 30 min later (2). 12347
DOI: 10.1021/acs.langmuir.7b02906 Langmuir 2017, 33, 12344−12350
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Figure 5. (A) Strain-dependent variation in the storage modulus, G′ (solid symbols), and the loss modulus, G″ (empty symbols), for the hydrogels formed at CCNC = 50 mg/mL and CGQD = 10 mg/mL (squares), at CCNC = 50 mg/mL and CGQD = 5 mg/mL (triangles), and at CCNC = 20 mg/mL and CGQD = 10 mg/mL (circles). (B) Variation in G′ for the hydrogel formed at CCNC = 50 mg/mL and CGQD = 5 mg/mL upon strain cycling between 1 and 50%. The vertical dashed lines are presented for eye guidance. The arrow direction represents the increase (down) and decrease (up) in strain. The strain was increased from 1 to 50% within 12 min. The storage modulus recovery time was 12 min.
strain, the mixed suspension with CCNC = 50 mg/mL and CGQD = 5 mg/mL formed a hydrogel with G = 80 Pa; however, when the strain value increased to 50%, the value of G′ decreased to 2 Pa and became smaller than the G″ value, signifying gel liquefication. Within 12 min, the G′ value completely recovered at the end of each cycle. The G″ values remained invariant during the strain increase and decrease cycles. The representative SEM images of the GQD-CNC hydrogel samples are shown in Figure 6. The hydrogels prepared at
propensity of CNCs and GQDs to form a nanofibrillar hydrogel. To utilize the shear-thinning behavior of the CNC-GQD hydrogels, we examined the hydrogel performance as an injectable material (Figure 7A). We used a 3D printer to
Figure 7. (A) 3D printing of the CNC-GQD hydrogel formed at CCNC = 50 mg/mL and CGQD = 10 mg/mL. The scale bar is 1 cm. Inset: Optical fluorescence microscopy image of the hydrogel thread. λexc = 365 nm. The scale bar is 1 cm. (B, C) Polarized optical microscopy images of the hydrogel thread and a dry thread, respectively, formed as in (A). Scale bars in (B) and (C) are 1 mm.
extrude the hydrogel prepared at CCNC = 50 mg/mL and CGQD = 10 mg/mL from the syringe on a horizontal polystyrene surface. Upon extrusion, the hydrogel retained its threadlike shape and formed a predesigned pattern. The hydrogel thread was fluorescent upon excitation at 365 nm (Figure 7A, inset) as a result of the photoluminescence properties of GQDs. We note that the CNCs exhibited weak autofluorescence upon excitation at this wavelength, which presumably originated from traces of lignin remaining after CNC preparation.52 The results of control experiments presented in Figure S4 show that a major contribution to the photoluminescence of CNC-GQD hydrogels stemmed from the fluorescence properties of GQDs. The extruded hydrogel exhibited birefringence, when imaged using polarized optical microscopy (Figure 7B), due to the shear-induced alignment of the CNCs along the direction of the thread during extrusion, which was preserved upon CNC cross-linking. The anisotropic structure of the hydrogels underlines the biomimetic morphology in which many tissues, such as striated muscle, cartilage, and cornea, have anisotropic morphologies that are important for cell guidance, proliferation, and differentiation as well as mass transport. The birefringence of the thread was preserved in the dried thread after water evaporation (Figure 7C).
Figure 6. SEM images of the CNC-GQD hydrogel at (A) CCNC = 50 mg/mL and CGQD = 5 mg/mL. (B) CCNC = 50 mg/mL and CGQD = 10 mg/mL. (C) CCNC = 30 mg/mL and CGQD = 7 mg/mL. (D) CCNC = 20 mg/mL and CGQD = 10 mg/mL. Each scale bar in A−D is 5 μm.
varying CNC and GQD contents exhibited a nanofibrillar structure. At CCNC = 50 mg/mL, the increase in CGQD from 5 to 10 mg/mL resulted in significantly thicker pore walls, that is, 57 ± 21 and 99 ± 35 nm, respectively (Figure 6A,B, respectively). This effect suggested that GQDs promoted lateral CNC crosslinking. On the other hand, at CGQD = 10 mg/mL, the size of the pores was affected by the CNC content (Figure 6B,D). Larger pores formed in the hydrogels prepared at a lower CCNC of 20 mg/mL, compared to the dense hydrogels prepared at CCNC = 50 mg/mL. A comparison of Figure 6B,D also suggested that the increase in CGQD/CCNC diminished the 12348
DOI: 10.1021/acs.langmuir.7b02906 Langmuir 2017, 33, 12344−12350
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CONCLUSIONS
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ASSOCIATED CONTENT
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We developed an injectable fluorescent nanofibrillar hydrogel formed by rod-like CNCs and disc-shaped GQDs. The hydrogel formed as a result of the hydrogen bonding between carboxyl groups of GQDs and hydroxyl groups of CNCs and hydrophobic interactions between the basal planes of GQDs and hydrophobic regions of CNCs, which overcame the electrostatic repulsion between the negatively charged carboxyl groups of GQDs and half-ester sulfate groups of CNCs. The attraction between two negatively charged types of nanoparticles is unusual and interesting. The ability to fine tune these three types of forces by changing the ambient conditions (e.g., by heating the system or by changing its ionic strength) may pave the way for the reversible gelation−liquification behavior of the system as well as the capability to control the gel properties, thus making it a stimulus-responsive material. The resulting hydrogels exhibited shear-thinning behavior. Hydrogel fluorescence was a very useful property, which suggested potential applications of the CNC-GQD in drug delivery, tissue engineering, bioimaging, and biosensing. The mechanical properties of the hydrogels were tuned by varying the GQD and CNC concentrations. The gel was utilized as an injectable material for 3D printing. The ease of the hydrogel preparation process, its fluorescence, the nanofibrillar structure, the shear-thinning behavior, and the control over the mechanical properties as well as the use of biocompatible constituent components make this hydrogel a promising candidate for biomedical applications.
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S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b02906. Characterization of GQDs by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), variation in fluorescence of the GQD aqueous solutions with changing GQD and CNC concentrations, the size distribution of GQDs, and frequency-dependent variation of storage and loss moduli of CNC-GQD hydrogels (PDF)
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Article
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Eugenia Kumacheva: 0000-0001-5942-3890 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful for the financial support of this work by NSERC Canada (Strategic and Discovery grants). E.K. thanks the Canada Research Chair program (NSERC). M.A. is grateful for an NSERC Vanier Canada graduate scholarship. The authors also thank Ilya Gourevich for assistance with electron microscopy imaging. M.T. thanks the Alexander von Humboldt Foundation for a Feodor Lynen research fellowship. 12349
DOI: 10.1021/acs.langmuir.7b02906 Langmuir 2017, 33, 12344−12350
Article
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DOI: 10.1021/acs.langmuir.7b02906 Langmuir 2017, 33, 12344−12350
