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Temperature-Responsive Nanofibrillar Hydrogels for Cell Encapsulation Héloïse Thérien-Aubin, Yihe Wang, Katja Nothdurft, Elisabeth Prince, Sangho Cho, and Eugenia Kumacheva Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00979 • Publication Date (Web): 10 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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Temperature-Responsive Nanofibrillar Hydrogels for Cell Encapsulation Héloïse Thérien-Aubin,1 Yihe Wang,1 Katja Nothdurft,1 Elisabeth Prince,1 Sangho Cho,1 Eugenia Kumacheva.1,2,3,* 1

Department of Chemistry, University of Toronto, 80 Saint George street, Toronto, Ontario, M5S 3H6, Canada

2

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College street, Toronto, Ontario M5S 3E5, Canada.

3c

The Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, Toronto, Ontario M5S 3G9, Canada

Abstract

Natural extracellular matrices often have a filamentous nature, however, only a limited number of artificial extracellular matrices have been designed from nanofibrillar building blocks. Here we report the preparation of temperature-responsive nanofibrillar hydrogels from rod-shaped cellulose nanocrystals (CNCs) functionalized with a copolymer of N-isopropylacrylamide and N,N’-dimethylaminoethylmethacrylate. The composition of the copolymer was tuned to achieve

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gelation of the suspension of copolymer-functionalized CNCs at 37 oC in cell culture medium and gel dissociation upon cooling it to room temperature. The mechanical properties and the structure of the hydrogel were controlled by changing copolymer composition and the CNC-tocopolymer mass ratio. The thermoreversible gels were used for the encapsulation and culture of fibroblasts and T-cells and showed low cytotoxicity. Following cell culture, the cells were released from the gel by reducing the temperature, thus enabling further cell characterization. These results pave the way for the generation of injectable temperature-responsive nanofibrillar hydrogels. The release of cells following their culture in the hydrogels would enable enhanced cell characterization and potential transfer in a different cell culture medium.


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Introduction Great interest in man-made nanofibrillar polymer hydrogels is largely motivated by the abundance of nature-derived filamentous networks, which are formed in a multi-step hierarchical process.1-3 Individual molecules associate into discrete high-aspect ratio supramolecular structures that subsequently and gradually form larger nanofibrils with diameters in the order of tens of nanometers and the length from hundreds of nanometers to several micrometers. The nanofibrils associate, branch and/or entangle to organize into a three-dimensional network. This behavior is observed for a variety of biopolymers - from cellulose in the cell walls of plants to collagen in the extracellular matrix of animal tissues (ECM) 3,4 to filamentous networks of actin, fibrin, or spectrin. In comparison with hydrogels formed by molecules, nanofibrillar hydrogels have a larger pore size, a higher stiffness of the filamentous building blocks and interesting strain-dependent mechanical properties.5 Nanofibrillar hydrogels are very promising candidates for a broad range of applications requiring specific mechanical or transport properties. The biological diversity of nanofibrillar hydrogels has stimulated research efforts to reproduce their structure and properties in man-made hydrogels formed by fiber-like supramolecular building blocks. Synthetic nanofibrillar hydrogels have been prepared by the hierarchical organization of worm-like micelles of block copolymers6-9 and amphiphilic peptide fibers.10,11 In these systems, the association of individual molecules in nanofibrils and subsequently, of nanofibrils into water-swollen networks could be triggered by changing temperature, polymer concentration, or the ionic strength of the medium. Alternatively, nanofibrillar gels can be prepared by the association and/or entanglement of biocompatible filamentous nanoparticles, which would alleviate the need for the rigorous synthesis of block copolymers or peptide amphiphiles. More specifically, rod-shaped cellulose


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nanocrystals12,13 and nanofibers14 show promising applications in the preparation of nanofibrillar gels. Cellulose nanocrystals (CNCs), nanoparticles with an average diameter of 5-70 nm and an average length of 100-500 nm,15,16,17-19 have recently attracted great interest as the structural components of filamentous hydrogels.13,20-23 Gelation of CNC suspensions has been triggered by adding salts,13,24 and by chemical20,21 or physical12 crosslinking. The individual CNCs25-28 and the hydrogels formed by the mixture of CNCs and polyschacarides20 were biocompatible and non-cytotoxic. The applications of nanofibrillar hydrogels in cell biology are currently impeded by scattering of light in the visible spectral range, due to the relatively large size of hydrogel pores.3 Light scattering interferes with the characterization of the encapsulated cells using optical techniques, e.g., fluorescence assays. To circumvent this problem, following cell culture in the gel, the cells are released by enzyme-mediated gel digestion29 or by chemical gel treatment,30-32 however these techniques may be harmful to cell integrity. A rational design and utilization of reversible temperature-responsive nanofibrillar hydrogels for cell culture is a more attractive approach, in which a suspension of cells and nanofibrils can form a gel at the temperature of cell culture. After cell culture experiments are complete, the cell-laden hydrogel can be disintegrated "on demand" by reducing the temperature, thereby making cells amenable to characterization using fluorescence assays, PCR, or flow cytometry. This approach would also enable enhanced quantification of the release of factors secreted by the cells, which can be otherwise entrapped and retained in the hydrogel.33 Furthermore, in addition to the ability to use temperatureresponsive gels as injection systems, the reversible gelation behavior offers the capability to grow cell colonies in the temperature-responsive hydrogel and subsequently, transfer them into a new cell culture environment.


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The development and utilization of polymer molecules for the generation of stimuli-responsive hydrogels for cell encapsulation and tissue engineering has persisted over many decades.34-41 Nanofibrillar thermoresponsive gels have been prepared by the entanglement of cylindrical block copolymer micelles6,8 or by the self-assembly of peptide amphiphiles fibers.42 Here we report temperature-responsive nanofibrillar CNC-derived hydrogels and their use for the encapsulation and three-dimensional culture of fibroblasts and T-cells. The CNCs were surface-functionalized with temperature-responsive copolymer of N-isopropylacrylamide and dimethylaminoethylmethacrylate, with the copolymer composition enabling hydrogel formation at 37 oC in the cell culture medium and gel disintegration upon cooling it to room temperature. We note that examples of polymer-grafted thermoresponsive CNCs have been reported,43-50 however they have not yet been utilized as the building blocks of nanofibrillar hydrogels for cell culture. Figure 1 illustrates the approach developed in the present work. A mixed suspension of the copolymer-functionalized CNCs and cells in a cell culture medium is heated to 37 oC to form a cell-laden nanofibrillar hydrogel. The cells are cultured in the hydrogel for a particular time. Subsequently, upon cooling of the gel to room temperature, the nanofibrillar network is disintegrated, leading to cell release and enabling further characterization of the cells and cell metabolites. We describe the rational design of the CNC-derived temperature-responsive hydrogels and show that the temperature of sol-gel transition, as well as the structure and the mechanical properties of the hydrogels, can be controlled by fine-tuning the composition of the copolymer grafts. The CNC-derived hydrogels were used for the encapsulation and culture of fibroblasts and T-cells and showed biocompatibility and low cytotoxicity.


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Figure 1. Schematics of the formation of thermoresponsive nanofibrillar cell-laden gel from copolymer-modified CNC building blocks. (a) Mixing of copolymer-functionalized CNCs and cells in the cell culture medium at room temperature. (b) Formation of a cell-laden hydrogel at 37 oC. (c) Cell culture at 37 oC. (d) Gel dissociation and cell release at room temperature.

Experimental Section Material. All chemicals, unless specified, were received from Sigma-Aldrich Canada. Nisopropylacrylamide

(NIPAM)

was

purified

by

recrystallization

in

toluene,

N,N-

dimethylaminoethyl methacrylate (DMAEMA) was purified by filtration over basic alumina oxide powder. Azobisisobutyronitrile (AIBN) was purified by recrystallization in methanol. All other chemicals were used without purification. Cellulose nanocrystals obtained by the acid hydrolysis of wood pulp, were supplied by the USDA Forest Product Laboratory. The average length and diameter of the CNCs were 180 and 13 nm, respectively (Figure S1). Functionalization of CNCs with chain transfer agent. An aqueous suspension of CNCs was dialyzed against dimethylformamide (DMF) for 1 week with the solvent being replaced every


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24 h. Following transfer to DMF, the CNCs were functionalized with 4-cyano-4[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, a RAFT chain transfer agent (CTA). To 5 g of CNCs suspended in 100 mL of DMF, 0.805 g of CTA was added, followed by the addition of 0.495 g of dicyclohexylcarbodiimide, 0.340 g of ethyl (hydroxyimino)cyanoacetate and 0.024 g of 4-(dimethylamino)pyridine. The suspension was stirred for 2 h at room temperature and, subsequently, for 48 h at 50 oC. The resulting CNCs functionalized with CTA (CNC-g-CTAs) were purified by dialysis in DMF for 1 week. Surface-initiated polymerization of copolymer grafts. Polymer-modified CNCs (mCNCs) were synthesized by RAFT copolymerization of NIPAM and DMAEMA on the surface of the CNC-gCTAs in DMF. Several mCNC samples were prepared with a varying weight concentration, p, of the

comonomers

in

the

reaction

mixture,

calculated

as

p=(mNIPAM+mDMAEMA)/

(mNIPAM+mDMAEMA+mCNC-g-CTA)100, where m is the mass of NIPAM, DMAEMA or CNC-gCTA in the reaction mixture. The molar concentration, c, of DMAEMA in the comonomer feed mixture varied from 0 to 20 mol%. The molar concentration was calculated as c=nDMAEMA/(nNIPAM+nDMAEMA)100, where n is the number of moles of the NIPAM or DMAEMA comonomer in the feed mixture. The mCNC samples were labeled as mCNC(p,c). For example, the sample mCNC(85,4) was prepared at 15 wt% of CNCs and 85 wt% of the comonomers in the reaction system, with 4 mol% of DMAEMA and 96 mol% of NIPAM. The recipes of the formulations used for the mCNC synthesis are given in Table S1 (Supporting Information). The resulting mCNCs were purified by precipitation in a 1:3 mixture of hexane and diethylether. Subsequently, the mCNCs were dispersed in water at the concentration of ∼1 wt% and dialyzed against water for 1 week with water being changed every 24 h.


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Characterization of mCNCs. The apparent hydrodynamic diameter of mCNCs, defined as a hydrodynamic diameter of a spherical particle with the same hydrodynamic drag as the nonspherical mCNC, was determined using dynamic light scattering (Malvern Nano ZS-ZEN3600) at a laser wavelength of 633 nm. Infrared spectra of the CNCs were recorded after each step of their functionalization on an infrared spectrometer (Vertex 70, Bruker) using a diamond crystal (MIRacle, Pike Technologies) with 32 scans at 10 kHz scan speed with 4 cm–1 spectral resolution. The composition of the mCNCs was analyzed by 1H NMR (DD2 500 MHz, Agilent). Rheology. The mechanical properties of mCNC suspensions and gels were characterized using a rheometer with a cone and plate geometry (AR-1000, TA Instruments). Strain sweep experiments were performed to determine the linear viscoelastic regime with an oscillation frequency of 1 Hz and amplitude varying between 0.1 and 50 % strain. Frequency sweep experiments were performed at an amplitude of 1% strain at the frequency varying from 1 to 100 Hz. The temperature was controlled with an integrated Peltier plate. Solvent evaporation was minimized using a solvent trap. The frequency and strain sweep experiments were performed at 25 oC. Subsequently, the sample was equilibrated at 37 oC for 15 min and the frequency and strain sweep experiments were repeated. Electron microscopy of hydrogels. Due to the cononsolvency of PNIPAM in the water/methanol mixture,51 the supercritical critical point drying method for the preparation of samples for imaging could not be used. The samples were prepared using freeze-drying in liquid propane. An mCNC gel was incubated for 30 min at 40 oC and instantly frozen in liquid propane, which was used to suppress crystallization of water affecting the structure of mCNC gels.52,53 After freezing, the water was removed by freeze-drying for 2 days. The dry mCNCs aerogels were freeze-fractured and coated with gold using a SC7640 High Resolution Sputter Coater


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(Quorum Technologies) for 15 s at 2.0 kV. Scanning Electron Microscopy (SEM) imaging was carried out on the Quanta FEI Scanning Electron Microscope. Cell culture. Prior to cell culture in the mCNC hydrogels, NIH 3T3 fibroblast cells were cultured in Dulbecco’s Modified Eagle Medium with 4.5 g/L glucose, L-glutamine, and sodium pyruvate (DMEM, GIBCO) supplemented with 10 % (v/v) fetal bovine serum (FBS, Invitrogen) and 1% (v/v) Penicillin/Streptomycin. The cells were cultured in plastic flasks placed in an incubator supplied with 5% CO2 at 37 ˚C. A 0.25 wt.% Trypsin-EDTA solution (GIBCO) was used to detach the cells from the plastic flask. After cell detachment, fresh nutrition medium was added, and the cell suspension was centrifuged at 1000 rpm for 3min. The supernatant was then removed, and the cells were redispersed in fresh medium. For cell passaging, 5 vol.% of the cell suspension was transferred to the fresh medium every 3 days. Prior to cell culture in mCNC hydrogels, EL4 T-cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, GIBCO) with 10% (v/v) fetal bovine serum and 1% (v/v) Penicillin/Streptomycin and incubated at 37 ˚C and at 5% CO2. Before cell passaging, T-cells were washed with Hank’s Balanced Salt Solution (HBSS, GIBCO). The cells were then centrifuged and dispersed in fresh cell culture medium, as described above. Cytotoxicity assay. The cytotoxicity of the thermoresponsive mCNC gels for 3T3 fibroblasts and EL4 T cells was determined using AlamarBlue® assay in a 96-well cell culture plate. A suspension of mCNCs in water at 6 wt% mCNC concentration was filtered (1 µm pore size, Life Sciences) and subsequently, sterilized by exposure to UV light (Sterilaire Lamp, 254 nm, 345 µW/cm2) for 10 min. The mCNC suspension was first, mixed with sterilized deionized water and concentrated HBSS (10X), then the cell suspension was added. The final concentration of mCNC in suspension was either 3, or 4 wt%, the final concentration of HBSS was 1X and the final


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concentration of fibroblasts or T cells was 8 × 105 cell/mL. The resulting suspension was introduced into a 96-well plate (30 µL/well, 6 × 103 cells/well) and incubated at 37 oC for 24 h. Cell-free mCNC gels were prepared as a control. After 24 h of cell culture, the cell-laden gels were dissociated by cooling them to room temperature for 1.5 h. The released cells were stained by the addition of AlamarBlue® (Invitrogen) to each well at a 10 vol% of the total volume of the cell suspension and incubated at 25 °C for 4 h. After incubation, the Relative Fluorescence Unit (RFU) in each well was measured using a plate reader (CLARIOstar, Mandel) at the excitation and emission wavelengths of 560 and 590 nm, respectively. Cell-free mCNC gels were used as a control. To construct calibration graphs, a series of cell suspensions (3T3 fibroblasts or EL4 T cells) in the cell culture medium was prepared at cell densities varying from 500 to 16,000 cell/well. A solution of AlamarBlue® was added to each well at a 10 vol% of the total volume of cell suspension. A cell-free medium for 3T3 fibroblasts or T cells was used as a control. The cell suspension was introduced in a 96-well plate (100 µL/well) and maintained at 25 °C for 4 h. After incubation, the RFU of cells in each well was measured using a plate reader at an excitation and emission wavelengths of 560 and 590 nm, respectively. The density of cells in suspension was measured by using a hemocytometer (Bright-Line, Haussar Scientific). The proliferation index of the fibroblasts and T cells encapsulated in the mCNC gels was measured as the ratio between the number of live cells on Day 0 and the number of live cells on Day 1. All experiments were carried out in triplicates. Live/dead assay and fluorescence staining of cells. After 1 day culture, live cells were identified by staining them with Calcein AM (Invitrogen, Carlsbad; green fluorescence) and dead cells were identified by staining with Ethidium Homodimer I (Invitrogen, Carlsbad; red


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fluorescence). After 24 h culture of cells in the mCNC gels, a solution of Calcein AM (4 µM) and Ethidium Homodimer I (4 µM) in HBSS was added to the wells containing cell-laden mCNC gels. Then, the cell-laden gels were maintained at 25 °C for 1.5 h. The suspension was then diluted by the addition of a five-fold larger volume of HBSS, transferred into a centrifuge tube filter (0.45 µm pore size, Corning) and centrifuged at 1100 g for 3 min. The cells deposited on the filter and separated from the mCNCs were redispersed by adding 1 mL of HBSS to the filter. Subsequently, the cells were centrifuged at 1100 g for 3 min and redispersed in a 100 µL medium. The suspension was transferred to a 96-well plate for imaging. The live and dead cells stained with fluorescence dyes were imaged by fluorescence microscopy (Nikon). Long-term cell culture. The cell culture of fibroblasts and T cells was carried out for 7 days at 37 oC and at 5% CO2. After 7-day culture, fibroblast cells or T cells were released from the mCNC gels at room temperature and the number of cells was analyzed as described above by AlamarBlue® assay and Live/dead assay.

Results The

surface

of

the

CNCs

was

functionalized

with

a

random

copolymer

of

N-isopropylacrylamide (NIPAM) and N,N-dimethylaminoethyl methacrylate (DMAEMA) synthesized using Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization. First, a RAFT chain transfer agent (CTA) was grafted to the CNC surface to form CNC-g-CTAs (Step 1, Figure 2). The surface grafting density of CTA on the CNCs was determiend using inductively coupled plasma atomic emission spectroscopy to measure the sulfur loading of CNCs and CNC-g-CTAs (Table S2, Supporting Information). The CNC-g-CTAs were then used as a


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substrate for the copolymerization of NIPAM and DMAEMA (Step 2, Figure 2), thus yielding a copolymer poly(NIPAM-co-DMAEMA) tethered to the CNCs. Functionalization of the CNC with the copolymer was confirmed in dynamic light scattering, infrared spectroscopy and NMR experiments (Table S3 and S4 and Figures S2-S4). The apparent hydrodynamic diameter of the mCNC increased, in comparison with pristine CNCs, from 85±7 to 205±5 nm (Figure S2). The appearance of strong peaks corresponding to the carbonyl stretching at ∼1600 cm-1 typical of the poly(NIPAM-co-DMAEMA) in the infrared spectra also confirmed the grafting of the copolymer molecules to the CNCs (Figure S3). The ratio of the comonomers in the copolymer grafts was similar to their feed ratio, confirmed by 1H NMR (Table S3 and Figure S4). Furthermore, NMR was used to perform end-group analysis to calculate the molecular weight of the copolymer (Table S3), which was comparable to its theoretical molecular weight (Table S4) calculated from the monomer conversion. The average conversion of the comonomers was 94%, thus enabling control of the degree of polymerization of the copolymers from 340 to 6500 (Table S3).

Figure 2. Surface functionalization of CNCs with grafted poly(NIPAM-co-DMAEMA) chains. Step 1. Functionalization of CNCs with chain transfer agent. Step 2. Surface-initiated polymerization of copolymer molecules.


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The mCNCs dispersed in an aqueous suspension exhibited a thermoresponsive behavior, which was first, characterized by measuring the lower critical aggregation temperature (LCAT) of mCNCs in their dilute (0.1 wt%) suspension in deionized water and in HBSS. Figure 3A shows the results of the measurements of turbidity of the suspension of mCNCs. The suspension exhibited a drastic temperature-dependent change in extinction. While at room temperature, the suspension had low extinction and was clear, upon an increase in temperature, the suspension became turbid and displayed a sharp increase in extinction. The change occurred due to the strong scattering of light by mCNC aggregates forming at elevated temperature (Figure S5). The value of LCAT (determined as the mid-point of the region of sharp transition in the extinction curve) shifted toward higher temperatures when the content of the hydrophilic DMAEMA comonomer in the copolymer was increased. Upon cooling, the turbid suspension became clear, due the dissociation of the mCNC aggregates. The heating-cooling induced mCNC aggregationdissociation exhibited hysteresis of ∼1-5 oC in the LCAT values. The LCAT behavior of the temperature-responsive mCNCs in dilute suspensions translated into the gelation of more concentrated mCNC suspensions. The insets in Figure 3A show that a 5 wt% suspension of mCNCs transformed from a clear suspension at 25 oC to a reversible turbid gel upon heating to 35 oC. The effect of the composition of copolymer molecules on the values of LCAT of mCNCs is shown in greater detail in Figure 3B. When the DMAEMA content in the copolymer chains increased from 0 to 20 mol%, the LCAT of mCNCs in deionized water was shifted from 32 to 54 o

C, due to an increased hydrophilicity of the copolymer (and thus of mCNCs). Furthermore,

Figure 3B shows the influence of the surrounding medium on the LCAT values of mCNCs. A decrease of 6±2oC in LCAT was observed when mCNCs modified with copolymer chains of


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different compositions were transferred from deionized water into HBSS, due to the increase in the ionic strength of the medium and the salting-out effect caused by the buffer.54 Importantly, in Figure 3B, the blue line represents the target temperature of 37 oC for gelation of mCNC suspensions, in order to use the resultant hydrogels for cell encapsulation and culture. More specifically, the suspensions of mCNCs functionalized with copolymer chains with 4-5 mol% of DMAEMA and 96-95 mol% of NIPAM displayed an LCAT at physiological temperature. The values of LCAT were also measured for mCNCs dispersed in the cell culture medium (Figure S6). No difference in LCAT values was observed for the mCNCs dispersed in HBSS and in the cell culture medium. This result indicated that the presence of protein(s) in the cell culture medium had no noticeable effect on the colloidal stability of the mCNCs. Furthermore, cells and cell metabolites did not influence the sol-gel transition of the suspension of mCNCs (Figure S8).


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Figure 3. Variation in the lower critical aggregation temperature (LCAT) of mCNCs. (a) Turbidimetry of 0.1 wt% suspension of mCNC(85,0) (black), mCNC(85,5) (red), and mCNCs(85,15) (blue) in water, upon the heating (solid line) and cooling (dashed line). (b) Effect of DMAEMA content in the copolymer grafts on the LCAT of mCNCs in water (black) and in HBSS (red). The horizontal blue line shows the target gelation temperature of 37 oC. The insets show inversion tests performed on 5 wt% suspension of mCNC(85,5) at 20 oC (left) and 45oC (right).


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The mechanical properties of the mCNC hydrogel in HBSS buffer were characterized in oscillatory shear experiments (Figure 4). Gelation of the mCNC suspension was evidenced by the variation of mechanical properties below and above LCAT (Figure 4A). At room temperature a 4 wt% suspension of mCNCs (85,4) displayed a viscous behavior, with the loss modulus, G'', being larger than the storage modulus G'; however, when this suspension was heated to 37 oC (above the LCAT of the mCNCs), the value of G' exhibited a 10-fold increase, with G' being larger that G'', thus indicating the formation of a hydrogel. At a temperature higher or equal to LCAT, the mCNC hydrogels with varying compositions exhibited two regimes in strain sweep experiments (Figure 4A and B). At low (10%) strain, a nonlinear viscoelastic behavior was observed, in which an initial increase in G'' was followed by the decrease in both G' and G''. This viscous regime was caused by the disruption of the gel network, when the yield strain of the network was exceeded. Figure 4B shows the reinforcement effect of mCNCs on the copolymer network. Gelation of the mCNC-free copolymer solution resulted in the formation of a weaker gel, in comparison with the gels formed by the mCNCs tethered with copolymer chains of the same composition. More specifically, the storage modulus of the poly(NIPAm-co-DMAEMA) gel was 16 times lower than that of the mCNC gel and the yield strain of the poly(NIPAm-co-DMAEMA) gel was only 5%, compared to 42% for the mCNC gel. Furthermore, although non-modified CNCs also formed gels in the presence of salts at concentrations comparable to physiological conditions or at the ionic strength of HBSS,13 those gels did not exhibit a thermoresponsive behavior (Figure


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S9). The hydrogels formed with mCNCs behaved differently than non-modified CNCs. In particular, the yield strain of the mCNC gels was larger than that of non-modified CNCs, which suggested the formation of a more extensive and a more entangled network by mCNCs, in comparison with gels formed by non-modified CNCs. Frequency-dependent experiments were performed for mCNC gels at low strain in the elastic regime (Figure 4C). In the applied frequency range, the value of G' was, at least, 10-fold larger than G'', indicating the formation of a network structure. The G'' was frequency-dependent and displayed a moderate increase with increasing frequency, with the slope more pronounced for weaker gels. The frequency dependence of G'' has been previously observed for concentrated colloidal dispersions and associative polymers and has been attributed to their structural relaxation and thermal fluctuations.56,57 The effect of the content of the copolymer in the mCNC sample was studied by examining the rheological properties of the hydrogels formed by mCNC(75,4), mCNC(85,4) and mCNC(95,4) samples (Figure 4C). The gels formed by mCNC(85,4) were stronger than those formed by mCNC(95,4) hydrogel, due to the weaker reinforcement effect of CNCs in the latter system (the CNC content was only 5 wt% in mCNC(95,4) vs. 15 wt% in the mCNC(85.4)

sample).

However, mCNC(75,4) displayed a lower value of G′ than the gels formed by both mCNC(85,4) and mCNC(95,4). This behavior of the gel formed by mCNC(75,4) was attributed to the limited association and entanglement between the copolymer grafts, due to their lower degree of polymerization. The mechanical properties of the hydrogels formed by mCNCs were also controlled by changing the concentrationof mCNCs, CmCNC, in the precursor suspension (Figure 4D). The complex modulus, G*, of the gel scaled as G*∼ CmCNC 2, indicating the formation of a more


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stable and stiffer network when the concentration of mCNCs in suspension was increased.

Figure 4. Variation in the mechanical properties of mCNC gels in HBSS buffer. (a) Straindependent variation of the storage modulus G' (solid lines) and loss modulus, G'' (dashed lines) of 4 wt% suspension of mCNC(85,4) at 25oC (black lines) and a gel at 37oC (red lines). (b) Strain-dependent measurement of a 4 wt% suspension of non-modified CNCs (black line), poly(NIPAm-co-DMAEMA) (red lines) and mCNC(85,4) (blue lines). (c) Frequency-dependent variation of G' and G'' of hydrogels mCNC(75,4) (black lines), mCNC(85,4) (red lines), and mCNC(95,4) (blue lines). (d) Variation of the complex modulus of the gel with the mCNC concentration at 37 oC.

Scanning electron microscopy was used to image the microstructure of the hydrogels. Figure 5


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shows representative images of the nanofibrillar structure of the mCNC gels. The mean fiber diameter in the hydrogels formed by mCNC(95,4), mCNC(85,4) and mCNC(75,4) was 91±3, 80 ± 5 and 67 ± 3 nm, respectively, suggesting that the copolymer grafts promoted side-by-side aggregation of mCNCs. The size of pores was notably larger in the hydrogels formed by mCNC(95,4).

Figure 5. SEM images and fiber width analysis for hydrogels made of mCNC(75,4) (a), mCNC(85,4) (b) and mCNC(95,4) (c). The scale bars are 1 µm. (d) Fiber width distribution in hydrogels made of mCNC(75,4) (grey), mCNC(85,4) (red) and mCNC(95,4) (blue). The distribution of the fiber width was evaluated for more than 450 fibers per sample.

To explore potential applications of mCNC hydrogels for cell encapsulation, we examined hydrogel cytotoxicity for two cell lines, namely, 3T3 fibroblasts and EL4 T cells, that is, for adherent and non-adherent cells, respectively. Figure 6 shows the images of 3T3 fibroblasts and


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EL4 T cells at 37 and 23 oC, that is, before and after cell release from the mCNC gels, following 1 day culture. The background of the images in Figure 6a and 6b was blurry, due to the light scattered by the nanofibrillar mCNC gels. The scattering of the gel was also noticeable in optical transmission microscopy images (Figure S13). The quality of fluorescence images was substantially higher for both types of cells after gel disintegration and cell release at 23 °C (Figures 6a' and b'). Fluorescence images of live/dead stained fibroblasts and T cells before and after release from the hydrogels formed by mCNC(95,4) and mCNC(85,4) at 3 and 4 wt% are shown in Figures S14 and S15. Figure 6c shows the variation of the proliferation index of the cells in the hydrogels formed by mCNCs with different compositions and at a different mCNC concentration. For both cell types, the proliferation index reduced at a higher mCNC concentration and was higher for cells cultured in the hydrogels formed by mCNC(95,4) than those formed by mCNC(85,4). Overall, T cells exhibited the proliferation index higher than 2 in all types of hydrogels, while the proliferation index of 3T3 fibroblasts was above 0.85 for all gels, except 4 wt% mCNC(85,4), which displayed the proliferation index of 0.6. Thus, we conclude that all the gels exhibited low cytotoxicity for both T cells and fibroblasts. A lower proliferation index for adherent fibroblasts than for non-adherent T cells can be attributed to the lack of cell-adhesive moieties on the surface of mCNCs.


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Figure 6. Effect of mCNC gel composition on cell culture. (a, a') Fibroblasts stained by Calcein AM (green) and Ethidium Homodimer I (red) after 1-day culture in gels formed by 3% mCNC(85,4) at 37 oC (a) and at 23 oC (a'). (b, b') T cells stained by Calcein AM and Ethidium Homodimer I after 1-day culture in the hydrogels formed by 3 wt% mCNC(85,4) at 37 oC (b) and at 23 oC (b'). (c) Cell proliferation index of 3T3 fibroblasts (gray bars) and EL4 T cells (red bars) in mCNC gels formed by mCNC(95,4) and mCNC(85,4). Scale bars are 250 µm.

Figure 7 shows that when the cell culture was prolonged to 7 days, the proliferation index of T


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cell increased by 1500 % in the hydrogels formed by mCNC(95,4), compared to the proliferation index after 24 h cell culture. The proliferation index of the fibroblasts in the same gel increased more moderately by 40% over 7 days (Figure S16). The live/dead assay showed that for both cell lines, the number of dead cells in the hydrogels increased after 7 day culture, in comparison with 24 h cell culture (Figure S17). This phenomenon may be ascribed to the overcrowding of the cells in the gel, the limited amount of nutrients, or the accumulation of cells metabolic wastes.

Figure 7. Cell proliferation index of 3T3 fibroblasts (gray bars) and EL4 T cells (red bars) in mCNC gels formed by mCNC(95,4) and mCNC(85,4) after 7 days of culture

Conclusions By the functionalization of CNCs with surface-grafted thermoresponsive copolymer molecules, we developed a new building block for the generation of temperature-responsive nanofibrillar gels for cell encapsulation and culture. The temperature-responsive nature of these building blocks enabled on demand hydrogel formation and dissociation under physiological conditions. The mCNC hydrogels exhibited controllable variation in structure and mechanical


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properties and were stronger and had a larger pore size than the hydrogels formed by the corresponding copolymer. The temperature-responsive hydrogels used for the encapsulation and culture of fibroblasts and T cells exhibited low cytotoxicity toward both cell lines and significantly promoted the proliferation of T cells (suspension cells) with a proliferation index of 2-4.5 after one day of cell culture. Due to the low cytotoxicity, biocompatibility and tunable properties, the temperatureresponsive hydrogels formed by copolymer-modified CNC can be used as injectable gels and as an artificial extracellular matrix for three-dimensional culture. The mCNCs represent a new family of nanofibrillar materials that can be used for the preparation of environments for cell culture, followed by cell release for their detailed and precise characterization or reencapsulation in a new environment to create more complex biological structures. In comparison with other man-made nanofibrillar hydrogels, mCNC hydrogels with tunable biophysical properties can be prepared in a robust, cost-efficient and easily scalable manner. Furthermore, the biofunctionalization of the copolymer with peptides promoting cell adhesion can be used to improve the culture of adherent cells such as fibroblasts and endothelial cells.

ASSOCIATED CONTENT Supporting Information. The supporting information contains supplemental details about the synthesis of the modified CNCs, as well as their in-depth characterization. The Supporting Information (PDF) files is available free of charge

AUTHOR INFORMATION


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Corresponding Author *Email: [email protected] Funding Sources The authors acknowledge funding from NSERC Canada under Discovery and CREATE programs.

ACKNOWLEDGMENT The authors thank NSERC Canada (Discovery, CREATE and Canada Research Chair programs) for financial support of this work. The authors thank Ilya Gourevich from the Center of Nanostructure Imaging for his assistance in electron microscopy experiments

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