Properties and Structure of Cellulose Nanocrystal Hydrogels for

Potential Applications as Three-Dimensional Artificial

Extracellular Matrices

By:

Shivanthi Easwari Sriskandha

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Chemistry

University of Toronto

© Copyright by Shivanthi Easwari Sriskandha, 2015

ii

Properties and Structure of Cellulose Nanocrystal Hydrogels for

Potential Applications as Three-Dimensional Artificial Extracellular

Matrices

Shivanthi Easwari Sriskandha

Master of Science

Graduate Department of Chemistry

University of Toronto

2015

Abstract

This thesis describes the preparation of hydrogels of cellulose nanocrystals (CNCs) into

environments that would support the growth of cells either by (i) adding Hank’s Balanced Salt

Solution to CNC suspensions to induce gelation or (ii) by modifying the surface of CNCs with a

thermoresponsive polymer to stimulate gelation in situ.

Fibroblast cells were grown within suspensions of CNCs of varying concentrations and the

mechanical properties and structure of the resulting suspensions were examined. The viability of

the cells cultured within the cellulose nanocrystal matrix was evaluated using two spectroscopic

techniques: UV-Vis absorption and fluorescence confocal microscopy.

The synthesis of thermoresponsive CNCs was conducted via an atom-transfer radical-

polymerization-based living radical polymerization. The thermoresponsive polymers, poly(N-

isopropylacrylamide) and poly(N-isopropylacrylamide-co-polyethylene glycol methacrylate)

were polymerized from the surface of initiator-modified CNCs. The resulting polymer was

characterized by attenuated total reflectance Fourier transform infrared spectroscopy, proton

nuclear magnetic resonance and dynamic light scattering.

iii

Acknowledgments

My heartfelt gratitude and appreciation goes to my supervisor Professor Eugenia Kumacheva for

her constant guidance and dedication to my work. I am thankful for her kind and patient

mentorship throughout my graduate studies.

I would especially like to thank Mokit Chau of the Kumacheva group, who has not only

been my mentor and lab partner but also my good friend. My sincerest thanks goes to Dr. Héloïse

Thérien-Aubin for her ceaseless support and assistance. I would also like to thank Dr. Yunfeng

Li and Yihe Wang for their generous help with my cell studies. Many thanks goes to Dr. Lindsey

Fiddes for technical help with the AFM. I also wish to acknowledge and thank the members of

the Kumacheva group for their friendship and encouragement. Finally, I am grateful to the

University of Toronto for supporting my research.

In closing, I would like to thank my parents and my sister, Malathi, without whom I could

not have completed my degree. Their support, care and love during this time has been invaluable

and greatly cherished by me.

iv

Table of Contents

Acknowledgments .......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Tables ................................................................................................................................. vi

List of Figures ............................................................................................................................... vii

Chapter 1 Introduction .................................................................................................................... 1

1.1 Artificial Extracellular Matrices and their Applications ..................................................... 1

1.2 Cellular Studies of Two-Dimensional and Three-Dimensional Environments .................. 6

1.3 Self-Assembled Nanofibrillar Polymer (Biological) Hydrogels ......................................... 7

1.3.1 Hydrogels formed by Cellulose Nanocrystals ...................................................... 11

1.4 Thermoresponsive Polymer Grafting on Cellulose Nanocrystals ..................................... 14

1.5 Summary ........................................................................................................................... 17

1.6 References ......................................................................................................................... 18

Chapter 2 Materials and Methods ................................................................................................. 25

2.1 Materials ........................................................................................................................... 25

2.1.1 Cellulose Nanocrystals (CNCs) ............................................................................ 25

2.1.2 Cellular Studies of Aqueous Suspensions and Gels formed by CNCs ................. 25

2.1.3 Surface Modification of Cellulose Nanocrystals .................................................. 26

2.2 Methods ............................................................................................................................. 26

2.2.1 Preparation and Characterization of CNC Suspensions ........................................ 26

2.2.2 Evaluating the Cytotoxicity of CNC Suspensions and Gels ................................. 29

2.2.3 Surface Modification of CNCs ............................................................................. 32

2.3 References ......................................................................................................................... 36

Chapter 3 Evaluating the Cytotoxicity of Cellulose Nanocrystal Suspensions ............................ 37

v

3.1 Introduction ....................................................................................................................... 37

3.2 Properties of Cellulose Nanocrystal Suspensions ............................................................. 39

3.3 Characterization of the Structural and Mechanical Properties of CNC-HBSS Suspensions ....................................................................................................................... 41

3.4 In Vitro Cytotoxicity of CNC-HBSS Suspensions ........................................................... 47

3.5 Discussion and Future Work ............................................................................................. 55

3.6 References ......................................................................................................................... 58

Chapter 4 Thermoresponsive Hydrogels of Cellulose Nanocrystals ............................................ 61

4.1 Introduction ....................................................................................................................... 61

4.2 Preparation and Characterization of CNC-g-poly(NIPAm) ............................................. 63

4.3 Preparation and Characterization of CNC-g-poly(NIPAm-co-PEGMA) ......................... 68

4.4 Conclusions and Future Work .......................................................................................... 71

4.5 References ......................................................................................................................... 72

Chapter 5 Conclusions and Future Work ...................................................................................... 74

5.1 Conclusions ....................................................................................................................... 74

5.2 Future Work ...................................................................................................................... 76

5.3 References ......................................................................................................................... 77

vi

List of Tables

Chapter 3 Evaluating the Cytotoxicity of Cellulose Nanocrystal Suspensions

Table 3.1 Summary of CNC properties ........................................................................................ 40

Table 3.2 Rheological Properties of CNC-HBSS Suspensions* ................................................... 44

Chapter 4 Thermoresponsive Hydrogels of Cellulose Nanocrystals

Table 4.1 Molar Percent Content of Unmodified CNC, CNC-g-poly(NIPAm) and CNC-g-

poly(NIPAm-co-PEGMA) determined by Elemental Analysis .................................................... 65

vii

List of Figures

Chapter 1 Introduction

Figure 1.1 Schematic illustration of the “bottom-up” assembly of nanofibrillar hydrogels.

Individual molecules typically assemble in nanofibrils, which further associate and/or entangle to

form a 3D network swollen with water. In biopolymers, the polymer chains are globally oriented

parallel to the long axis of the nanofibril whereas in synthetic polymers, polymer chains are

oriented perpendicularly to the main axis. Triggers such as changes in temperature and pH, and

increases in polymer concentration or in ionic strength govern attraction forces such as hydrogen

bonding or hydrophobic interactions, to name a few, that encourage the self-assembly of

molecules into nanofibrils and subsequently, the formation of nanofibrillar hydrogels. ............... 9

Figure 1.2 Structure and properties of nanocellulose. a) Schematic portraying the organization of

cellulose molecules into CNC fibers.69 Adapted and reproduced with permission from ref. 69.

Copyright 2012 Elsevier. b) Photo taken between crossed polarizers of a biphasic 8.78 wt% CNC

suspension.70 Adapted and reproduced with permission from ref. 70. Copyright 1996 American

Chemical Society. c) Polarized-light micrograph of CNC suspension. Scale bar is 200 µm.71

Adapted and reproduced with permission from ref. 71. Copyright 2000 American Chemical

Society. .......................................................................................................................................... 12

Figure 1.3 Schematic illustration of the thermoresponsive sol-gel behaviour of a poly(N-

isopropyl acrylamide) (pNIPAm) grafted cellulose nanocrystals (CNCs). .................................. 17

Chapter 3 Evaluating the Cytotoxicity of Cellulose Nanocrystal Suspensions

Figure 3.1 TEM images of CNC suspensions from two sources (a) FP Innovations, and (b) Forest

Products Laboratory. ..................................................................................................................... 39

Figure 3.2 Size distribution plots of the (a, c) diameter and (b, d) length of individual CNC

particles from (a, b) CNC1 and (c, d) CNC2. Approximately 100 individual CNC fibrils were

measured for each CNC source using ImageJ software. ............................................................... 40

viii

Figure 3.3 Strain amplitude sweeps for CNC-HBSS gels of CNC-1 for concentrations of 4

(▼, ) 3 (●, ○), 2(▲, ∆) and 1 (■, □) % w/w. The open and closed symbols are used to represent

data of the storage (G’) and loss (G”) moduli, respectively. ........................................................ 42

Figure 3.4 Frequency dependence of dynamic storage modulus, G′ (filled symbols) and loss

modulus, G′′ (open symbols) of 4 (▼, ), 3 (●, ○), 2(▲, ∆) and 1 (■, □) % w/w CNC-HBSS

hydrogels prepared from CNC1 at 0.5 % strain. ........................................................................... 43

Figure 3.5 Strain amplitude sweeps for CNC-HBSS suspension of CNC2 for concentrations of 4

% w/w. .......................................................................................................................................... 45

Figure 3.6 Frequency dependence of dynamic storage modulus (G′) and loss modulus (G′′) of 4

% w/w for CNC-HBSS hydrogel (prepared from CNC2) at 0.5 % strain. ................................... 45

Figure 3.7 Scanning electron microscopy images of CNC-HBSS hydrogels prepared from CNC-

1 with concentrations of a) 1, b) 2, c) 3 and d) 4 % w/w. Samples were prepared for imaging by

supercritical point drying. Scale bars are 100 µm. ....................................................................... 46

Figure 3.8 Schematic illustration of cells encapsulated within a CNC gel using Method 2. The

hydrogel suspension was prepared using an aqueous suspension of CNCs and a buffer (in this

case, HBSS). The cells in this schematic are not to scale. ............................................................ 47

Figure 3.9 Representative calibration curve for (■) 1 % w/w CNC1-HBSS, (▲) 1 % w/w CNC2-

HBSS and (♦) DMEM. A similar calibration curve was constructed for each experiment and

specified for 2, 3 and 4 % w/w CNC-HBSS mixtures. Calibration curves were used to extrapolate

the number of cells per well with a given fluorescence intensity. ................................................ 48

Figure 3.10 Controls for Live/Dead assay cell experiments illustrating negative controls

(containing no cells) and positive controls (containing cells). Fluorescent microscopy images

depict CNC-free environments as controls for the medium, cells in medium, cells in autoclaved

water and cells in medium killed by saponin, respectively. Scale bars are 200 µm. .................... 50

Figure 3.11 (a) Optical fluorescence microscopy images of stained cells containing live (green)

and dead (red) NIH-3T3 fibroblasts encapsulated using Method 1 in 2 % w/w CNC-HBSS

suspensions from CNC1 (ii., iv.) and CNC2 (i., iii.). The results of a positive control experiment

ix

containing membrane-compromised cells suspended in 2 % w/w CNC-HBSS suspensions are

shown in iii. and iv. Scale bars are 200 µm. (b) The number of proliferating cells per well

containing NIH-3T3 fibroblasts encapsulated using Method 1 in CNC-HBSS hydrogels of 2 %

w/w measured via Alamar Blue assay. The CNC used have an average ζ-potential of -41 mV and

an average pH of 3.7. .................................................................................................................... 52

Figure 3.12 (a) Fluorescence microscopy images of stained cells containing live (green) and

dead (red) NIH-3T3 fibroblasts encapsulated using Method 2 in CNC-HBSS suspensions of 1 (i.,

ii.), 2 (iii., iv.), 3 (v., vi.) and 4 (vii., viii.) % w/w. CNC-HBSS suspensions are made from CNC1

and CNC2. Dead cells are circled in red for greater clarity. Scale bars are 200 µm. (b) The

number of viable cells per well containing NIH-3T3 fibroblasts encapsulated using Method 2 in

CNC1-HBSS suspensions of 1, 2, 3, and 4 % w/w measured by AlamarBlue assay. (c)

Fluorescence microscopy images depicting 3D z-stacks of stained cells containing live (green)

and dead (red) NIH-3T3 fibroblasts encapsulated within CNC-HBSS hydrogels of (i.) 3 and (ii.)

4 % w/w from CNC1. The height of the images is 2200 µm. Scale bars are 200 µm. ................. 54

Chapter 4 Thermoresponsive Hydrogels of Cellulose Nanocrystals

Figure 4.1 Schematic illustration of the grafting of poly(NIPAm) to the surface of cellulose

nanocrystals (CNC) showing the formation of a bromine-initiator modified CNC in the first step

and then the polymerization of NIPAm in the second step. ......................................................... 62

Figure 4.2 FTIR-ATR spectra of unmodified CNCs, initiator-modified CNC (a) and CNC-g-

poly(NIPAm) (b). .......................................................................................................................... 64

Figure 4.3 1H NMR spectrum of pNIPAm cleaved from the surface of CNC-g-poly(NIPAm) via

saponification. ............................................................................................................................... 66

Figure 4.4 Variation in effective hydrodynamic diameter as a function of temperature for

unmodified CNCs ( ), CNC-g-Br initiator ( ) and CNC-g-pNIPAm (♦). .................................... 67

x

Figure 4.5 Schematic illustration of the grafting of poly(NIPAm-co-PEGMA) to the surface of

CNCs that have previously been modified with BriB initiator. A SET-LRP-based mechanism

was used. ....................................................................................................................................... 69

Figure 4.6 FTIR-ATR spectra of unmodified CNCs, initiator-modified CNC and CNC-g-

poly(NIPAm-co-PEGMA). ........................................................................................................... 70

1

Chapter 1

Introduction

The following chapter has, in part, been reproduced with permission from ref. 1. Copyright 2015

Springer Publishing Company.

1

1.1 Artificial Extracellular Matrices and their Applications

The extracellular matrix (ECM) is a heterogeneous, self-assembled network of biological

macromolecules existing in a structural hierarchy.2 The ECM surrounds cells within multicellular

organisms and has great influence over cell function; guiding both the spatially and temporally

complex processes of tissue formation and regeneration.3 Three major classes of components

constitute the ECM: fibrous elements such as collagen and elastin; specialized connector proteins

such as fibrillin, fibronectin and laminin; and space-filling molecules such as

glycosaminoglycans.4 All components of the ECM undergo self-assembly to form a highly

hydrated three-dimensional (3D) network. This 3D scaffold, to which cells can adhere,

differentiate and proliferate, can resist tensile stress due to the nanofibrillar structure and can

resist compressive stress as a result of the hydrated network.4 Ultimately, the structural decisions

of a cell to differentiate, proliferate, migrate, apoptose, etc., are a coordinated response to the

molecular interactions the cell has with the ECM components.3

The importance of the ECM resides in its function as a microenvironment that impacts

cell adhesion and movement, and signals cell morphogenesis and differentiation.4 Understanding

cell behavior within these multicellular tissues requires studying cells within a model

microenvironment. By mimicking the in vivo ECM, researchers may significantly reduce its

complexity.3

2

Existing model ECMs are multicomponent matrices derived from natural cells or tissues.

One of the most widespread scaffolds is Matrigel: a solubilized basement membrane preparation

extracted from Englebreth-Holm-Swarm mouse tumors that is enriched with laminin, collagen

IV and enactin. Other model systems include matrices composed of individually purified or

recombinantly produced ECM proteins, modified versions of the ECM components, and

proteolytic or recombinant fragment such as reconstituted collagen I (e.g. Vitrogen®) and

reconstituted fibrin gels. Collagen and fibrin are also U.S. Food and Drug Administration (FDA)-

approved for wound healing, treating burns and for use as tissue sealants.3 Despite their wide-

spread usage, the matrices described above have many disadvantages. Matrigel is not a well-

classified matrix and may introduce a source of variability in experimental results.5 Fibrous

natural ECMs such as fibrin and collagen I exhibit cellular contraction and can detach matrices

from their surroundings, thus destroying the intended cell geometry.6 Other natural systems can

cause batch-to-batch variation in materials isolated from tissues and may raise concerns about

disease transmission, especially in those materials isolated from mammalian sources.7 The

biochemical and biophysical differences between natural ECMs and these experimental mimics

could lead to altered cell behavior due to changes in signal transduction.8 Furthermore, natural

ECM proteins have a restricted flexibility in terms of material properties and cannot be tailored

to suit the needs of individual cell lines. With the goal of studying (and modifying) the effects of

substrate properties on cell fate, it would be important to control substrate properties

independently from each other without additionally altering other properties.

The ideal 3D ECM model would have the following characteristics: its fabrication must

be efficient, reproducible and cost-effective and be produced via crosslinking reactions that are

harmless to the cell. It must be transparent for optical analysis via microscopy. It should be

viscoelastic with tunable mechanical properties and preferably, possess a nanofibrillar

3

architecture that will more genuinely mimic the natural ECM. Finally, the material itself should

be biocompatible and nontoxic, and possibly contain surface properties that render it bioactive,

for example cell-adhesion ligands, cytokines and growth factors or morphogens.8

Most currently used artificial ECM are 3D gel networks that mimic the composition,

viscoelasticity and 3D nature of natural ECMs. They can be sourced from either natural, or

artificial materials and can be prepared in organic or aqueous media, although for use as

biomaterials, hydrogels (water-swollen networks) are the most widely studied.9 Materials from

natural sources are advantageous because of their inherent bioactive properties allowing for cell

recognition via receptor-binding ligands, and susceptibility to cell-triggered proteolytic

degradation and remodeling. Examples of hydrogels from natural sources generally include

collagen, gelatin, hyaluronate, fibrin, alginate, agarose, chitosan or silk, including others.10 Gels

derived from synthetic compounds can be divided into polymer or molecular constituents.

Examples of polymeric building blocks include poly(acrylic acid), poly(ethylene oxide),

poly(vinyl alcohol), polyphosphazene and polypeptides, to name a few examples. Generally, low

molecular weight hydrogelators possess an amphiphilic structure, including amino acids,

saccharides, nucleosides, nucleotides, bile acids,11 small organic molecules, surfactants or

globular proteins.12

In light of creating an artificial ECM that mimics natural ECMs found in vivo, most

applications are targeted towards developing biomaterials for bioengineering, cell culture, drug

delivery and cosmetics. A large number of gels composed of nanofibrillar biopolymers have

been used for cell encapsulation.13 For example, fibrin gels offer a good 3D scaffold for cell

studies because fibrin contains cell-binding sites and has tunable mechanical properties.14 It has

been used to control the differentiation and proliferation rates of tumor cell subsets, thereby

4

providing a mechanical method for selecting tumor cells.13 Much research has been conducted on

agarose hydrogels to seed chondrocytes for fabricating tissue constructs or to study chondrogenic

differentiation of adult human stem cells. 13,15 The utilization of composite gels enables a rational

design of hydrogels with the desired properties. For example, bone marrow stromal cells were

encapsulated in a fibrin-alginate gel, in which the fibrin enhanced the bioactivity of the

composite and the alginate provided a long encapsulation period.16 Human mesenchymal stem

cells were encapsulated within a spherical hydrogel consisting of collagen Type I and agarose,

with the goal of inducing osteoblastic differentiation. Cell viability post-encapsulation was 75-

90% over 8 days in culture.17

Tissue engineering aims to repair, regenerate or replace tissue or organ function.18

Scaffolds that mimic the natural ECM should provide structural integrity for cell differentiation,

proliferation and biodegradability, in order to be removed after tissue is grown.18 The advantage

of self-assembled nanofibrillar hydrogels over crosslinked molecular gels in tissue engineering

applications is that they possess mechanical properties similar to the replaced tissues.19

Biological fiber-forming polymers such as collagen, fibrin, agarose, alginate and chitosan were

used to regenerate or repair damaged tissues and organs.10 Studies on collagen hydrogels placed

between the stumps of a transected spinal cord demonstrate axons emerging from the spinal

tissue interface and growing into the collagen bioimplant after only one month.20,21 The surface

of an alginate polysaccharide was modified with RGD-containing peptides and then seeded with

mouse skeletal myoblasts. Myoblasts adhered to the GRGDY-modified alginate surface and

proliferated into nucleated myofibrils that exhibited heavy-chain myosin which is a

differentiation marker for skeletal muscle.22

5

On the other hand, significant progress has been achieved in the utilization of synthetic

gels, for example, gels formed by peptide amphiphiles.23 The antimicrobial properties of PAs

make them ideal candidates for scaffolds in tissue engineering and regenerative medicine.23 One

of the earliest studies revealed that a 3D nanofibrillar hydrogel of peptide amphiphiles

(containing a pentapeptide that is found within laminin) induced the selective in vitro

differentiation of murine neural progenitor cells into neurons.24 In many cases, short peptide

sequences such as RGD were attached to the peptide backbone of an amphiphilic peptide

polymer to imbue passive hydrogels with biological activity.25

In addition, reversible “on-demand” sol-gel transitions, that is, assembly and disassembly

of nanofibrillar gels would enable their programmed response to external stimuli, which would

be useful in cell encapsulation, delivery of drugs or clearance from the body.26 Such materials

could improve the implantation of bulky medical devices into the human body via minimally

invasive surgery.7 These “smart” gels also include those that can alter their shape in response to

temperature changes.27 For example, in biomedical applications, the required sterilization of

hydrogels can benefit from the thermoresponsive nature of poly(glycerol momomethacrylate)-

block-poly(2-hydroxypropyl methacrylate) copolymer. In aqueous solutions, this copolymer self-

assembles into worm-like particles at 21 °C and forms a free-standing hydrogel above ambient

temperature (21-25 °C), but liquifies upon cooling to 4 °C with the formation of spherical

micelles.28 Ultrafiltration of these micelles led to the complete removal of micrometer-sized

bacteria. When designing smart materials, it is necessary to address the reversibility of the

material alteration and the tunability of its functions and properties.29 The synthesis of a similar

stimulus-responsive material utilizing cellulose nanocrystals will be discussed in an upcoming

section.

6

1.2 Cellular Studies of Two-Dimensional and Three-Dimensional Environments

Currently, cell culture is conducted on two-dimensional (2D) surfaces such as microwell

plates, tissue culture flasks and petri dishes because of the ease, convenience and high cell

viability of 2D cell culture.30 However, in in vivo environments, many cell types, e.g.,

osteoblasts, hepatocytes and lymphocytes, are embedded in a unique three-dimensional (3D)

environment whose biochemistry and topology strongly affect cell fate. Cells inhabiting a 2D

rigid substrate must adapt to this adverse environment, thus altering cell metabolism, reducing

functionality and misrepresenting experimental results.

Superficially, the difference between 2D and 3D cell culture is that cells in 2D are grown

on top of a surface, whereas cells in 3D are fully encased by the matrix material. This difference

affects many aspects of the cellular environment.31 Most notably, in 2D, cell adhesion and

spreading are restricted to the horizontal plane, which leads to the forced polarity of cells that is

undesirable in many cases. Alternatively, encapsulated cells can adhere to multiple surfaces

thereby retaining a rounded morphology which is not polarized by cell-matrix interactions. Yet,

fully embedded cells are sterically hindered in their spreading and migration due to their

confinement by the surrounding matrix. Movement throughout the environment differs from

those cells adhered on 2D matrices.32 Finally, the mechanical forces imposed on cells cultured

three-dimensionally versus two-dimensionally are very different. Polymer or glass substrates for

cells are usually stiff with Young’s modulus on the order of GPa. By comparison, hydrogels are

softer environments that may contain discrete matrix fibrils and have a Young’s modulus on the

order of kilopascals.31 Since natural tissues are soft, e.g., brain tissue has E ~0.1,33 the 3D culture

of cells within a hydrogel more accurately imitates the mechanical environment.34 This trend has

been shown in the differentiation of human pluripotent stem cells whose self-renewable

7

properties are insensitive to ECM stiffness, yet its differentiation into neural ectoderm is

mechanosensitive.35

Since the 1970s, morphological differences have been observed in cells cultured in 2D

versus 3D substrates. Fibroblasts plated on glass exhibited a spread morphology with prominent

cellular extensions in two-dimensions whereas those embedded within 3D collagen matrices

favoured the spindle or stellate shape.36 In 1997, Bissell showed that malignant breast cancer

cells grown in 3D culture become non-cancerous and lost their abnormal shapes and patterns of

growth when an antibody against beta-integrin was added to the system. Such behavior had

never been observed for cells cultured in 2D environments.37 Furthermore, fibroblasts

encapsulated in a 3D environment moved and divided more quickly than in a 2D environment,

adopting the characteristic asymmetric shape that fibroblasts have in living tissues.38 It has

therefore been shown that cells grown in 3D more precisely mimic the behavior of those grown

in vivo and therefore artificial ECMs should be designed three-dimensionally rather than two-

dimensionally.

1.3 Self-Assembled Nanofibrillar Polymer (Biological) Hydrogels

In order for tissue engineering to be successful, biomaterials should be engineered to

recapitulate the hierarchical organization of natural ECMs based on evidence that its anisotropic,

fibrillar architecture has consequences for cell behavior.39 A hydrogel network can be formed by

non-reversible covalent crosslinking or by reversible physical crosslinking via hydrogen

bonding, hydrophobic forces, electrostatic interactions, or host-guest interactions. The "building

blocks" of the hydrogels can be individual molecules or supramolecular objects containing many

molecules. In the latter case, gelation is caused by either the association of shape-isotropic

8

molecular aggregates, e.g., spherical micelles, or shape-anisotropic supramolecular species, e.g.,

high-aspect ratio nanofibrils that associate via reversible physical connections. In biological

systems, the nanofibrils are critical to the mechanical properties, structure and signaling of

extracellular matrices, the cellular cytoskeleton, axons and dendrites, to name a few examples.

The unique properties and broad range of applications of naturally derived supramolecular

nanofibrillar hydrogels have motivated the design of their synthetic analogues, that is, hydrogels

formed by worm-like block copolymer micelles40 and amphiphilic peptides.41 Nanofibrillar

dimensions depend on the type of constituent polymer, but typically, their diameter is on the

order of tens of nanometers. The length can be up to hundreds of nanometers and under some

conditions, can reach micrometers. Generally, hydrogel formation is a hierarchical several-step

process commencing with the association of individual molecules into discrete high-aspect ratio

supramolecular structures, the association of these species into larger nanofibrils; and

subsequently, the formation of a three-dimensional (3D) network (Figure 1.1). Importantly, all of

the steps during gel formation can be controlled thermodynamically and kinetically, yielding

hydrogels with varying structures. A variety of diving forces are involved in the association of

nanofibrils into a 3D network, including a change in temperature, increase in polymer

concentration, increase in ionic strength, or addition of ions oppositely charged to the

nanofibrillar building blocks. Nanofibrillar gels can also be formed by the entanglement

mechanism. This mechanism is especially important for synthetic nanofibrillar hydrogels.

9

Figure 1.1 Schematic illustration of the “bottom-up” assembly of nanofibrillar hydrogels.

Individual molecules typically assemble in nanofibrils, which further associate and/or entangle to

form a 3D network swollen with water. In biopolymers, the polymer chains are globally oriented

parallel to the long axis of the nanofibril whereas in synthetic polymers, polymer chains are

oriented perpendicularly to the main axis. Triggers such as changes in temperature and pH, and

increases in polymer concentration or in ionic strength govern attraction forces such as hydrogen

bonding or hydrophobic interactions, to name a few, that encourage the self-assembly of

molecules into nanofibrils and subsequently, the formation of nanofibrillar hydrogels.

Extensive studies of nanofibrillar hydrogels are greatly motivated by their biological

relevance. For example, biophysical properties of filamentous collagenous hydrogels affect

interactions between cells and the extracellular matrix,42 while the mechanical properties of

fibrin gel influences the obstruction of blood vessels with blood clots.43 Fundamental

understanding of the structure-property relationship in nature-derived nanofibrillar gels is

important for more efficient applications in tissue engineering and drug delivery. On the other

hand, applications of synthetic nanofibrillar gels as scaffolds in tissue engineering, artificial

extracellular matrices for cell encapsulation, and drug carriers in pharmaceutics and cosmetics

necessitate fundamental and applied research in the area of nanofibrillar hydrogels. Nanofibrillar

hydrogels are also gaining interest as intelligent (smart) soft matter materials that respond to

external triggers, e.g., changes in pH, temperature, or light.44 These nanofibrillar building blocks

10

can be isolated from natural resources such as algae, wood, or chitin and offer cost-efficient and

sustainable materials. However, recent progress in the design and self-assembly of synthetic

supramolecular structures can greatly benefit the field of nanofibrillar hydrogels. In addition,

there are a variety of methods to shape nanofibrillar gels into potentially useful morphologies. In

the past decade, fabrication of micrometer-size hydrogel modules has benefited from the

microfluidic generation of micrometer-size hydrogel particles that can be used as vast

combinatorial libraries of instructive extracellular matrices, and as building blocks in tissue

engineering and in drug delivery.45

Recently, much interest in the literature has been focused on nanofibrils obtained from

the cellulose. Cellulose is one of the most abundant natural polysaccharides that can be extracted

from plants, e.g., wood or cotton, bacteria, fungi, algae and marine animals, e.g., tunicates.46

Linear cellulose molecules consist of β-1,4,-linked-anhydro-D-glucose repeat units. Depending

on the source, elementary fibrils have widths of 3-50 nm19 and lengths greater than 10,000 nm.47

In wood, elementary fibrils contain 36 glucan chains giving a diameter of 3.5 nm.48–50 These

elementary fibrils are ubiquitous structures of cellulose derived from all natural sources

including wood, cotton, ramie, jute, and bacteria.51 The fibrils have alternating crystalline and

amorphous regions. The crystalline regions of the elementary fibrils can be isolated by the

selective degradation of the amorphous regions by acid hydrolysis (Figure 1.2a) yielding

cellulose nanocrystals (CNC) or alternatively, nanocrystalline cellulose and cellulose

nanowhiskers.52–56 Nanofibrillar hydrogels formed by CNCs are described in the section below.

11

1.3.1 Hydrogels formed by Cellulose Nanocrystals

In 1951, Rånby conducted a sulfuric acid hydrolysis of mercerized cellulose fibers from

spruce wood to degrade the amorphous regions of the microfibers and obtain small cellulose

nanocrystals (CNCs) with an average diameter of 5-70 nm and a length of 100-250 nm.57,58

Different cellulose sources such as wood or algae yield CNCs with different dimensions even

under similar preparation conditions.59 For example, cotton and wood produce highly crystalline

CNCs with narrow size distribution, whereas tunicin and algae generate CNCs with larger

dispersities and lengths in the range of 100 nm to several micrometers.60 During hydrolysis,

sulfuric acid reacts with surface hydroxyl groups on CNCs allowing the grafting on of anionic

sulfate ester groups. The stability of aqueous CNC suspensions results from an electrostatic

repulsion between individual CNCs that counteracts their attraction due to van der Waals forces

and hydrogen bonding.61,62

Low-concentration CNC suspensions are clear isotropic fluids, while beyond a critical

concentration a birefringent chiral nematic liquid crystalline phase is formed, which exists in

equilibrium with the isotropic phase (Figure 1.2b).58 As the CNC content is further increased, the

entire suspension forms a chiral nematic liquid-crystalline phase with a characteristic fingerprint

pattern shown in Figure 1.2c.63 The origin of this effect is not entirely clear and is proposed to be

a result of the helicoidal structure of CNCs.46 At a higher CNC content, a gel is formed. The

aspect ratio of the CNCs is a key variable in determining CNC gelation and phase separation

during the formation of a liquid crystalline phase.46,64,65 For example, suspensions of long CNCs

tend to gel before attaining the liquid crystalline structure.58 It has also been shown that the

degree of sulfation of CNCs determines the surface charge density and significantly effects the

critical concentration at which the transition isotropicliquid crystal gel takes place. It was

12

observed that lower degrees of sulfation decrease electrostatic repulsion and lead to gel

formation at lower CNC concentrations than samples with higher degrees of sulfation.66,67 Earlier

work in the field revealed the formation of a birefringent gel when a suspension of CNC was

heated on a steam bath.68

Figure 1.2 Structure and properties of nanocellulose. a) Schematic portraying the organization of

cellulose molecules into CNC fibers.69 Adapted and reproduced with permission from ref. 69.

Copyright 2012 Elsevier. b) Photo taken between crossed polarizers of a biphasic 8.78 wt% CNC

suspension.70 Adapted and reproduced with permission from ref. 70. Copyright 1996 American

Chemical Society. c) Polarized-light micrograph of CNC suspension. Scale bar is 200 µm.71

Adapted and reproduced with permission from ref. 71. Copyright 2000 American Chemical

Society.

13

Gelation of CNC suspensions is also induced by suppressing the electrostatic repulsion

between CNCs either by decreasing the surface density of charged sulfate ester groups, or by

increasing the ionic strength of the aqueous medium. For example, shear thinning (thixotropic)

CNC hydrogels were obtained through the desulfation of CNCs with glycerol.60 Alternatively,

the addition of NaCl was used to control the rheological behavior of CNC suspensions in the

isotropic, chiral nematic and gel states over a range of CNC and NaCl concentrations.72 For

biphasic samples (above the threshold of isotropic-to-chiral nematic transition), increasing the

ionic strength to 5 mM NaCl decreased the size of chiral nematic domains and resulted in an

increase of sample viscosity at low shear rates. In the case of CNC gels, the addition of NaCl

decreased gel viscosity.

Surface modification of CNCs results in a decrease of surface negative charge and

enables the formation of gels due to the change in temperature, pH or ionic strength.73,74 For

example, cationic surface functionalization of CNCs resulted in the formation of thixotropic gels

at CNC concentrations of 3.5 % w/w or greater.75 In addition, functionalization of the CNC

surface with carboxylic acid or amine groups rendered the CNCs pH-responsive.76 Sol-gel

transitions occurred at pH values corresponding to neutral or weakly charged CNCs, thereby

allowing hydrogen bonding to dominate.

Cellulose nanocrystals have been used in synthetic polymer matrices such as poly(vinyl

acrylic acid)77, poly(2-hydroxyethylmethacrylate) and polyacrylamide78, as well as natural

polymer matrices such as regenerated cellulose79, agarose and cyclodextrin to prepare physically

crosslinked hydrogels.80 Recent work has been done to evaluate the cytotoxicity of CNC

composites such as polysaccharide hydrogels reinforced with CNC81 and melt-drawn poly(lactic

acid)-CNC fibers.82

14

Hydrogels of CNCs have many desirable properties including their low cost, nontoxic

behaviour, hydrophilicity, biocompatibility and biodegradability, all of which contribute to their

potential applications in bioengineering and biomedicine. For example, CNC dispersed in a

solution of cellulose, sodium hydroxide and urea formed a gel that steadily released bovine

serum albumin into a simulated body fluid.83 Nanocomposites of CNC and polyvinyl alcohol, a

hydrophilic biocompatible polymer, exhibited a broad range of mechanical properties that could

be tuned to mimic those of cardiovascular tissues and therefore, have potential use as

cardiovascular implants.84

While extensive research has been conducted on using CNCs as fillers in composite

materials, their individual use in pure or birefringent gels has not been extensively explored.

1.4 Thermoresponsive Polymer Grafting on Cellulose Nanocrystals

In order to use hydrogels formed by CNCs for cell encapsulation, it would be

advantageous to induce a controlled sol-gel transition into the CNC aqueous suspension laden

with cells. Forming a hydrogel in situ would permit more feasible applications in

macromolecular drug delivery, tissue barriers and tissue engineering.85 Such a transition can be

achieved by grafting a thermoresponsive polymer onto the CNC surface and using the lower

critical solution temperature (LCST) transition of the polymer to induce reversible association of

the CNCs at ~37 °C to form a gel and control its disintegration. Below the LCST, hydrogen

bonding between water molecules and the polar groups of the polymer formation lead to

dissolution.85 As the temperature increases above the physiological temperature, water becomes

a poor solvent for the polymer and polymer-polymer attraction would dominate under these

conditions, resulting in gel. If cells are encapsulated within a CNC gel, reducing the temperature

15

below the LCST of the polymer can release the cells for subsequent analysis. The grafted

polymer should be hydrophilic, non-cytotoxic and have an LCST at or below the physiological

temperature of 37°C.86

Several studies have been conducted to graft thermoresponsive polymers with LCSTs in

the physiological range to the surface of CNC. Polymer grafting on cellulose can occur via

“grafting-to” or “grafting-from” methods. In the grafting-to method, pre-synthesized polymer

chains are attached to cellulose hydroxyl groups. Steric hindrance may counteract polymer

attachment, since polymer chains must diffuse through already grafted “brushes” to reach the

surface reactive sites. As such, the grafting-to method is limited to a low surface density of the

polymer grafts. The grafting-from method increases grafting density by polymerizing chains in

situ from initiators attached to the cellulose surface.87 Polymerization can be achieved by

conventional radical, ionic and ring-opening polymerizations.88

The liquid-crystalline phase behaviour of poly(N,N-dimethylaminoethyl methacrylate)

(PDMAEMA)-grafted CNCs was investigated. In solution, this polymer has an LCST range of

32-53 °C, depending on the molecular weight, pH and salt concentration, and has the ability to

interact with DNA, enzymes and polyanion drugs via electrostatic attraction.89 Temperature

changes in the aqueous thermosensitive polymer brush-grafted CNC induced changes in the

fingerprint texture of the suspension. These disruptions in the chiral-nematic phase of the

polymer-grafted CNC are due to an LCST-type phase separation of the PDMAEMA gel.

Jeffamines, or commercially available statistical copolymers of ethylene oxide (EO) and

propylene oxide (PO) were also grafted onto CNC via a TEMPO-catalyzed oxidation. The LCST

varied from 16 to 80 °C depending on the EO/PO ratio.90 Thermoreversible growing aggregates

were formed when heated above the LCST.

16

The most commonly used thermoresponsive polymer is poly(N-isopropyl acrylamide)

(pNIPAm),91 because of its biocompatibility, hydrophilicity92 and an LCST in the range of 30 to

35 °C.93,94 As such, it has many biological controlled-release applications, including drug

delivery95, cell encapsulation96 and tissue engineering97 as a linear polymer, copolymer or

hydrogel. Close to its LCST, pNIPAm exhibits a conformational transition from an expanded

coil to a compact globule due to the formation of a hydrogen-bonded network at high

temperatures.29 Several groups have succeeded in grafting pNIPAm onto the surface of CNC

(Figure 1.3) via an atom-transfer radical polymerization (ATRP)-based mechanism utilizing

Cu(0) mediated single-electron transfer living radical polymerization (SET-LRP).87 The

difference between the latter technique and ATRP is the low activation energy in the outer-

sphere electron transfer mechanism and the rapid disproportionation of Cu(I) with nitrogen-

containing ligands in polar solvents. Thin films were produced by spin-coating aqueous

dispersions of CNCs and CNC-g-pNIPAm onto silicon wafers, followed by drying at room temp

overnight. Above the LCST, rheological measurements of aqueous dispersions showed increased

viscosities of the polymer-grafted CNC.98 The hydrophobic nature of pNIPAm dominated inter-

particle attractions among the CNC and resulted in aggregation of the polymer-grafted CNC

particles. Yet, these aggregates did not re-disperse at reduced temperatures indicating an

irreversible thermoresponsive behavior.99 Therefore, the formation of a bulk, homogeneous

thermoreversible polymer-grafted CNC hydrogel has yet to be synthesized.

17

Figure 1.3 Schematic illustration of the thermoresponsive sol-gel behaviour of a poly(N-

isopropyl acrylamide) (pNIPAm) grafted cellulose nanocrystals (CNCs).

1.5 Summary

The focus of this thesis is to develop an artificial ECM that will support the growth,

proliferation, differentiation and other vital processes of cells found within natural ECMs. The

biological ECM is a self-assembled hydrated 3D network composed of nanofibrillar structures

that provide structural and mechanical cues to surrounding cells. The hierarchical supramolecular

fibers of CNCs form gels that can mimic the nanofibrillar architecture of natural ECMs.

For these reasons, the following work describes the preparation of CNCs as matrices to

support cell growth and examines its cytotoxicity for NIH 3T3 mouse embryonic fibroblast cells,

a typical cell line. We evaluate the viability of cells grown in suspensions of CNCs mixed with a

biological buffer, Hank’s Balanced Salt Solution, that has been observed to increase the viscosity

of the CNCs and in some cases, induce gelation. Cells were cultured in 2D and 3D formats as a

means of moving towards their encapsulation in artificial environments.

18

Furthermore, to facilitate cell culture within hydrogels of CNCs, the surface was

modified with a thermoresponsive polymer to induce a sol-gel transition at physiological

temperature. Based upon current literature, an ATRP-based mechanism was used to graft

poly(NIPAm) and poly(NIPAM-co-PEG methacrylate) onto the surface of initiator-modified

CNCs.

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25

Chapter 2

Materials and Methods

2

2.1 Materials

2.1.1 Cellulose Nanocrystals (CNCs)

Cellulose nanocrystals (CNCs) were prepared by acid hydrolysis of wood pulp product. The

following types of CNC suspensions were used in the present work: (i) 6.43 % w/w CNC

suspension provided by FP Innovations (Pointe-Claire, QC, Canada) and (ii) 11.5 % w/w CNC

suspension purchased from the Forest Products Laboratory (Madison, WI, U.S.A.). In Chapter

3: Evaluating the Cytotoxicity of Cellulose Nanocrystal Suspensions, CNCs from FP Innovations

will be known as CNC1, and CNCs from Forest Products Laboratory will be known as CNC2. In

Chapter 4: Thermoresponsive Hydrogels of Cellulose Nanocrystals, CNCs are only used from

Forest Products Laboratory and therefore are unlabeled.

2.1.2 Cellular Studies of Aqueous Suspensions and Gels formed by CNCs

Dulbecco’s Modified Eagle Medium (DMEM, 1X), Hank’s Balanced Saline Solution (HBSS,

1X) and trypsin (1X, 0.25% with ethylenediaminetetraacetic acid (EDTA)) were purchased from

Gibco®. Cell viability was analyzed using LIVE/DEAD® Viability/Cytotoxicity kit for

mammalian cells that was purchased from Molecular ProbesTM. 7-Aminoactinomycin D (7-

AAD) was generously donated by Professor Zandstra from the University of Toronto Institute of

Biomaterials and Biomedical Engineering. AlamarBlue Cell Viability Reagent was purchased

from InvitrogenTM. Saponin was purchased from Bio Basic Inc. Syringe filters (Acrodisc Syringe

Filters, 0.45 µm Supor membrane) were purchased from Pall Corporation (Ann Arbor, MI,

26

U.S.A.). Milli-Q water (ultrapure water) was used and obtained from a Millipore water filtration

system (Millipore Corporation).

2.1.3 Surface Modification of Cellulose Nanocrystals

2-Bromoisobutyryl bromide (BriB), 4-dimethylaminopyridine (DMAP), dimethylformamide

(DMF), methanol (>99.8%), N-isopropylacrylamide (NIPAm), copper(I) bromide, and

N,N,N’,N”,N”-pentamethyldiethylenetriamine (PMDETA) were purchased from Sigma-Aldrich.

Triethylamine (TEA) and sodium hydroxide pellets were purchased from ACP Chemicals.

Hexanes were purchased from Caledon Laboratory Chemicals, toluene was purchased from

Fisher Scientific. Hydrochloric acid (1.0 N), poly(ethylene glycol) methacrylate (Mn ~360 g/mol)

and 4,4’-dipyridyl (98%) were purchased from Aldrich Chemical Company. Dialysis membrane

(MWCO: 6000) was supplied by Spectrum Laboratories, Inc. (Spectra/Por, Rancho Dominguez,

CA).

2.2 Methods

2.2.1 Preparation and Characterization of CNC Suspensions

2.2.1.1 Preparation of CNC Suspensions

Aqueous suspensions with CNC concentrations of 6.43 % w/w and 11.5 % w/w (CNC1 and

CNC2, respectively), were diluted in a 1:4 (v/v) ratio with Milli-Q water, and dialyzed against

Milli-Q water for four days with solvent changes every 24 h. Following dialysis, the CNCs were

filtered using No. 41 and 42 Whatman filter papers. The filtered suspension was re-concentrated

by partially drying under ambient conditions to concentrate solutions from ~1 to 6 % w/w or

higher. The resulting suspension from CNC1 and CNC2 had a concentration of 5.53 and 7.04 %

w/w, respectively, both determined gravimetrically. Cellulose nanocrystal suspensions of CNCs

27

with concentration of 5.33, 4.00, 2.67, 1.33 and 0.67 % w/w were obtained by diluting these

stock solutions with Milli-Q water.

Suspensions of CNC (11 % w/w) purchased from the Forest Products Laboratory were also

diluted in a 1:4 v/v ratio with Milli-Q water and dialyzed against N,N-dimethylformamide

(DMF) for four days with solvent changes every 24 h. The resulting suspension had a

concentration of 4.34 % w/w determined gravimetrically. These CNCs suspended in DMF will

be used in the surface modification of CNC with a thermoresponsive polymer.

2.2.1.2 Preparation of CNC-HBSS Suspensions

Suspensions of CNC1 and CNC2 with the concentrations of 5.33, 4.00, 2.67 and 1.33 % w/w

were added to Hank’s Balanced Salt Solution (HBSS) in a 4:1 v/v mixture. The final CNC

concentrations were 4, 3, 2 and 1 % w/w, respectively. In future, these systems will be known as

CNC1-HBSS and CNC2-HBSS, to identify their source. The inversion test was used to

qualitatively test the strength of the gel after 3 h. If the CNC-HBSS mixture flowed upon vial

inversion, the system was identified as "liquid-like." If the CNC-HBSS mixture remained at the

bottom of the vial, when it was inverted, the system was identified as a "gel."

2.2.1.3 Acidity of CNC Suspensions

The pH of dialyzed CNC suspensions was measured using a pH meter (Istek, Inc., Ecomet

pH/mV/TEMP meter P25).

2.2.1.4 Measurements of the Electrokinetic Potential of CNCs

The electrokinetic potential of the CNCs was measured using a Zetasizer (Malvern, Nano ZS).

Measurements were conducted of 2 % w/w aqueous CNC suspensions at 25 °C in triplicate using

the Smoluchowski model. The attenuator value varied from 5 to 10.

28

2.2.1.5 Transmission Electron Microscopy (TEM)

A JEOL JEM-2010 high resolution transmission electron microscope (TEM) was used to image

CNC1 and CNC2 at an acceleration voltage of 200 kV. The length and diameter of individual

CNC fibrils was measured using ImageJ software.

2.2.1.6 Environmental Scanning Electron Microscopy (ESEM)

The structure of the CNC suspensions prepared with HBSS was imaged using a Quanta FEI 250

environmental scanning electron microscope (5 kV). In order to prevent collapse of the hydrogel,

a supercritical point drying method was used to prepare the gel samples. The gels were placed in

microporous specimen capsules (30 μm pore size, Canemco-Marivac). The hydrogels were

solvent-exchanged by immersing them in methanol/water mixtures, and the methanol constant

was increased step-wise to reach 20, 40, 60, 80, and 100 % v/v. Following this step, the capsules

containing the solvent-exchanged CNC hydrogels were supercritically dried using an

Autosamdri-810 Tousimis critical point drier. The methanol within each hydrogel sample was

exchanged with liquid CO2, which was subsequently brought to a supercritical state and removed

with slow venting. The dried gels were sputter-coated with gold and then imaged using the

ESEM.

2.2.1.7 Rheology

The rheological properties of CNC hydrogels were studied using an ARES rheometer (TA

Instruments) with a parallel-plate geometry (plate diameter 50 mm). The gap between the plates

was 1 mm. A dynamic frequency sweep test was performed with oscillatory frequencies from 0.1

to 100 rad/s at a constant strain of 0.5% to determine the dynamic storage modulus (G’) of each

hydrogel at ambient temperature.

29

2.2.2 Evaluating the Cytotoxicity of CNC Suspensions and Gels

2.2.2.1 Cell Culture

Mouse embryonic fibroblast (NIH 3T3) cells were generously donated by Prof. Zandstra from

the University of Toronto Institute of Biomaterials and Biomedical Engineering. Cells were

cultured under sterile conditions in Dulbeco’s Modified Eagle Media (DMEM, 1X)

supplemented with 10% v/v fetal bovine serum (FBS) (purchased from Sigma-Aldrich) and 1%

v/v penicillin (purchased from Sigma-Aldrich). Cells were passaged every four days with trypsin

(purchased from Life Technologies) and maintained in a 5% CO2 humidified incubator (Incu-

Safe, Sanyo Scientific) at 37 °C. Passaging included splitting or transferring a small number of

cells into a new vessel, in order to culture cells for a longer period of time. Passaged cells used

for cytotoxicity experiments were counted using a hemocytometer (Bright-Line, Hausser

Scientific, Horsham PA).

2.2.2.2 Culturing NIH-3T3 Fibroblasts within CNC-HBSS Suspensions

Experiments were conducted in 96 microwell plates in a triplicate format. Hank’s Balanced

Saline Solution (HBSS) was added to each well in the perimeter of the well-plate to prevent

evaporation of the medium. Gels and suspensions of CNCs were prepared with a 1:4 v/v mixture

of HBSS and CNC1 or CNC2. The final concentration of CNC in each suspension was 1, 2, 3

and 4% w/w. Prior to mixing with HBSS, all CNC suspensions were filtered and sterilized using

syringe filters (0.45 μm pore size Supor membrane). An additional decontamination was

conducted on 4.0 and 5.33 % w/w CNC2 suspensions using UV radiation (248 nm) for 10 min to

combat bacterial contamination. Suspensions of CNCs were added to the well-plate using a glass

100 µL needle rinsed with 70% ethanol and autoclaved water prior to use.

30

To introduce cells into CNC hydrogels, two methods were used. In the first method (Method 1),

HBSS and CNC were mixed in a 96-plate wells and incubated for 1 h (to induce gel formation).

Cells suspended in DMEM at a concentration of 3 x 104 cells/mL were seeded on top of the gel

at a density of 3000 cells/well. The total volume of the cell culture medium in the well was 200

µL. The experiment was incubated in 5% CO2 at 37 °C for three days. Analysis was conducted

on Day 4 using either AlamarBlue or Live/Dead assays (see below).

In the second method (Method 2), cells were suspended in HBSS, first, and then added to each

well at a density of 3000 cells/well (1.5 x 105 cells/mL). Suspensions of CNCs were then added

to the cell suspension in the well in a 1:4 v/v ratio (cells in HBSS to CNC suspension) and mixed

thoroughly with a pipette. After incubation for 1 h to induce gel formation, DMEM was added to

each well. The total volume of all components in the well was 200 µL. The experiment was

incubated in 5% CO2 at 37 °C for three days. Analysis was conducted on the Day 4 using either

AlamarBlue or Live/Dead assays (see below).

In both series of experiments, several control systems were studied. Negative control systems

included cell culture medium only and cell-free CNC gel in cell culture medium. Positive control

systems included cells in cell culture medium only and cells in a water/HBSS mixture.

2.2.2.3 AlamarBlue Quantitative Cytotoxicity Experiment and Analysis

In addition to the experimental controls listed above, a calibration curve was prepared on Day 4

of the experiment to determine the number of viable cells per well. To construct the calibration

curve, cells were added to five wells in concentrations of 0, 1750, 3500, 7000, and 14 000

cells/well, in medium-only environments and in CNC gel environments with concentrations of 1,

2, 3 and 4% w/w. Cells and CNC gels were plated in 2D or 3D formats, as described above. In

this case, however, the cells were incubated for a minimal period of time before AlamarBlue dye

31

was added. For Method 1 experiments, cells were incubated for a maximum of 1 h before

AlamarBlue dye was added. For Method 2 experiments, AlamarBlue was added immediately

after the calibration curve was prepared. AlamarBlue dye was added to each sample at 10 vol. %

of the total volume of the suspension in the well (20 µL) and incubated for 3-5 h in 5% CO2 at 37

°C. Plates were covered in aluminum foil prior to analysis, in order to prevent the dye from

photobleaching. Experiments were measured using a UV-vis plate reader (Molecular Devices,

SpectraMAX GeminiXS, SOFTmax Pro 3.1.2. software). Fluorescence emission measurements

were acquired at 560 nm (excitation) and 590 nm (emission) for the samples (the λmax for

AlamarBlue dye).

After the raw data were collected, the number of viable cells was determined based on the

appropriate calibration curve. All results were presented as averages, with error bars representing

a standard deviation.

2.2.2.4 Live/Dead Qualitative Cytotoxicity Experiment

As mentioned in section 2.2.2.2, in addition to several control experiments described above, two

more positive control experiments were conducted with dead cells in medium only and dead cells

in a CNC suspension or gel. To prepare these positive controls, live cells were added to the wells

containing medium or CNC gel. On Day 4, 5 µL of 5% w/w saponin solution, sterilized and

filtered using a syringe filter (0.45 µm pore size Supor membrane), was added to the wells

containing medium or CNC gel in order to induce cell death. After 30 min, the Live/Dead assay

was added to ensure that all cells were dead. The Live/Dead cell viability/cytotoxicity kit for

mammalian cells is a two-colour assay containing a green-fluorescent Calcein, AM (Ca-AM) dye

to indicate intracellular esterase activity and a red-fluorescent Ethidium Homodimer-1 (EthD-1)

dye to indicate loss of cell membrane integrity. When these dyes are used concurrently, they

32

simultaneously discriminate live cells from dead cells. On Day 4, Ca-AM and EthD-1 were

added to each sample at a final concentration of 2 µM per well (HBSS was used as the solvent)

and incubated for 45 min to 1 h in 5% CO2 at 37 °C. Alternatively, 7-aminoactinomycin D (7-

AAD) was added to each sample in place of EthD-1 at a final concentration of 0.2 µg/mL per

well and incubated under identical conditions. 7-aminoactinomycin D was used as a replacement

for EthD-1, which electrostatically interacted with sulfonate groups on the CNC surface resulting

in a diminished fluorescence signal. 7-aminoactinomycin D was prepared at a starting

concentration of 1 mg/mL via the following procedure: add 5 mL of purchased 7-AAD to

methanol, vortex the solution, then add 950 mL phosphate-buffered saline (PBS) and mix. The

plates were covered in aluminum foil prior to analysis to prevent the dyes from photobleaching.

Fluorescence microscopy images of the encapsulated cells were taken using an Olympus

Confocal Laser Scanning Biological Microscope FV1000 with 10x objective magnification and

UV/Visible laser light (Center Valley, Pennsylvania, U.S.A.). The lasers used were 488 nm,

fluorescein isothiocyanate (FITC, 490 nm/525 nm) for Ca-AM and 568 nm, AlexaFluor568 (578

nm/603 nm) for EthD-1 and 7-AAD.

2.2.3 Surface Modification of CNCs

2.2.3.1 Preparation of Initiator-Modified CNCs

The procedure for grafting the initiator from the CNC surface was adapted as described

elsewhere.1 The procedure was as follows: 1 g of CNCs (6.17 mmol of anhydroglucose units,

equivalent to 18.5 mmol OH groups) were placed in a two-neck 250 mL round-bottomed flask

containing 100 mL dimethylformamide (DMF) while continuously stirred. To this was added

18.5 mmol 4-dimethylaminopyridine (DMAP) and 18.65 mmol triethylamine (TEA). The

solution was stirred under nitrogen for approximately 5 min. Lastly, 100 mmol of 2-

33

Bromoisobutyryl bromide (BriB) was added dropwise and the mixture was left to react under

nitrogen for 24 h at room temperature. The resulting solution was centrifuged and redispersed

three times in DMF at 10 ⁰C and 7200 rcf for 30 min (Eppendorf Microcentrifuge 5430 R).

These rinsing steps insured that free initiator had been removed from the CNC suspension. An

aliquot of the CNC-g-Br initiator conjugate was dried in a vacuum oven at 40 ⁰C for 24 h and

subsequently, characterized by attenuated total reflectance Fourier transform infrared

spectroscopy (ATR-FT-IR).

2.2.3.2 Single-Electron Transfer Living Radical Polymerization (SET-LRP)

of Poly(NIPAm) and Poly(NIPAm)-co-poly(ethylene glycol) methacrylate from

Initiator-Modified CNCs

The thermoresponsive polymer poly(N-isopropylacrylamide) (pNIPAm) was polymerized from

the surface of initiator-modified CNCs by surface-initiated single-electron transfer living radical

polymerization as previously described.1 The procedure was modified as follows. A suspension

of initiator-modified CNCs was centrifuged once at 10 ⁰C and 7200 rcf for 30 min (Eppendorf

Microcentrifuge 5430 R) and the supernatant was replaced with 50 mL Milli-Q H2O and

sonicated for 30 min. The aqueous suspension of CNC-g-Br was added to a 250 mL Schlenk

flask with 50 mL of methanol, 1 mmol of PMDETA and 100 mmol of NIPAm recrystallized in a

1:2 v/v toluene:n-hexane mixture. In a second 250 mL Schlenk flask, 1 mmol of copper(I)

bromide was added. Three freeze-pump-thaw cycles were conducted. Following this step, the

suspension was transferred into the Schlenk flask containing Cu(I)Br. The mixture was left under

nitrogen to react for 20 h at 45 ⁰C. The resulting modified CNC suspension was centrifuged and

redispersed in methanol three times at 10 ⁰C and 21000 g for 15 min (Thermo Scientific,

Heraeus Multifuge X1R Centrifuge).

34

Using a similar procedure, NIPAm was copolymerized with poly(ethylene glycol) methacrylate

(PEG-MA) utilizing with ratios of 10 mol% PEG-MA (Mn ~360) and 90 mol% NIPAm as

previously specified.2 The polymerization was conducted in water with 10 wt% total monomer

concentration. The resulting modified CNC suspension was centrifuged and redispersed in Milli-

Q water three times at 10 ⁰C and 21000 g for 15 min (Thermo Scientific, Heraeus Multifuge

X1R Centrifuge).

2.2.3.3 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FT-IR) was used

to confirm the presence of 2-Bromoisobutyryl bromide initiator, poly(NIPAm) and poly(NIPAm-

co-PEGMA) polymerized from the surface of CNCs. Samples of unmodified CNCs, CNC-g-Br

initiator, and complexes of CNC-g-poly(NIPAm) and CNC-g-poly(NIPAm-co-PEG-MA) were

dried in a vacuum oven at 40 ⁰C for 24 h and subsequently analyzed using a Bruker Vertex 70

ATR-FT-IR spectrometer.

2.2.3.4 Saponification of CNC-g-poly(NIPAm) and CNC-g-poly(NIPAm-co-PEG-MA)

The saponification of CNC-g-poly(NIPAm) was performed to cleave the grafted polymer with

the purpose of analyzing its structure and determining its molecular weight. The procedure was

adapted from a previous work.1 Briefly, 25 mL of 2% NaOH solution was added to 150 mg

CNC-g-poly(NIPAm) and stirred for 48 hrs. Afterwards, the basic solution was brought to

neutral pH with 1N HCl and centrifuged three times for 1 h at 10 °C at 7200 rcf to isolate the

cleaved pNIPAm. The cleaved polymer was dialyzed (MWCO: 6-8 KD) against Milli-Q H2O for

one week with water changed every day was followed by lyophilisation.

35

2.2.3.5 NMR Spectroscopy

NMR spectroscopy was used to determine the presence of the grafted poly(NIPAm). Dried CNC-

g-Poly(NIPAm) was redispersed in CD3OD via sonication and analyzed by 1H NMR with an

(Agilent DD2 500) spectrometer at a frequency of 500 MHz in a 3 mm o.d. tube with 1024

scans. Poly(NIPAm) chains cleaved from the CNC surface were also characterized by 1H and 13C

NMR using the same conditions stated.

2.2.3.6 Dynamic Light Scattering (DLS)

Dynamic light scattering (DLS) was used to determine the change in effective size of modified

CNCs in response to an increase in temperature. Using a concentration of 0.2 % w/w, aqueous

suspension of unmodified CNCs, CNC-g-Br initiator and CNC-g-poly(NIPAm) were analyzed

with a Zetasizer (Malvern, Nano ZS) with one degree increments from 20 to 60 ⁰C.

Measurements were repeated three times.

2.2.3.7 Inductively Coupled Plasma Atomic Optical Emission Spectroscopy (ICP-AOES)

Inductively coupled plasma atomic optical emission spectroscopy (ICP-AOES) was used to

determine the amount of sulfur (µg/mL) present on the surface of modified CNC. An aqueous

solution of 5 mg/L (~5 ppm) unmodified CNC and CNC-g-poly(NIPAm) was measured using an

ICP-AES (Optima 7300). The detection limit was 0.3 µg/mL.

2.2.3.8 Elemental Analysis

Elemental analysis was used to determine the presence of carbon, hydrogen and nitrogen on

unmodified CNC (from FP Innovations and from Forest Products Laboratory) and CNC-g-

poly(NIPAm). Aqueous samples of CNC were evaporated to dryness and the resulting solid

products were analyzed using a 2400 Series II CHNS Analyzer.

36

2.3 References

(1) Zoppe, J. O.; Habibi, Y.; Rojas, O. J.; Venditti, R. A.; Johansson, L.; Efimenko, K.;

Monika, O. Biomacromolecules 2010, 11, 2683–2691.

(2) Pollock, J. F.; Healy, K. E. Acta Biomater. 2010, 6, 1307–1318.

37

Chapter 3

Evaluating the Cytotoxicity of Cellulose Nanocrystal Suspensions

The following work was done in collaboration with Mokit Chau of Professor Kumacheva’s

group, Department of Chemistry, University of Toronto.

3

3.1 Introduction

Hydrogels of CNCs have many desirable properties including their low cost and

biodegradability, which can be beneficial in bioengineering and biomedicine. Studies have

already been conducted to determine the cytotoxicity and impact of cellulose nanomaterials on

biological systems. Such works include electrospun1,2 and melt-drawn3 CNC-composite fibers

that were evaluated as potential scaffolds for tissue engineering. In these systems, CNCs were

prepared with polymer materials to form scaffolds that were cut to disc-like shapes before a

suspension of cells was added on top. In each case, a variety of assays and microscopy

experiments revealed that cells successfully attached and proliferated onto the scaffolds with no

cytotoxic implications. Bio-composite materials reinforced with CNCs were also prepared.4,5 In

one such study, films of polyvinyl alcohol reinforced with CNCs and poly(lactide-co-glycolide)

were prepared through solvent-casting in water. The viability of mesenchymal stem cells was

evaluated on substrates of various compositions.5 In another work, a hydrogel of dextran and

modified cellulose were extruded from a double-barrel syringe containing 1 wt % CNCs. Cells

were cultured in tissue culture flasks, after which hydrogels were placed on top. The hydrogels

showed no significant cytotoxicity towards NIH-3T3 fibroblasts cells as determined by MTT

assay.4 Hydrogels were prepared from carboxylated cellulose nanofibers crosslinked with metal

cations and were subsequently investigated as tissue culture substrates for C3H10T1/2

38

fibroblasts. The crosslinked hydrogels were modified with fibronectin to improve cellular

adhesion. Fibroblasts were seeded 2D on top of the gels for five days and were found to

successfully support the growth of cells on the surface of the gel.6

Of all the works previously mentioned, only one study has been conducted using CNC

suspensions that have not undergone surface modification or mechanical processing, and were

not components of a composite system. The toxicity of CNCs suspensions of various

concentrations was tested against two cells lines: NIH-3T3 murine embryo fibroblasts and

HCT116 colon adenocarcinoma that were cultured on a tissue culture well-plate. The authors

concluded that cytotoxicity is independent of CNC size and concentrations below 250 µg/mL.7

However, in this case cells were cultured on a 2D substrate, which may impact their behaviour.

The results of these experiments are not instructive of the true performance of cells in in

vivo environments where they are embedded in a 3D manner. Cellulose nanofibers (CNFs), an

analogue of CNCs, have been explored as 3D scaffolds for tissue engineering.8 Cellulose

nanofibers isolated from wood pulp were constructed as hydrogels to act as artificial ECMs and

were determined to be noncytotoxic to human hepatic cells cultured in 3D within the CNF

hydrogel.10 Three-dimensional cell culture was prepared by mixing CNF hydrogels with

suspensions of liver progenitor cells. The hydrogels were found to improve upon hepatocyte

differentiation.9

The objective of the present work was to evaluate the use of CNCs for the preparation of

artificial ECMs. We examined hydrogel structure, mechanical properties and cytotoxicity. Since

no previous works have been conducted on the 3D encapsulation of cells within unmodified

CNC hydrogels, we examined the efficacy of CNC hydrogels as three-dimensional environments

39

for the NIH-3T3 adherent cell line. Furthermore, we compared CNCs obtained from two sources

and evaluated two methods in preparing CNC scaffolds for cellular experiments.

3.2 Properties of Cellulose Nanocrystal Suspensions

Aqueous suspensions of CNC1 and CNC2 were extensively dialyzed, as described in

section 2.2, in order to remove impurities (e.g., sulfuric acid from the hydrolysis of the wood

pulp and sodium ions from the neutralization step). Figure 3.1 shows Transmission Electron

Microscopy (TEM) images of the individual CNCs cast from their dialyzed suspensions. Both

types of CNCs had a well-defined whisker-like shape. Based on the analysis of TEM images, we

determined the size-distribution of CNC length and diameter (Figure 3.2). The CNCs obtained

from FP Innovations (CNC1) had an average length and diameter of 219 ± 94 and 18 ± 7 nm,

respectively, while CNCs obtained from FPL (CNC2) have average dimensions of 180 ± 70 nm

by 13 ± 4 nm. Overall, CNC1 had a broader distribution in dimensions than CNC2.

Figure 3.1 TEM images of CNC suspensions from two sources (a) FP Innovations, and (b)

Forest Products Laboratory.

40

Figure 3.2 Size distribution plots of the (a, c) diameter and (b, d) length of individual CNC

particles from (a, b) CNC1 and (c, d) CNC2. Approximately 100 individual CNC fibrils were

measured for each CNC source using ImageJ software.

The electrokinetic potential (ζ-potential) of CNC1 and CNC2 in aqueous suspensions

mixed with HBSS was -39 ± 1 mV (CNC1) and -43 ± 2 mV, with no discernible difference

distinguishing the CNCs from either source. A summary of the properties of CNC suspensions

can be found in Table 3.1.

Table 3.1 Summary of CNC properties

Notation Source

Average

Dimensions