# Grafting Polymers from Cellulose Nanocrystals: Synthesis, Properties, and Applications

Sandra Wohlhauser, Gwendoline Delepierre, Marianne Labet, Gaëlle Morandi, Wim Thielemans, Christoph Weder, Justin Zoppe

► To cite this version:

Sandra Wohlhauser, Gwendoline Delepierre, Marianne Labet, Gaëlle Morandi, Wim Thielemans, et al. Grafting Polymers from Cellulose Nanocrystals: Synthesis, Properties, and Applications. Macromolecules, 2018, 51 (16), pp.6157-6189. 10.1021/acs.macromol.8b00733. hal-02353644

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https://hal.science/hal-02353644

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# Grafting Polymers from Cellulose Nanocrystals: Synthesis, Properties, and Applications

Sandra Wohlhauser,† Gwendoline Delepierre,† Marianne Labet,ℓ Gaëlle Morandi,‡ Wim Thielemans,ℓ,∫ Christoph Weder,†∫ and Justin O. Zoppe*‡,∫

†Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland

ℓRenewable Materials and Nanotechnology Research Group, Chemical Engineering, KU Leuven, Campus Kulak Kortrijk, Etienne Sabbelaan 53, 8500 Kortrijk, Belgium

‡Laboratoire Polymères, Biopolymères, Surfaces, Normandie Université, INSA de Rouen, Avenue de l'Université, 76801 Saint-Étienne-du-Rouvray Cedex, France

<figure><img src="image_2.png" /></figure>

Supporting Information

**ABSTRACT:** Over the past 10 years, the grafting of polymers from the surface of cellulose nanocrystals (CNCs) has gained substantial interest in both academia and industry due to the rapidly growing number of potential applications of surface-modified CNCs, which range from building blocks in nano-composites and responsive nanomaterials to antimicrobial agents. CNCs are rod-like nanoparticles that can be isolated from renewable biosources and which exhibit high crystallinity, tunable aspect ratio, high stiffness, and strength. Upon drying, the abundance of surface hydroxyl groups often leads

to a degree of irreversible aggregation, as a result of strong hydrogen bonding. Moreover, their relatively hydrophilic character renders CNCs incompatible with hydrophobic media, e.g., nonpolar solvents and polyolefin matrices. By grafting macro-molecules from their surface, CNCs can be imparted with surface characteristics and other physicochemical properties that are reminiscent of the grafted polymer. This has allowed the design of nanoscale building blocks whose readily tunable properties are useful for the formation of both colloidal dispersions and solid state materials. In this Perspective, we provide an overview of the morphology and surface chemistry of CNCs and detail various techniques to manipulate their surface chemistry via polymer grafting *from* approaches. Moreover, we explore the most common polymerization techniques that are used to graft polymers from the surface and reducing end groups of CNCs, including surface-initiated ring-opening polymerization (SI-ROP), surface-initiated free (SI-FRP), and controlled (SI-CRP) radical polymerization. Finally, we provide insights into some of the emerging applications and conclude with an outlook of future work that would benefit the field.

<figure><img src="image_3.png" /></figure>

## 1. INTRODUCTION

During the last 3 decades, nanomaterials have emerged to play an ever increasing role in the material science field and are nowadays commonly found in our daily lives.¹ Nanomaterials display interesting and often unique mechanical, optical, and/or electronic properties, as a result of their high surface area as well as their specific structural features, that are not available in micro- and macroscale analogues. Cellulose-based nanocrystals and -fibrils have emerged as a particularly widely used class of building blocks for structural as well as advanced functional materials.²⁻⁸ Cellulose, a linear homopolysaccharide composed of β-D-anhydroglucopyranose units, is the most abundant organic polymer on Earth and a promising renewable resource for the future production of biobased materials and fuels.⁹ Cellulose is found in a wide variety of species, such as higher plants, tunicates, algae, fungi, bacteria, invertebrates, and amoeba. In nature, cellulose is found in hierarchically structured multicomponent materials, which at the lowest hierarchical level feature assemblies that contain individual cellulose in a uniaxially ordered

(semi)crystalline organization. Careful disintegration schemes permit accessing cellulose nanocrystals (CNCs) and cellulose nanofibrils (CNFs), whose properties (e.g., crystallinity, morphology, aspect ratio, and surface chemistry)¹⁰ depend on the requirements for use in polymer nanocomposites, colloidal liquid crystals, hydrogels, and multiresponsive materials.²,⁸,¹¹⁻¹⁴

CNCs are rod-like nanoparticles that can be isolated from renewable biosources, and which exhibit high crystallinity, tunable aspect ratio, and high stiffness and strength. Typical dimensions are lengths in the range from 50 nm to more than 1 µm and widths of 5–50 nm, depending on the source material.⁵,¹⁰ In contrast to many other nanorods, CNCs appear to be physiologically benign.¹⁵⁻¹⁷ CNCs are commonly obtained by strong acid-catalyzed hydrolysis, with sulfuric acid as the most commonly used acid, which selectively degrades the amorphous regions of

Received: April 6, 2018

Revised: June 28, 2018

Published: July 26, 2018

<figure><img src="image_4.png" /></figure>
<figure><img src="image_2.png" /></figure>

# Grafting Polymers from Cellulose Nanocrystals: Synthesis, Properties, and Applications

Sandra Wohlhauser,† Gwendoline Delepierre,† Marianne Labet,ℓ Gaëlle Morandi,‡ Wim Thielemans,ℓ,∫ Christoph Weder,†∫ and Justin O. Zoppe*,†∫

†Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland

ℓRenewable Materials and Nanotechnology Research Group, Chemical Engineering, KU Leuven, Campus Kulak Kortrijk, Etienne Sabbelaan 53, 8500 Kortrijk, Belgium

‡Laboratoire Polymères, Biopolymères, Surfaces, Normandie Université, INSA de Rouen, Avenue de l'Université, 76801 Saint-Étienne-du-Rouvray Cedex, France

<figure><img src="image_3.png" /></figure>

## Supporting Information

**ABSTRACT:** Over the past 10 years, the grafting of polymers from the surface of cellulose nanocrystals (CNCs) has gained substantial interest in both academia and industry due to the rapidly growing number of potential applications of surface-modified CNCs, which range from building blocks in nano-composites and responsive nanomaterials to antimicrobial agents. CNCs are rod-like nanoparticles that can be isolated from renewable biosources and which exhibit high crystallinity, tunable aspect ratio, high stiffness, and strength. Upon drying, the abundance of surface hydroxyl groups often leads

to a degree of irreversible aggregation, as a result of strong hydrogen bonding. Moreover, their relatively hydrophilic character renders CNCs incompatible with hydrophobic media, e.g., nonpolar solvents and polyolefin matrices. By grafting macromolecules from their surface, CNCs can be imparted with surface characteristics and other physicochemical properties that are reminiscent of the grafted polymer. This has allowed the design of nanoscale building blocks whose readily tunable properties are useful for the formation of both colloidal dispersions and solid state materials. In this Perspective, we provide an overview of the morphology and surface chemistry of CNCs and detail various techniques to manipulate their surface chemistry via polymer grafting from approaches. Moreover, we explore the most common polymerization techniques that are used to graft polymers from the surface and reducing end groups of CNCs, including surface-initiated ring-opening polymerization (SI-ROP), surface-initiated free (SI-FRP), and controlled (SI-CRP) radical polymerization. Finally, we provide insights into some of the emerging applications and conclude with an outlook of future work that would benefit the field.

<figure><img src="image_4.png" /><figcaption>NANOCOMPOSITES</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>LC BEHAVIOR</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>STIMULI-RESPONSIVENESS</figcaption></figure>

## 1. INTRODUCTION

During the last 3 decades, nanomaterials have emerged to play an ever increasing role in the material science field and are nowadays commonly found in our daily lives.¹ Nanomaterials display interesting and often unique mechanical, optical, and/or electronic properties, as a result of their high surface area as well as their specific structural features, that are not available in micro- and macroscale analogues. Cellulose-based nanocrystals and -fibrils have emerged as a particularly widely used class of building blocks for structural as well as advanced functional materials.²⁻⁸ Cellulose, a linear homopolysaccharide composed of β-D-anhydroglucopyranose units, is the most abundant organic polymer on Earth and a promising renewable resource for the future production of biobased materials and fuels.⁹ Cellulose is found in a wide variety of species, such as higher plants, tunicates, algae, fungi, bacteria, invertebrates, and amoeba. In nature, cellulose is found in hierarchically structured multicomponent materials, which at the lowest hierarchical level feature assemblies that contain individual cellulose in a uniaxially ordered

(semi)crystalline organization. Careful disintegration schemes permit accessing cellulose nanocrystals (CNCs) and cellulose nanofibrils (CNFs), whose properties (e.g., crystallinity, morphology, aspect ratio, and surface chemistry)¹⁰ depend on the requirements for use in polymer nanocomposites, colloidal liquid crystals, hydrogels, and multiresponsive materials.²,⁸,¹¹⁻¹⁴

CNCs are rod-like nanoparticles that can be isolated from renewable biosources, and which exhibit high crystallinity, tunable aspect ratio, and high stiffness and strength. Typical dimensions are lengths in the range from 50 nm to more than 1 µm and widths of 5–50 nm, depending on the source material.⁵,¹⁰ In contrast to many other nanorods, CNCs appear to be physiologically benign.¹⁵⁻¹⁷ CNCs are commonly obtained by strong acid-catalyzed hydrolysis, with sulfuric acid as the most commonly used acid, which selectively degrades the amorphous regions of

Received: April 6, 2018

Revised: June 28, 2018

Published: July 26, 2018

<figure><img src="image_7.png" /></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Schematic representation of CNC transformation into polymer-modified CNCs, which are useful for the formation of nanocomposites containing one or multiple components, display LC behavior, or exhibit stimuli-responsive behavior. (The center graphic in the bottom row was adapted from ref 31. Copyright 2017 Wiley-VCH Verlag GMbH & Co. KGaA.)</figcaption></figure>

macroscopic cellulose fibers, while the crystalline domains stay intact. Hydrolysis with sulfuric acid also introduces anionic sulfate half-ester groups on the surface of the CNCs, which are essential for their colloidal stability in water, on account of electrostatic repulsions. This effect also likely plays an important role in their spontaneous assembly into chiral nematic (a.k.a. cholesteric) colloidal liquid crystal (LC) phases, in addition to their inherent chirality.<sup>18-20</sup> CNCs hydrolyzed by sulfuric acid typically show, however, a limited thermal stability, which is apparent by significant coloration when processed at elevated temperatures and presents an obstacle for the fabrication of polymer nanocomposites.<sup>21,22</sup> This problem can be overcome by hydrolyzing cellulose pulp with phosphoric acid, which affords CNCs with a low concentration of phosphate surface groups and which offer an attractive combination of adequate dispersibility in polar solvents and improved thermal stability.<sup>21</sup> The production of CNCs has also evolved at the industrial scale in some countries such as Canada, the United States, Israel, and Sweden, even though it is still developing.<sup>23</sup>

The CNC surfaces also contain many hydroxyl (–OH) groups, which allow many types of chemical modifications and enable a broad range of nanomaterials that cover a wide spectrum of properties and functions, ranging from stimuli-responsiveness to antimicrobial capabilities.<sup>8,12</sup> In addition, surface chemical modifications allow transforming the rather hydrophilic CNCs into hydrophobic particles, and thus facilitate their use in hydrophobic media.<sup>24-26</sup> One of the primary challenges in the chemical modification of CNCs is to carry out successful reactions without impacting their intrinsic properties, notably the dimensions, crystallinity, and intrinsic mechanical properties.<sup>7</sup>

Surface modification by graft polymerization proved to be a robust approach, in which some of the physical and chemical properties can be tailored at will.<sup>4,27-29</sup> Graft polymerization of surfaces can be performed by using “grafting to”, “grafting from”, or “grafting through” methods. The grafting through method involves the functionalization of solid substrates with polymerizable functional groups, e.g., acrylic moieties, followed by polymerization in situ. While the grafting to method involves the connection of preformed polymeric chains with the surface through reactive end groups, the grafting from strategy concerns the growth of polymer chains via surface-initiated polymerization either from a substrate containing functional groups capable of initiating the polymerization reaction, or by immobilization of an active initiator species to the substrate surface. Due to the steric hindrance often encountered while utilizing the grafting to approach, the grafting from approach has the advantage of well-controlled polymer graft lengths and high polymer tether grafting density.

As the latter strategy has been widely employed to modify CNCs toward multicomponent colloids and, more recently, one component-nanocomposites (OCNs), in which no polymer matrix is required and the final materials do not usually suffer from phase separation issues, this will be the focus of our Perspective (Figure 1). Our aim is to provide a concise overview of the field of grafting polymers from the surface of CNCs, while highlighting some noteworthy contributions over the past decade, since the pioneering work of Habibi et al. in 2008,<sup>30</sup> who introduced ε-caprolactone grafting from CNCs via surface-initiated ring-opening polymerization (SI-ROP). In the following sections, the physicochemical properties of CNCs, various chemical modification pathways known to graft polymers
<figure><img src="image_8.png" /><figcaption>**Figure 1.** Schematic representation of CNC transformation into polymer-modified CNCs, which are useful for the formation of nanocomposites containing one or multiple components, display LC behavior, or exhibit stimuli-responsive behavior. (The center graphic in the bottom row was adapted from ref 31. Copyright 2017 Wiley-VCH Verlag GMbH & Co. KGaA.)</figcaption></figure>

macroscopic cellulose fibers, while the crystalline domains stay intact. Hydrolysis with sulfuric acid also introduces anionic sulfate half-ester groups on the surface of the CNCs, which are essential for their colloidal stability in water, on account of electrostatic repulsions. This effect also likely plays an important role in their spontaneous assembly into chiral nematic (a.k.a. cholesteric) colloidal liquid crystal (LC) phases, in addition to their inherent chirality.<sup>18-20</sup> CNCs hydrolyzed by sulfuric acid typically show, however, a limited thermal stability, which is apparent by significant coloration when processed at elevated temperatures and presents an obstacle for the fabrication of polymer nanocomposites.<sup>21,22</sup> This problem can be overcome by hydrolyzing cellulose pulp with phosphoric acid, which affords CNCs with a low concentration of phosphate surface groups and which offer an attractive combination of adequate dispersibility in polar solvents and improved thermal stability.<sup>21</sup> The production of CNCs has also evolved at the industrial scale in some countries such as Canada, the United States, Israel, and Sweden, even though it is still developing.<sup>23</sup>

The CNC surfaces also contain many hydroxyl (–OH) groups, which allow many types of chemical modifications and enable a broad range of nanomaterials that cover a wide spectrum of properties and functions, ranging from stimuli-responsiveness to antimicrobial capabilities.<sup>8,12</sup> In addition, surface chemical modifications allow transforming the rather hydrophilic CNCs into hydrophobic particles, and thus facilitate their use in hydrophobic media.<sup>24-26</sup> One of the primary challenges in the chemical modification of CNCs is to carry out successful reactions without impacting their intrinsic properties, notably the dimensions, crystallinity, and intrinsic mechanical properties.<sup>7</sup>

Surface modification by graft polymerization proved to be a robust approach, in which some of the physical and chemical properties can be tailored at will.<sup>4,27-29</sup> Graft polymerization of surfaces can be performed by using “grafting to”, “grafting from”, or “grafting through” methods. The grafting through method involves the functionalization of solid substrates with polymerizable functional groups, e.g., acrylic moieties, followed by polymerization in situ. While the grafting to method involves the connection of preformed polymeric chains with the surface through reactive end groups, the grafting from strategy concerns the growth of polymer chains via surface-initiated polymerization either from a substrate containing functional groups capable of initiating the polymerization reaction, or by immobilization of an active initiator species to the substrate surface. Due to the steric hindrance often encountered while utilizing the grafting to approach, the grafting from approach has the advantage of well-controlled polymer graft lengths and high polymer tether grafting density.

As the latter strategy has been widely employed to modify CNCs toward multicomponent colloids and, more recently, one component-nanocomposites (OCNs), in which no polymer matrix is required and the final materials do not usually suffer from phase separation issues, this will be the focus of our Perspective (Figure 1). Our aim is to provide a concise overview of the field of grafting polymers from the surface of CNCs, while highlighting some noteworthy contributions over the past decade, since the pioneering work of Habibi et al. in 2008,<sup>30</sup> who introduced ε-caprolactone grafting from CNCs via surface-initiated ring-opening polymerization (SI-ROP). In the following sections, the physicochemical properties of CNCs, various chemical modification pathways known to graft polymers
from the surface of the CNCs and applications of polymer-
modified CNCs (CNC-g-polymer) will be described and the
opportunities and challenges that have emerged in the field will
be discussed.

## 2. MORPHOLOGY AND SURFACE CHEMISTRY OF CNCS

The semicrystalline nature of cellulose fibers, in which the crystalline regions are held together by strong inter- and intra-molecular hydrogen bonds, gives rise to their unique mechanical properties and is the naturally selected main structural component of the primary cell wall in higher plants.⁹ Although less understood from a structural standpoint due to their disordered morphology, the noncrystalline, or “amorphous,” regions of cellulose are thought to be advantageous in terms of water transport in plants.³² This inherent architecture of cellulose fibers is what ultimately allows the nanoscale crystalline regions to be selectively extracted from macroscopic fibers through acid-catalyzed hydrolysis. Thus, before moving forward into methodologies to modify the surface of CNCs with polymer grafts, it is imperative to first discuss the structure of cellulose, its crystalline domains and the various extraction methods to produce CNCs, which lead to strikingly different surface chemistries and reactivity.

**2.1. Morphology.** Cellulose is the trivial name for the linear homopolysaccharide polymer composed of β-D-anhydroglucopyranose and linked by β(1-4′) glycosidic (ether) bonds. The repeating anhydroglucopyranose unit (referred to as AGU)³³ in cellulose is a six-membered closed ring form of D-glucose in the chair conformation with the anomeric carbon (C1) in the β-position (Figure 2).³⁴ The β(1-4′) glycosidic bond between

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Molecular structure of cellulose, with the anhydroglucose repeating unit in the chair conformation, the glycosidic bond, as well as reducing and nonreducing chain ends.</figcaption></figure>

the different AGUs results in a twist along the length of the
chain, which can be defined by the glycosidic torsion angles $\phi = H1-C1-O4'-C4'$ and $\psi = C1-O4'-C4'-H4'$.³⁵ It has to be
noted that the definition of these angles is not standardized in the
literature, so comparisons of values reported for different sources
needs to take into account the angle definitions used.³⁵⁻³⁷

While the ring structure of the AGU is confined to the chair conformation with the C-O bonds in the equatorial position, free rotation can occur around the C5–C6 bond, resulting in different conformations of the hydroxymethyl group relative to the glucose ring. The position of the hydroxymethyl group relative to the glucopyranose ring can be described by the angles $\chi = O5–C5–C6–O6$ and $\chi’ = C4–C5–C6–O6$.⁵,³⁸ Three energetic minima can be found when the angles are both 60° (gauche–gauche conformation, gg), 60°–180° (gauche–trans, gt), and 180°–60° (trans–gauche, tg).⁵,³⁶,³⁷ The free rotation around the C5–C6 bond with the resulting freedom of positioning of O6 enables different hydrogen bonding patterns, including in cellulose crystals.³⁷,³⁹

Until recently, seven different crystal allomorphs of cellulose were thought to exist: $I_\alpha$, $I_\beta$, II, III$_D$, III$_{II}$, IV$_D$, and IV$_{II}$. While cellulose II is thermodynamically the most stable form, native cellulose was thought to exist in polymorphs $I_\alpha$ (found in bacterial cellulose and algae, triclinic unit cell) and $I_\beta$ (higher plants, monoclinic unit cells).⁴⁰,⁴¹ Recent findings from solid-state NMR spectroscopic investigations suggest, however, that native plant cellulose is structurally polymorphic and differs greatly from $I_\alpha$ and $I_\beta$ as evidenced by seven unique sets of $^{13}$C chemical shifts.⁴²,⁴³ While five of these sets are attributed to interior cellulose chains within microfibrils, two are assigned to surface chains and such structural complexity arises from interactions with matrix polysaccharides unique to higher plant celluloses. Nevertheless, cellulose $I_\alpha$ has been found to be metastable and can be partially transformed into $I_\beta$ by annealing above 260 °C or treatment with gases and organic solvents.⁴⁴⁻⁴⁶ Cellulose II is obtained by recrystallization of cellulose after dissolution, thus attaining the thermodynamic minimum. The other allomorphs are local minima in the highly regular hydrogen bonded network of cellulose chains. All allomorphs, despite some having parallel and others antiparallel chain alignment, have a pseudo 2$_1$ helical conformation of cellulose chains as a result of intrachain O3H–O5' hydrogen bonding.⁴⁷ Disruption of any cellulose crystal structure can thus be expected to be linked to disruption of this intrachain hydrogen bond, a fact supported by the ability of cellulose to attain a random coil conformation when dissolved.⁴⁸ The various cellulose polymorphs have unique X-ray diffraction signatures corresponding to the spatial crystal arrangements in their structure.⁹ The crystal packing of cellulose's polymorphs differ in the unit cell dimensions and, may present variations in the chain polarity, which result in changes in the properties of cellulose, such as its rigidity, thermal stability, solubility, or reactivity.⁴⁹

Cellulose chains are synthesized by cellulose synthase enzymes arranged in complexes located at the plasma membrane. This cellulose synthase complex (CSC) produces elementary cellulose protofibrils with a width of 3–4 nm.⁵⁰ These CSCs sit at the end of the growing fibrils.⁵¹,⁵² The CSC can take different forms with three rows of ordered particles, as seen in the algae *Oocytis apiculata*,⁵¹ other linear complexes, as in tunicates and the algae *Valonia* and *Erythrocladia*,⁵³ while in higher plants as well as the green algae *Micrasterias* a hexameric rosette complex containing six particles, each featuring six cellulose synthases, have been reported.⁵³,⁵⁴ The protofibrils are arranged into semicrystalline microfibrils with diameters up to 20 nm.⁵⁵,⁵⁶ The ratio of crystalline to amorphous domains and the ratio of $I_\alpha$ to $I_\beta$ found in the crystalline domains is dependent on the source of cellulose.⁵,⁷,³⁹,⁵⁷

CNCs are commonly represented as ribbons with either a
parallelepiped or a hexagonal cross-section (Figure 3); the shape
and the number of individual cellulose molecules varies greatly
with the cellulose source. Native cellulose microfibrils in plants
and bacteria have long been thought to consist of 36 chains, but
recent advances in the structural characterization of CSCs sug-
gest that an 18-chain model may be a more accurate depiction.⁵⁸
On the other hand, solid-state NMR spectroscopic experiments
indicate that primary cell wall cellulose microfibrils are com-
posed of at least 24 chains, which may be due to coalescence of
multiple microfibrils synthesized by neighboring CSCs.⁴²,⁵⁹ The
exact number of cellulose chains within native cellulose micro-
fibrils still remains a highly debated research topic.

As the main differences between the $I_\alpha$ and $I_\beta$ structures have been shown to lie in the conformation of the AGU and the $\beta(1-4')$ linkages by NMR spectroscopy and X-ray and neutron
from the surface of the CNCs and applications of polymer-
modified CNCs (CNC-g-polymer) will be described and the
opportunities and challenges that have emerged in the field will
be discussed.

## 2. MORPHOLOGY AND SURFACE CHEMISTRY OF CNCS

The semicrystalline nature of cellulose fibers, in which the crystalline regions are held together by strong inter- and intra-molecular hydrogen bonds, gives rise to their unique mechanical properties and is the naturally selected main structural component of the primary cell wall in higher plants.⁹ Although less understood from a structural standpoint due to their disordered morphology, the noncrystalline, or “amorphous,” regions of cellulose are thought to be advantageous in terms of water transport in plants.³² This inherent architecture of cellulose fibers is what ultimately allows the nanoscale crystalline regions to be selectively extracted from macroscopic fibers through acid-catalyzed hydrolysis. Thus, before moving forward into methodologies to modify the surface of CNCs with polymer grafts, it is imperative to first discuss the structure of cellulose, its crystalline domains and the various extraction methods to produce CNCs, which lead to strikingly different surface chemistries and reactivity.

**2.1. Morphology.** Cellulose is the trivial name for the linear homopolysaccharide polymer composed of β-D-anhydroglucopyranose and linked by β(1-4′) glycosidic (ether) bonds. The repeating anhydroglucopyranose unit (referred to as AGU)³³ in cellulose is a six-membered closed ring form of D-glucose in the chair conformation with the anomeric carbon (C1) in the β-position (Figure 2).³⁴ The β(1-4′) glycosidic bond between

<figure><img src="image_9.png" /><figcaption>**Figure 2.** Molecular structure of cellulose, with the anhydroglucose repeating unit in the chair conformation, the glycosidic bond, as well as reducing and nonreducing chain ends.</figcaption></figure>

the different AGUs results in a twist along the length of the
chain, which can be defined by the glycosidic torsion angles $\phi = H1-C1-O4'-C4'$ and $\psi = C1-O4'-C4'-H4'$.³⁵ It has to be
noted that the definition of these angles is not standardized in the
literature, so comparisons of values reported for different sources
needs to take into account the angle definitions used.³⁵⁻³⁷

While the ring structure of the AGU is confined to the chair conformation with the C-O bonds in the equatorial position, free rotation can occur around the C5–C6 bond, resulting in different conformations of the hydroxymethyl group relative to the glucose ring. The position of the hydroxymethyl group relative to the glucopyranose ring can be described by the angles $\chi = O5–C5–C6–O6$ and $\chi’ = C4–C5–C6–O6$.⁵,³⁸ Three energetic minima can be found when the angles are both 60° (gauche–gauche conformation, gg), 60°–180° (gauche–trans, gt), and 180°–60° (trans–gauche, tg).⁵,³⁶,³⁷ The free rotation around the C5–C6 bond with the resulting freedom of positioning of O6 enables different hydrogen bonding patterns, including in cellulose crystals.³⁷,³⁹

Until recently, seven different crystal allomorphs of cellulose were thought to exist: $I_\alpha$, $I_\beta$, II, III$_D$, III$_{II}$, IV$_D$, and IV$_{II}$. While cellulose II is thermodynamically the most stable form, native cellulose was thought to exist in polymorphs $I_\alpha$ (found in bacterial cellulose and algae, triclinic unit cell) and $I_\beta$ (higher plants, monoclinic unit cells).⁴⁰,⁴¹ Recent findings from solid-state NMR spectroscopic investigations suggest, however, that native plant cellulose is structurally polymorphic and differs greatly from $I_\alpha$ and $I_\beta$ as evidenced by seven unique sets of ¹³C chemical shifts.⁴²,⁴³ While five of these sets are attributed to interior cellulose chains within microfibrils, two are assigned to surface chains and such structural complexity arises from interactions with matrix polysaccharides unique to higher plant celluloses. Nevertheless, cellulose $I_\alpha$ has been found to be metastable and can be partially transformed into $I_\beta$ by annealing above 260 °C or treatment with gases and organic solvents.⁴⁴⁻⁴⁶ Cellulose II is obtained by recrystallization of cellulose after dissolution, thus attaining the thermodynamic minimum. The other allomorphs are local minima in the highly regular hydrogen bonded network of cellulose chains. All allomorphs, despite some having parallel and others antiparallel chain alignment, have a pseudo 2₁ helical conformation of cellulose chains as a result of intrachain O3H–O5' hydrogen bonding.⁴⁷ Disruption of any cellulose crystal structure can thus be expected to be linked to disruption of this intrachain hydrogen bond, a fact supported by the ability of cellulose to attain a random coil conformation when dissolved.⁴⁸ The various cellulose polymorphs have unique X-ray diffraction signatures corresponding to the spatial crystal arrangements in their structure.⁹ The crystal packing of cellulose's polymorphs differ in the unit cell dimensions and, may present variations in the chain polarity, which result in changes in the properties of cellulose, such as its rigidity, thermal stability, solubility, or reactivity.⁴⁹

Cellulose chains are synthesized by cellulose synthase enzymes arranged in complexes located at the plasma membrane. This cellulose synthase complex (CSC) produces elementary cellulose protofibrils with a width of 3–4 nm.⁵⁰ These CSCs sit at the end of the growing fibrils.⁵¹,⁵² The CSC can take different forms with three rows of ordered particles, as seen in the algae *Oocytis apiculata*,⁵¹ other linear complexes, as in tunicates and the algae *Valonia* and *Erythrocladia*,⁵³ while in higher plants as well as the green algae *Micrasterias* a hexameric rosette complex containing six particles, each featuring six cellulose synthases, have been reported.⁵³,⁵⁴ The protofibrils are arranged into semicrystalline microfibrils with diameters up to 20 nm.⁵⁵,⁵⁶ The ratio of crystalline to amorphous domains and the ratio of $I_\alpha$ to $I_\beta$ found in the crystalline domains is dependent on the source of cellulose.⁵,⁷,³⁹,⁵⁷

CNCs are commonly represented as ribbons with either a
parallelepiped or a hexagonal cross-section (Figure 3); the shape
and the number of individual cellulose molecules varies greatly
with the cellulose source. Native cellulose microfibrils in plants
and bacteria have long been thought to consist of 36 chains, but
recent advances in the structural characterization of CSCs sug-
gest that an 18-chain model may be a more accurate depiction.⁵⁸
On the other hand, solid-state NMR spectroscopic experiments
indicate that primary cell wall cellulose microfibrils are com-
posed of at least 24 chains, which may be due to coalescence of
multiple microfibrils synthesized by neighboring CSCs.⁴²,⁵⁹ The
exact number of cellulose chains within native cellulose micro-
fibrils still remains a highly debated research topic.

As the main differences between the $I_\alpha$ and $I_\beta$ structures have been shown to lie in the conformation of the AGU and the $\beta(1-4')$ linkages by NMR spectroscopy and X-ray and neutron
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Schematic representation of idealized cellulose cross sections showing the faces of cellulose nanocrystals with the crystal planes (t = triclinic, m = monoclinic unit cell) and the crystal structure. Each gray box represents the cross-section of a cellulose chain viewed in the direction of its long axis. Representations for CNC extracted from (a) wood, (b) tunicate, (c) *Valonia*, (d) *Micrasterias*, (e) reference bacterial cellulose from *Acetobacter*, and (f) bacterial cellulose from *Acetobacter* produced in the presence of mannan. Reproduced from ref 7 with permission. Copyright 2011 The Royal Society of Chemistry.</figcaption></figure>

diffraction,⁵⁷,⁶⁰ one could assume that the availability and reactivity of the surface -OH groups between both allomorphs are similar. Crystal structure diffraction studies indicate the hydroxymethyl conformation to occur in the *tg* conformation with hydrogen bonding to exist between O3H–O5' and O2H–O6' of cellulose chain aligned next to each other, whereas hydrogen bonding between stacked chains is virtually nonexistent.⁵⁷ The 2-fold helical twist of the cellulose chain due to the $\beta(1-4')$ linkage (Figure 2) results in an alternation of primary and secondary -OH groups pointing out of the crystal along a single cellulose chain.

To assess surface reactivity, one needs to consider that surface groups are not as confined as the same groups within the CNCs. Indeed, ¹³C solid state NMR studies suggest that the hydroxymethyl groups on the surface chains pointing outward display a different conformation (*gg*) than the hydrogen-bonded primary -OH groups inside the CNCs (*tg*).³⁸,⁶⁰ One can also envision that the ability to change the conformation of the hydroxymethyl group will depend on the level of interference of the solvent in the hydrogen bonding of the hydroxymethyl group with other groups in the crystal surface.

Experimental observations suggest that the surface reactivity of the surface -OH groups follows O2 > O6 > O3 for native cotton and cotton-like (i.e., linen, ramie, sisal and wood) celluloses, whereas it follows O6 > O2 > O3 for *Valonia ventricosa* cellulose as a consequence of a higher degree of intramolecular hydrogen bonding between O3H and O5'H and the higher crystalline order leading to a reduction in the absolute availability of O2H by inaccessibility in the interior of the crystalline region (Figure 2).⁶¹ This may be considered further confirmation of the *gg* conformation of the surface hydroxymethyl group as the intramolecular O2H–O6 hydrogen bond is not possible in this conformation. Surface reactivity also needs to consider the reaction mechanism. After activating surface hydroxyl groups, the O2 position cannot react under S<sub>N</sub>2 conditions as the CNC crystal inhibits Walden inversion and only O6 can be expected to react. This can also be seen when surface alcohol groups have been replaced by chloride.⁶² Finally, the reactivity of the -OH groups at the surface of cellulose seems also to be affected by the reaction media, i.e., the -OH groups at position 6 are the most available for reaction in homogeneous

solutions following the order O6 > O2 > O3 due to steric reasons, while for heterogeneous modifications the -OH groups at position 2 become the most reactive species with the following order O2 > O6 > O3.⁶³

**2.2. Surface Chemistry.** Chemical reactions carried out at solid-liquid interfaces differ from homogeneous reactions in that adsorption is a prerequisite for the chemical reaction to occur and functional groups on surfaces have fewer degrees of freedom as discussed above.⁶⁴ With an understanding of cellulose crystal morphology and surface -OH group reactivity, one must also be aware of the factors involved in CNC processing that affect their surface chemistry.

By far the most common method to extract CNCs from cellulosic raw materials is sulfuric acid-catalyzed hydrolysis.²,³,⁵⁻⁷ After delignification of the native material and removal of hemi-celluloses, this typically involves exposing high purity cellulose fibers to a 55–65 wt % sulfuric acid solution for 30–60 min at temperatures ranging from 35 to 70 °C. During this process, sulfuric acid reacts with a fraction of the -OH groups at the surface of the crystalline regions to form sulfate half-esters, which are typically present at a concentration in the range of 150–400 mmol/kg.⁶⁵ Upon purification by multiple centrifugation steps and dialysis against deionized water, these sulfate half-ester groups are negatively charged, yielding highly stable CNC dispersions due to electrostatic repulsions. As mentioned above, CNCs hydrolyzed by sulfuric acid typically display a limited thermal stability.²¹,²² The sulfate half-esters can be removed by hydrolytic or solvolytic desulfation procedures,⁶⁶,⁶⁷ but this modification comes at the expense of a reduced dispersibility.

It is also possible to employ hydrochloric (HCl)⁶⁸ or hydrobromic (HBr)⁶⁹ acid for the hydrolysis. In some cases, CNCs with a very low surface charge are produced; these charges likely originate from a small amount of carboxylate groups (ca. 20–30 mmol/kg).⁶⁵ Accordingly, CNCs produced by HCl- or HBr-based hydrolysis methods also have the disadvantage of low colloidal stability due to the lack of sufficient electrostatic repulsive forces that consequently allow hydrogen bonding between adjacent nanoparticles. Such dispersion aggregation is obviously problematic for subsequent surface modification. Phosphoric acid hydrolysis, introduced by Camarero Espinosa et al.,²¹ affords
<figure><img src="image_10.png" /><figcaption>**Figure 3.** Schematic representation of idealized cellulose cross sections showing the faces of cellulose nanocrystals with the crystal planes (t = triclinic, m = monoclinic unit cell) and the crystal structure. Each gray box represents the cross-section of a cellulose chain viewed in the direction of its long axis. Representations for CNC extracted from (a) wood, (b) tunicate, (c) *Valonia*, (d) *Micrasterias*, (e) reference bacterial cellulose from *Acetobacter*, and (f) bacterial cellulose from *Acetobacter* produced in the presence of mannan. Reproduced from ref 7 with permission. Copyright 2011 The Royal Society of Chemistry.</figcaption></figure>

diffraction,⁵⁷,⁶⁰ one could assume that the availability and reactivity of the surface -OH groups between both allomorphs are similar. Crystal structure diffraction studies indicate the hydroxymethyl conformation to occur in the *tg* conformation with hydrogen bonding to exist between O3H–O5' and O2H–O6' of cellulose chain aligned next to each other, whereas hydrogen bonding between stacked chains is virtually nonexistent.⁵⁷ The 2-fold helical twist of the cellulose chain due to the $\beta(1-4')$ linkage (Figure 2) results in an alternation of primary and secondary -OH groups pointing out of the crystal along a single cellulose chain.

To assess surface reactivity, one needs to consider that surface groups are not as confined as the same groups within the CNCs. Indeed, ¹³C solid state NMR studies suggest that the hydroxymethyl groups on the surface chains pointing outward display a different conformation (*gg*) than the hydrogen-bonded primary -OH groups inside the CNCs (*tg*).³⁸,⁶⁰ One can also envision that the ability to change the conformation of the hydroxymethyl group will depend on the level of interference of the solvent in the hydrogen bonding of the hydroxymethyl group with other groups in the crystal surface.

Experimental observations suggest that the surface reactivity of the surface -OH groups follows O2 > O6 > O3 for native cotton and cotton-like (i.e., linen, ramie, sisal and wood) celluloses, whereas it follows O6 > O2 > O3 for *Valonia ventricosa* cellulose as a consequence of a higher degree of intramolecular hydrogen bonding between O3H and O5'H and the higher crystalline order leading to a reduction in the absolute availability of O2H by inaccessibility in the interior of the crystalline region (Figure 2).⁶¹ This may be considered further confirmation of the *gg* conformation of the surface hydroxymethyl group as the intramolecular O2H–O6 hydrogen bond is not possible in this conformation. Surface reactivity also needs to consider the reaction mechanism. After activating surface hydroxyl groups, the O2 position cannot react under S<sub>N</sub>2 conditions as the CNC crystal inhibits Walden inversion and only O6 can be expected to react. This can also be seen when surface alcohol groups have been replaced by chloride.⁶² Finally, the reactivity of the -OH groups at the surface of cellulose seems also to be affected by the reaction media, i.e., the -OH groups at position 6 are the most available for reaction in homogeneous

solutions following the order O6 > O2 > O3 due to steric reasons, while for heterogeneous modifications the -OH groups at position 2 become the most reactive species with the following order O2 > O6 > O3.⁶³

## 2.2. Surface Chemistry.
Chemical reactions carried out at solid-liquid interfaces differ from homogeneous reactions in that adsorption is a prerequisite for the chemical reaction to occur and functional groups on surfaces have fewer degrees of freedom as discussed above.⁶⁴ With an understanding of cellulose crystal morphology and surface -OH group reactivity, one must also be aware of the factors involved in CNC processing that affect their surface chemistry.

By far the most common method to extract CNCs from cellulosic raw materials is sulfuric acid-catalyzed hydrolysis.²,³,⁵⁻⁷ After delignification of the native material and removal of hemi-celluloses, this typically involves exposing high purity cellulose fibers to a 55–65 wt % sulfuric acid solution for 30–60 min at temperatures ranging from 35 to 70 °C. During this process, sulfuric acid reacts with a fraction of the -OH groups at the surface of the crystalline regions to form sulfate half-esters, which are typically present at a concentration in the range of 150–400 mmol/kg.⁶⁵ Upon purification by multiple centrifugation steps and dialysis against deionized water, these sulfate half-ester groups are negatively charged, yielding highly stable CNC dispersions due to electrostatic repulsions. As mentioned above, CNCs hydrolyzed by sulfuric acid typically display a limited thermal stability.²¹,²² The sulfate half-esters can be removed by hydrolytic or solvolytic desulfation procedures,⁶⁶,⁶⁷ but this modification comes at the expense of a reduced dispersibility.

It is also possible to employ hydrochloric (HCl)⁶⁸ or hydrobromic (HBr)⁶⁹ acid for the hydrolysis. In some cases, CNCs with a very low surface charge are produced; these charges likely originate from a small amount of carboxylate groups (ca. 20–30 mmol/kg).⁶⁵ Accordingly, CNCs produced by HCl- or HBr-based hydrolysis methods also have the disadvantage of low colloidal stability due to the lack of sufficient electrostatic repulsive forces that consequently allow hydrogen bonding between adjacent nanoparticles. Such dispersion aggregation is obviously problematic for subsequent surface modification. Phosphoric acid hydrolysis, introduced by Camarero Espinosa et al.,²¹ affords
CNCs that feature a small concentration of phosphate groups (ca. 11 mmol/kg). In this case, each phosphate group at the CNC surface provides two acidic –OH groups and the CNCs display an interesting combination of good colloidal and thermal stability.

Other means to prepare CNCs include endoglucanase-mediated hydrolysis,⁷⁰ mechanochemical activation, and phosphotungstic acid hydrolysis,⁷¹ ammonium persulfate (APS) oxidation,⁷² and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-mediated oxidation followed by cavitation.⁷³ The latter two methods yield CNCs functionalized with a high concentration of surface carboxylate groups (ca. 1.12–1.74 mmol/g for TEMPO-mediated oxidation),⁷³ which not only promote good dispersibility but also offer alternative surface chemical modification pathways.²⁴,²⁵

Regardless of the method utilized to produce CNCs, it is clear that anionic surface charges provide the colloidal stability necessary to prevent a certain degree of aggregation in both water and polar aprotic solvents, such as N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).⁷⁴ The surface charges also affect the ease at which CNCs can be freeze-dried, and at the same time, they use up some of the surface –OH groups that could be used for later functionalization. In addition, the counterions of the surface charges strongly influence the redispersibility of dried CNCs by ultrasonication, especially on material with a moisture content lower than 4 wt % due to the presence of stronger intermolecular hydrogen bonds between CNCs.⁷⁵,⁷⁶ Thus, using hydrolysis protocols that impart “just the right amount” of surface charges to promote colloidal stability appears advisable if subsequent surface modification reactions are to be carried out.

### 2.3. Pretreatments of CNCs for Surface Chemical Modification.
The presence of surface impurities, bound water, and excess counterions can have a significant impact on the success of surface functionalization reactions and their reproducibility, notably between different types or batches of CNCs.⁷⁷⁻⁷⁹ Furthermore, many reaction pathways suitable for the modification of –OH groups are intolerant to water, so that CNCs must either be dried and redispersed or transferred into a dry organic solvent by other means. It is possible to freeze-dry the CNCs⁸⁰⁻⁸⁵ or to exchange the solvent.³⁰,⁸³,⁸⁶⁻⁸⁹

If freeze-dried CNCs are to be surface-modified, the nanocrystals are often washed with organic solvents such as ethanol,⁷⁸,⁷⁹ acetone,⁷⁷ or n-pentane⁷⁷ to remove impurities. This is accomplished by either Soxhlet extraction⁷⁸,⁷⁹ or by several cycles of dispersing the CNCs in the solvent of choice and subsequent separation via centrifugation.⁷⁷ It was found that such pretreatments improved the reproducibility between grafting experiments.⁷⁷

Solvent exchanges usually involve mixing an aqueous dispersion of the CNCs with a water-miscible organic solvent, usually acetone, centrifugation, replacement of the solvent, and multiple repetitions of this cycle, until the solvent is changed to the organic solvent, in which surface-initiated polymerization is often performed.³⁰,⁸⁷,⁸⁸,⁹⁰,⁹¹ For example, Tian et al. compared the SI-ROP of ε-caprolactone (ε-CL) from freeze-dried CNCs and from solvent-exchanged CNCs and observed that the grafting density is higher from the solvent-exchanged nanocrystals than from those that were freeze-dried.⁸⁶ From these findings, it is clear that impurities adsorbed on the surface of CNCs are generally better removed by treatment with organic solvents. These impurities are likely byproducts formed by the hydrolysis of residual hemicellulose or lignin or perhaps the acid-catalyzed dehydration of mono- and oligosaccharides.²,⁷

## 3. SURFACE-INITIATED POLYMERIZATION FROM CNCS

Much of the research efforts directed toward the surface modification of CNCs have focused on tuning the physicochemical characteristics of their surface, while at the same time preserving their morphological integrity and crystallinity.²⁴,²⁵,⁹² Indeed, if the functionalization chemistry is chosen to selectively modify the surface or the reducing end groups, the intrinsic crystallinity and crystal structure of the CNCs should be retained, which can be checked by X-ray diffraction studies.²⁴,³⁰,⁸²,⁸³,⁸⁶,⁹³⁻⁹⁶ In the present section, the methods most often utilized to modify CNCs via the grafting from approach, i.e., surface-initiated and reducing end-initiated polymerization, will be presented.

### 3.1. Surface-Initiated Ring-Opening Polymerization.

Ring-opening polymerization (ROP) is a common technique to produce polymers from cyclic monomers such as lactones,⁹⁷,⁹⁸ dilactones,⁹⁹ lactams,¹⁰⁰ cyclic carbonates,¹⁰¹ cyclic ethers,¹⁰² and oxazolines¹⁰² (Figure 4). For some of these monomers, e.g.,

<figure><img src="image_8.png" /><figcaption>Figure 4. Examples of common monomers used for ROP.</figcaption></figure>

lactones and dilactones, the ROP can be initiated by –OH groups. Therefore, they are ideal candidates for the surface modification of not only CNCs, but also cellulose fibers and nanofibrils, as they also contain a high density of –OH surface groups. In this case, the cellulosic substrate acts as a macroinitiator and the polymerization process is most often referred to as SI-ROP.

CNC-g-polyesters. CNC-g-polyesters have been prepared by the SI-ROP of ε-caprolactone (ε-CL),³⁰,⁸⁰⁻⁸³,⁸⁶,¹⁰³⁻¹⁰⁵ L-lactide (L-LA),⁸⁴,⁸⁵,⁸⁷,⁸⁸,⁹⁰,⁹¹,¹⁰⁴,¹⁰⁶⁻¹⁰⁸ and D-lactide (D-LA)⁸⁹,¹⁰⁵,¹⁰⁹,¹¹⁰ from CNCs. The mechanism of the ROP of lactones and dilactones varies depending on the system used, i.e. the nature of the catalyst and the initiator.¹¹¹ The mechanism can be an anionic, a cationic, a monomer-activated or a coordination-insertion polymerization. In the case of SI-ROP from CNCs, the nanocrystals act as the initiator and the mechanism is only dependent on the catalyst. This reaction is water sensitive as water can also initiate the ROP of lactones. Therefore, the water, in which the nanocrystals are normally dispersed, needs to be removed as much as possible by means described in section 2.3.

Tin(II) 2-ethylhexanoate (Sn(Oct)₂)³⁰,⁷⁷,⁸⁰⁻⁸⁸,⁹⁰,⁹¹,¹⁰³,¹⁰⁵,¹⁰⁶,¹⁰⁸⁻¹¹⁰,¹¹² is the most commonly used catalyst for the SI-ROP of (di)lactones. The reaction is usually performed in toluene, however it has also been carried out in ionic liquids (1-allyl-3-methylimidazolium chloride, [AMIM]Cl¹¹³), in DMSO,¹⁰⁸ or in bulk.⁸⁴ Typical conditions further are temperatures between 80 and 130 °C and a reaction time of typically 24 h. The grafted polymers have also been prepared under microwave irradiation.⁸¹,¹¹² Benzyl alcohol is sometimes used as a sacrificial initiator to produce free polymer in solution, which is easier to characterize than the grafts.⁷⁷,¹⁰⁸ Of course, the comparison with free polymer that is produced “in parallel” is only sensible if one can safely assume that the kinetics of solution ROP are similar to SI-ROP from CNCs, which may or may not be the case.

In general, the nature of SI-ROP from CNCs makes the accurate characterization of grafted polymer molecular weight
CNCs that feature a small concentration of phosphate groups (ca. 11 mmol/kg). In this case, each phosphate group at the CNC surface provides two acidic –OH groups and the CNCs display an interesting combination of good colloidal and thermal stability.

Other means to prepare CNCs include endoglucanase-mediated hydrolysis,⁷⁰ mechanochemical activation, and phosphotungstic acid hydrolysis,⁷¹ ammonium persulfate (APS) oxidation,⁷² and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-mediated oxidation followed by cavitation.⁷³ The latter two methods yield CNCs functionalized with a high concentration of surface carboxylate groups (ca. 1.12–1.74 mmol/g for TEMPO-mediated oxidation),⁷³ which not only promote good dispersibility but also offer alternative surface chemical modification pathways.²⁴,²⁵

Regardless of the method utilized to produce CNCs, it is clear that anionic surface charges provide the colloidal stability necessary to prevent a certain degree of aggregation in both water and polar aprotic solvents, such as N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).⁷⁴ The surface charges also affect the ease at which CNCs can be freeze-dried, and at the same time, they use up some of the surface –OH groups that could be used for later functionalization. In addition, the counterions of the surface charges strongly influence the redispersibility of dried CNCs by ultrasonication, especially on material with a moisture content lower than 4 wt % due to the presence of stronger intermolecular hydrogen bonds between CNCs.⁷⁵,⁷⁶ Thus, using hydrolysis protocols that impart “just the right amount” of surface charges to promote colloidal stability appears advisable if subsequent surface modification reactions are to be carried out.

### 2.3. Pretreatments of CNCs for Surface Chemical Modification.
The presence of surface impurities, bound water, and excess counterions can have a significant impact on the success of surface functionalization reactions and their reproducibility, notably between different types or batches of CNCs.⁷⁷⁻⁷⁹ Furthermore, many reaction pathways suitable for the modification of –OH groups are intolerant to water, so that CNCs must either be dried and redispersed or transferred into a dry organic solvent by other means. It is possible to freeze-dry the CNCs⁸⁰⁻⁸⁵ or to exchange the solvent.³⁰,⁸³,⁸⁶⁻⁸⁹

If freeze-dried CNCs are to be surface-modified, the nanocrystals are often washed with organic solvents such as ethanol,⁷⁸,⁷⁹ acetone,⁷⁷ or n-pentane⁷⁷ to remove impurities. This is accomplished by either Soxhlet extraction⁷⁸,⁷⁹ or by several cycles of dispersing the CNCs in the solvent of choice and subsequent separation via centrifugation.⁷⁷ It was found that such pretreatments improved the reproducibility between grafting experiments.⁷⁷

Solvent exchanges usually involve mixing an aqueous dispersion of the CNCs with a water-miscible organic solvent, usually acetone, centrifugation, replacement of the solvent, and multiple repetitions of this cycle, until the solvent is changed to the organic solvent, in which surface-initiated polymerization is often performed.³⁰,⁸⁷,⁸⁸,⁹⁰,⁹¹ For example, Tian et al. compared the SI-ROP of ε-caprolactone (ε-CL) from freeze-dried CNCs and from solvent-exchanged CNCs and observed that the grafting density is higher from the solvent-exchanged nanocrystals than from those that were freeze-dried.⁸⁶ From these findings, it is clear that impurities adsorbed on the surface of CNCs are generally better removed by treatment with organic solvents. These impurities are likely byproducts formed by the hydrolysis of residual hemicellulose or lignin or perhaps the acid-catalyzed dehydration of mono- and oligosaccharides.²,⁷

## 3. SURFACE-INITIATED POLYMERIZATION FROM CNCS

Much of the research efforts directed toward the surface modification of CNCs have focused on tuning the physicochemical characteristics of their surface, while at the same time preserving their morphological integrity and crystallinity.²⁴,²⁵,⁹² Indeed, if the functionalization chemistry is chosen to selectively modify the surface or the reducing end groups, the intrinsic crystallinity and crystal structure of the CNCs should be retained, which can be checked by X-ray diffraction studies.²⁴,³⁰,⁸²,⁸³,⁸⁶,⁹³⁻⁹⁶ In the present section, the methods most often utilized to modify CNCs via the grafting from approach, i.e., surface-initiated and reducing end-initiated polymerization, will be presented.

### 3.1. Surface-Initiated Ring-Opening Polymerization.
Ring-opening polymerization (ROP) is a common technique to produce polymers from cyclic monomers such as lactones,⁹⁷,⁹⁸ dilactones,⁹⁹ lactams,¹⁰⁰ cyclic carbonates,¹⁰¹ cyclic ethers,¹⁰² and oxazolines¹⁰² (Figure 4). For some of these monomers, e.g.,

<figure><img src="image_11.png" /><figcaption>Figure 4. Examples of common monomers used for ROP.</figcaption></figure>

lactones and dilactones, the ROP can be initiated by –OH groups. Therefore, they are ideal candidates for the surface modification of not only CNCs, but also cellulose fibers and nanofibrils, as they also contain a high density of –OH surface groups. In this case, the cellulosic substrate acts as a macroinitiator and the polymerization process is most often referred to as SI-ROP.

CNC-g-polyesters. CNC-g-polyesters have been prepared by the SI-ROP of ε-caprolactone (ε-CL),³⁰,⁸⁰⁻⁸³,⁸⁶,¹⁰³⁻¹⁰⁵ L-lactide (L-LA),⁸⁴,⁸⁵,⁸⁷,⁸⁸,⁹⁰,⁹¹,¹⁰⁴,¹⁰⁶⁻¹⁰⁸ and D-lactide (D-LA)⁸⁹,¹⁰⁵,¹⁰⁹,¹¹⁰ from CNCs. The mechanism of the ROP of lactones and dilactones varies depending on the system used, i.e. the nature of the catalyst and the initiator.¹¹¹ The mechanism can be an anionic, a cationic, a monomer-activated or a coordination-insertion polymerization. In the case of SI-ROP from CNCs, the nanocrystals act as the initiator and the mechanism is only dependent on the catalyst. This reaction is water sensitive as water can also initiate the ROP of lactones. Therefore, the water, in which the nanocrystals are normally dispersed, needs to be removed as much as possible by means described in section 2.3.

Tin(II) 2-ethylhexanoate (Sn(Oct)₂)³⁰,⁷⁷,⁸⁰⁻⁸⁸,⁹⁰,⁹¹,¹⁰³,¹⁰⁵,¹⁰⁶,¹⁰⁸⁻¹¹⁰,¹¹² is the most commonly used catalyst for the SI-ROP of (di)lactones. The reaction is usually performed in toluene, however it has also been carried out in ionic liquids (1-allyl-3-methylimidazolium chloride, [AMIM]Cl¹¹³), in DMSO,¹⁰⁸ or in bulk.⁸⁴ Typical conditions further are temperatures between 80 and 130 °C and a reaction time of typically 24 h. The grafted polymers have also been prepared under microwave irradiation.⁸¹,¹¹² Benzyl alcohol is sometimes used as a sacrificial initiator to produce free polymer in solution, which is easier to characterize than the grafts.⁷⁷,¹⁰⁸ Of course, the comparison with free polymer that is produced “in parallel” is only sensible if one can safely assume that the kinetics of solution ROP are similar to SI-ROP from CNCs, which may or may not be the case.

In general, the nature of SI-ROP from CNCs makes the accurate characterization of grafted polymer molecular weight
<figure><img src="image_9.png" /><figcaption>Scheme 1. Mechanism of the SI-ROP of ε-CL from CNCs Using Sn(Oct)₂ as a Catalystᵃ</figcaption></figure>

ᵃKey: (A) Conversion of Sn(Oct)₂ into tin alkoxide. (B) Grafting of ε-CL from the surface of CNCs via coordination of tin alkoxide with the monomer.

and dispersity challenging, as the polymer must be degrafted before full molecular characterization is possible. Most degrafting schemes rely on hydrolytic cleavage of interfacial ester bonds, which, however, are also present in some of the polymer chains discussed here. This problem can potentially be addressed through the use of cleavable anchoring groups, such as those containing disulfides¹¹⁴ or UV-sensitive moieties,¹¹⁵ similar to the approached employed in the case of polymer-modified cellulosic substrates synthesized via surface-initiated controlled radical polymerization (SI-CRP).²⁸ Perhaps one could also take advantage of the high bond tension of the esters at the polymer-CNC interface, which are likely more sensitive to hydrolysis than esters along the polymer chain such that they could be cleaved by mechanochemical means, e.g., by ultrasound.¹¹⁶,¹¹⁷

Some researchers have attempted to estimate the length of the polymer grafts based on the melting temperature determined by differential scanning calorimetry (DSC) using a correlation curve,⁸⁹,¹⁰⁶,¹¹⁰ although this method does not account for potential differences in grafting density or effects of surface-confinement. Wu et al. proposed the use of FTIR data for poly(lactic acid) (PLA) brush quantification.⁸⁹ The approach relied on measuring FTIR spectra of physical mixtures of CNCs and PLA and creating a calibration curve based on the relative intensities of a characteristic band of PLA (i.e., 1454 cm⁻¹, corresponding to the CH₃ asymmetric stretching) and a characteristic band of the CNCs (i.e., 1428 cm⁻¹, corresponding to the CH₂ symmetric bending). The calibration curve was then employed to analyze CNC-g-PLA samples. While this method does not provide information on the molecular weight or grafting density of grafted polyesters, it permits to quantify the composition of PLA in CNC-g-PLA to be 25.3 wt % and estimate the molecular weight of the grafts to be 3.4 kDa.

Although the analysis of grafted CNCs in the liquid phase is usually impractical due to poor dispersibility in typical

solvents,¹⁰⁶ some researchers managed to make stable colloidal dispersion of surface-modified CNCs in CDCl₃¹⁰⁵ or DMSO-d₆¹⁰⁷,¹¹⁸ (mixed with small amount of chloroform) in order to perform ¹H and ¹³C NMR in well-stabilized dispersions. In this sense, the synthesis of CNC-g-PLA was evidenced by the appearance of resonance signals at 1.50–1.75 ppm (methyl protons),¹⁰⁵,¹⁰⁷ 5.00–5.50 ppm (methine proton),¹⁰⁵,¹⁰⁷ and 1.35 ppm (terminal methyl protons)¹⁰⁷ corresponding to PLA brushes. In the latter case, de Paula et al.¹⁰⁷ dispersed CNC-g-PLA in DMSO-d₆ with the addition of some chloroform and were able to directly calculate the degree of polymerization (DP) of PLA (=12) from the ¹H NMR spectra. This particular study highlights the limited DP one can achieve with SI-ROP from CNCs. In addition, the grafting of poly(ε-caprolactone) (PCL) from the surface of CNCs was confirmed in another study by the appearance of signals at 1.50–1.65 ppm, 2.23–2.32 ppm, and 4.07–4.11 ppm (methylene protons),¹⁰⁵ however, the DP of the polymer was not reported. It is important to note that the NMR results obtained from well-dispersed CNC-g-polymers may only provide partial information, which, unless appropriate separation protocols are used, is mainly related to free polymer molecules, while the polymer chains grafted from the dispersed contribute broader signals. Thus, the quantitative analysis of such NMR data is not always straightforward.

Regarding the mechanism of the SI-ROP catalyzed by Sn(Oct)₂, it is widely accepted that the reaction proceeds by a coordination-insertion mechanism. The first step is the conversion of the catalyst into a tin alkoxide to produce the active species, followed by the coordination of the monomer to the alkoxide through its insertion into the metal-oxygen bond (Scheme 1).¹¹¹

Other metal-based catalysts that have been tested for SI-ROP of cyclic esters from CNCs include magnesium hydride (MgH₂)¹⁰⁷ and zinc oxide.¹⁰⁸ Zinc oxide seems to give similar
<figure><img src="image_12.png" /><figcaption>Scheme 1. Mechanism of the SI-ROP of ε-CL from CNCs Using Sn(Oct)₂ as a Catalystᵃ</figcaption></figure>

ᵃKey: (A) Conversion of Sn(Oct)₂ into tin alkoxide. (B) Grafting of ε-CL from the surface of CNCs via coordination of tin alkoxide with the monomer.

and dispersity challenging, as the polymer must be degrafted before full molecular characterization is possible. Most degrafting schemes rely on hydrolytic cleavage of interfacial ester bonds, which, however, are also present in some of the polymer chains discussed here. This problem can potentially be addressed through the use of cleavable anchoring groups, such as those containing disulfides¹¹⁴ or UV-sensitive moieties,¹¹⁵ similar to the approached employed in the case of polymer-modified cellulosic substrates synthesized via surface-initiated controlled radical polymerization (SI-CRP).²⁸ Perhaps one could also take advantage of the high bond tension of the esters at the polymer-CNC interface, which are likely more sensitive to hydrolysis than esters along the polymer chain such that they could be cleaved by mechanochemical means, e.g., by ultrasound.¹¹⁶,¹¹⁷

Some researchers have attempted to estimate the length of the polymer grafts based on the melting temperature determined by differential scanning calorimetry (DSC) using a correlation curve,⁸⁹,¹⁰⁶,¹¹⁰ although this method does not account for potential differences in grafting density or effects of surface-confinement. Wu et al. proposed the use of FTIR data for poly(lactic acid) (PLA) brush quantification.⁸⁹ The approach relied on measuring FTIR spectra of physical mixtures of CNCs and PLA and creating a calibration curve based on the relative intensities of a characteristic band of PLA (i.e., 1454 cm⁻¹, corresponding to the CH₃ asymmetric stretching) and a characteristic band of the CNCs (i.e., 1428 cm⁻¹, corresponding to the CH₂ symmetric bending). The calibration curve was then employed to analyze CNC-g-PLA samples. While this method does not provide information on the molecular weight or grafting density of grafted polyesters, it permits to quantify the composition of PLA in CNC-g-PLA to be 25.3 wt % and estimate the molecular weight of the grafts to be 3.4 kDa.

Although the analysis of grafted CNCs in the liquid phase is usually impractical due to poor dispersibility in typical

solvents,¹⁰⁶ some researchers managed to make stable colloidal dispersion of surface-modified CNCs in CDCl₃¹⁰⁵ or DMSO-d₆¹⁰⁷,¹¹⁸ (mixed with small amount of chloroform) in order to perform ¹H and ¹³C NMR in well-stabilized dispersions. In this sense, the synthesis of CNC-g-PLA was evidenced by the appearance of resonance signals at 1.50–1.75 ppm (methyl protons),¹⁰⁵,¹⁰⁷ 5.00–5.50 ppm (methine proton),¹⁰⁵,¹⁰⁷ and 1.35 ppm (terminal methyl protons)¹⁰⁷ corresponding to PLA brushes. In the latter case, de Paula et al.¹⁰⁷ dispersed CNC-g-PLA in DMSO-d₆ with the addition of some chloroform and were able to directly calculate the degree of polymerization (DP) of PLA (=12) from the ¹H NMR spectra. This particular study highlights the limited DP one can achieve with SI-ROP from CNCs. In addition, the grafting of poly(ε-caprolactone) (PCL) from the surface of CNCs was confirmed in another study by the appearance of signals at 1.50–1.65 ppm, 2.23–2.32 ppm, and 4.07–4.11 ppm (methylene protons),¹⁰⁵ however, the DP of the polymer was not reported. It is important to note that the NMR results obtained from well-dispersed CNC-g-polymers may only provide partial information, which, unless appropriate separation protocols are used, is mainly related to free polymer molecules, while the polymer chains grafted from the dispersed contribute broader signals. Thus, the quantitative analysis of such NMR data is not always straightforward.

Regarding the mechanism of the SI-ROP catalyzed by Sn(Oct)₂, it is widely accepted that the reaction proceeds by a coordination-insertion mechanism. The first step is the conversion of the catalyst into a tin alkoxide to produce the active species, followed by the coordination of the monomer to the alkoxide through its insertion into the metal-oxygen bond (Scheme 1).¹¹¹

Other metal-based catalysts that have been tested for SI-ROP of cyclic esters from CNCs include magnesium hydride (MgH₂)¹⁰⁷ and zinc oxide.¹⁰⁸ Zinc oxide seems to give similar
<figure><img src="image_10.png" /><figcaption>Scheme 3. SI-ROP of 2-Ethyl-2-oxazoline from Tosylated CNCs</figcaption></figure>

<figure><img src="image_11.png" /></figure>

results in terms of monomer conversion and grafting yield as Sn(Oct)₂.¹⁰⁸ Organic catalysts such as 4-dimethylaminopyridine (DMAP),⁸³,¹¹³ citric acid,⁷⁸ and tartaric acid⁸³ have also been used for the SI-ROP of lactones from CNCs.

In the case of organic acids (citric acid and tartaric acid), the polymerization occurs through a monomer-activated mechanism,¹¹⁹,¹²⁰ in which the monomer is activated by a proton donated by the carboxylic acid. An -OH group of the cellulose, acting as a nucleophile, then reacts with this proton-activated monomer, leading to the formation of a hydroxyl-terminated ester via the ring-opening of the monomer. The propagation occurs as this newly formed hydroxyl-terminated species, acting as a new nucleophile, reacts with a new proton-activated monomer (Scheme 2). A few drops of HCl are sometimes added to the reaction medium at the end of the procedure to stop the reaction.³⁰,⁸⁰,⁸²,⁸⁸⁻⁹⁰ The grafted nanocrystals can be recovered by precipitation (in heptane,⁸⁰,⁸² deionized water,¹¹³ or methanol⁸¹,⁸⁹,⁹⁰,¹⁰⁵,¹⁰⁶,¹⁰⁹,¹¹²) and purified by Soxhlet extraction in toluene,⁸⁰,⁸² dichloromethane,⁸⁰,⁸³,⁸⁶ and/or tetrahydrofuran (THF)⁷⁷ to remove residual monomer, catalyst, and free homopolymer, in cases where a sacrificial initiator is used or impurities are present that initiate polymerization in solution.

Alternatively, they can be recovered by successive dispersion in methanol, ethanol, and acetone, each step followed by sedimentation via centrifugation.⁸⁷ Bitinis et al. noted that this second procedure allows the removal of the unwanted coproducts and unreacted reagents while losing fewer grafted nanocrystals in comparison to the purification performed via Soxhlet extractions.⁸⁷ However, when applying the same procedure with

toluene, Habibi et al. could not recover the grafted nanocrystals via centrifugation as they remained highly stable in the supernatant.³⁰ This is likely due to the higher grafting yield obtained by Habibi et al.,³⁰ which was on the order of 85% in comparison to the 70% yield obtained by Bitinis et al.;⁸⁷ however, the molecular weight and grafting density of the tethered polymers was unknown.

**CNC-g-polyoxazolines.** The grafting of oxazolines via SI-ROP follows a cationic mechanism, and thus cannot be initiated from the -OH groups at the surface of CNCs; indeed a modification of the CNC surface through tosylation substitutes the nucleophilic -OH groups with electrophilic tosyl moieties.¹²¹ In this case, tosylated CNCs act as the macroinitiator for the SI-ROP of 2-ethyl-2-oxazoline. It was shown that after tosylation, the modified CNCs can simply be mixed with the monomer and the SI-ROP readily takes place under microwave irradiation (completed in *ca.* 13 min). The reaction can be stopped by the addition of cold water and is subsequently washed with methanol (Scheme 3).

### 3.2. Surface-Initiated Free Radical Polymerization.
One of the most commonly used approaches for the grafting of vinyl polymers from the surface of CNCs is surface-initiated free radical polymerization (SI-FRP). The polymerization starts by the activation of the initiator species, which will, often via hydrogen abstraction, generate a reactive radical on the surface of CNCs, from which monomers add to a propagating chain.¹²² This method allows the facile growth of polymers at the CNC surface, along with free chains in solution, with relatively high molecular weight, even at low monomer conversions.
<figure><img src="image_13.png" /><figcaption>Scheme 3. SI-ROP of 2-Ethyl-2-oxazoline from Tosylated CNCs</figcaption></figure>

<figure><img src="image_14.png" /></figure>

results in terms of monomer conversion and grafting yield as Sn(Oct)₂.¹⁰⁸ Organic catalysts such as 4-dimethylaminopyridine (DMAP),⁸³,¹¹³ citric acid,⁷⁸ and tartaric acid⁸³ have also been used for the SI-ROP of lactones from CNCs.

In the case of organic acids (citric acid and tartaric acid), the polymerization occurs through a monomer-activated mechanism,¹¹⁹,¹²⁰ in which the monomer is activated by a proton donated by the carboxylic acid. An -OH group of the cellulose, acting as a nucleophile, then reacts with this proton-activated monomer, leading to the formation of a hydroxyl-terminated ester via the ring-opening of the monomer. The propagation occurs as this newly formed hydroxyl-terminated species, acting as a new nucleophile, reacts with a new proton-activated monomer (Scheme 2). A few drops of HCl are sometimes added to the reaction medium at the end of the procedure to stop the reaction.³⁰,⁸⁰,⁸²,⁸⁸⁻⁹⁰ The grafted nanocrystals can be recovered by precipitation (in heptane,⁸⁰,⁸² deionized water,¹¹³ or methanol⁸¹,⁸⁹,⁹⁰,¹⁰⁵,¹⁰⁶,¹⁰⁹,¹¹²) and purified by Soxhlet extraction in toluene,⁸⁰,⁸² dichloromethane,⁸⁰,⁸³,⁸⁶ and/or tetrahydrofuran (THF)⁷⁷ to remove residual monomer, catalyst, and free homopolymer, in cases where a sacrificial initiator is used or impurities are present that initiate polymerization in solution.

Alternatively, they can be recovered by successive dispersion in methanol, ethanol, and acetone, each step followed by sedimentation via centrifugation.⁸⁷ Bitinis et al. noted that this second procedure allows the removal of the unwanted coproducts and unreacted reagents while losing fewer grafted nanocrystals in comparison to the purification performed via Soxhlet extractions.⁸⁷ However, when applying the same procedure with

toluene, Habibi et al. could not recover the grafted nanocrystals via centrifugation as they remained highly stable in the supernatant.³⁰ This is likely due to the higher grafting yield obtained by Habibi et al.,³⁰ which was on the order of 85% in comparison to the 70% yield obtained by Bitinis et al.;⁸⁷ however, the molecular weight and grafting density of the tethered polymers was unknown.

**CNC-g-polyoxazolines.** The grafting of oxazolines via SI-ROP follows a cationic mechanism, and thus cannot be initiated from the -OH groups at the surface of CNCs; indeed a modification of the CNC surface through tosylation substitutes the nucleophilic -OH groups with electrophilic tosyl moieties.¹²¹ In this case, tosylated CNCs act as the macroinitiator for the SI-ROP of 2-ethyl-2-oxazoline. It was shown that after tosylation, the modified CNCs can simply be mixed with the monomer and the SI-ROP readily takes place under microwave irradiation (completed in *ca.* 13 min). The reaction can be stopped by the addition of cold water and is subsequently washed with methanol (Scheme 3).

## 3.2. Surface-Initiated Free Radical Polymerization.
One of the most commonly used approaches for the grafting of vinyl polymers from the surface of CNCs is surface-initiated free radical polymerization (SI-FRP). The polymerization starts by the activation of the initiator species, which will, often via hydrogen abstraction, generate a reactive radical on the surface of CNCs, from which monomers add to a propagating chain.¹²² This method allows the facile growth of polymers at the CNC surface, along with free chains in solution, with relatively high molecular weight, even at low monomer conversions.
<figure><img src="image_12.png" /><figcaption>Scheme 4. Synthesis of CNCs Grafted with PAA via SI-FRP Using KPS as Radical Initiator</figcaption></figure>

A wide variety of radical initiators have been demonstrated to be effective for the surface-initiated polymerization of vinyl monomers. Among the thermal initiators, potassium persulfate (KPS) is widely employed to decorate CNC surfaces with polymer chains, due to the stability of free radicals and compatibility with aqueous solutions. The radical formation starts in the aqueous phase by the thermal homolytic dissociation of the KPS peroxide bond, at temperatures ranging between 60 and 70 °C. The free sulfate radicals thus produced are capable of abstracting hydrogen atoms from the surface of the CNCs and thus generate the surface-bound initiating species (Scheme 4). Using this initiator, 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido)ethyl methacrylate (UPyMA) was successfully polymerized from the surface of CNCs, affording CNC-g-PUPyMA containing 6.9 wt % of polymer.¹²³ The presence of UPyMA units contributed significantly to the emulsion stabilizing ability of the nanoparticles (NPs) in comparison with pristine CNCs, which made them suitable to prepare high internal phase emulsions and macroporous hydrogels. Another example was reported by Wu et al., who used acrylic acid (AA) as the monomer.¹²⁴ The use of grafted poly(acrylic acid) (PAA) chains offered CNC-based hydrogels excellent water retention properties with a swelling ratio of 323 g/g in distilled water versus 33 g/g in sodium chloride (NaCl) solution.

A particular case of SI-FRP was described by Anirudhan et al., who reported the grafting of poly(methacrylic acid-co-vinylsulfonic acid) (P(MAA-co-VSA)) from the surface of magnetized CNCs.¹²⁵ First, magnetic CNCs were prepared by the chemical precipitation of magnetite (Fe₃O₄) onto the surface of CNCs. Then, the SI-FRP was carried out in the presence of KPS as initiator, the corresponding monomers and 2-(2-methylacryloyloxy)ethyl 2-methylacrylate (EGDMA) as cross-linker in water to form a three-dimensional network, which ultimately had a surface area that was five times larger than that of the parent CNCs.

Ammonium persulfate (APS) is another thermal initiator that has been used for the grafting from the surface of CNCs, in water, via SI-FRP; in this case, poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) was used as the monomer.¹²⁶ The final product contained 11.7 wt % of polymer chains, which imparted stimuli-responsive properties to the CNCs.

In addition to the radical formation via thermal homolytic decomposition of the peroxide bond, persulfate initiators can also effectively generate free radicals in the presence of a reducing agent, such as oxyacids of sulfur (e.g., sulfite, bisulfite, thiosulfate, metabisulfite and dithionate), organic acids

(e.g., ascorbic acid), metal ions (e.g., Fe²⁺ or Ag⁺) or amines (e.g., TMEDA: N,N,N',N'-tetramethylethane-1,2-diamine). This works by electron transfer from atoms or ions containing species that feature unpaired electrons and subsequent bond dissociation in the acceptor (persulfate) molecules.¹²⁷ These systems are referred to as redox initiators, redox catalysts or redox activators. They are often used at or below room temperature and are commonly employed in emulsion and aqueous polymerizations. This pathway has also been utilized for the formation of active radicals on the surface of CNCs by Zhou et al., where KPS and sodium bisulfite (SBS) were used as a redox initiator system to directly graft polyacrylamide (PAM) from the surface of CNCs dispersed in water.¹²⁸ The initiation reaction of this system is shown in Scheme 5.

<caption>Scheme 5. Radical Formation of the KPS/SBS Redox Initiator System</caption>

$$S_2O_8^{2-} + HSO_3^- \rightarrow SO_4^{2-} + SO_4^{2-} + HSO_3^- \quad (1)$$

$$SO_4^{2-} + H_2O \rightarrow OH^- + HSO_4^- \quad (2)$$

In this case, both radicals, formed through reaction 1 (Scheme 5), could activate CNCs via hydrogen abstraction at -OH groups on the surface. Upon addition of acrylamide (AM) monomer and N,N'-methylenebis(acrylamide) (NMBA) as a cross-linker, these surface radicals acted as the initiating sites for grafted PAM chains. Simultaneously, free radicals were also formed in solution promoting the polymerization of AM, which were also cross-linked by NMBA. Finally, a nanocomposite hydrogel based on CNC-g-PAM and PAM network was formed and showed well-dispersed modified-CNCs and enhanced interfacial adhesion between the two components.

The main advantage of using this type of initiator system is that the polymerization can be performed under mild conditions, as no heat is needed to promote the radical formation, which decreases the possibility of undesired side reactions while still obtaining high molecular weight polymers with a high yield. APS and TMEDA have likewise been used as a redox couple for the SI-FRP of N-isopropylacrylamide (NIPAM) from CNCs in order to form thermoresponsive hydrogels.¹²⁹ The initiation mechanism proceeds via a contact charge transfer complex and a cyclic transition state, from which two primary radicals are formed, one from the amine and another from persulfate, which both in turn initiate the polymerization process (Scheme 6).¹³⁰
<figure><img src="image_15.png" /><figcaption>Scheme 4. Synthesis of CNCs Grafted with PAA via SI-FRP Using KPS as Radical Initiator</figcaption></figure>

A wide variety of radical initiators have been demonstrated to be effective for the surface-initiated polymerization of vinyl monomers. Among the thermal initiators, potassium persulfate (KPS) is widely employed to decorate CNC surfaces with polymer chains, due to the stability of free radicals and compatibility with aqueous solutions. The radical formation starts in the aqueous phase by the thermal homolytic dissociation of the KPS peroxide bond, at temperatures ranging between 60 and 70 °C. The free sulfate radicals thus produced are capable of abstracting hydrogen atoms from the surface of the CNCs and thus generate the surface-bound initiating species (Scheme 4). Using this initiator, 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido)ethyl methacrylate (UPyMA) was successfully polymerized from the surface of CNCs, affording CNC-g-PUPyMA containing 6.9 wt % of polymer.¹²³ The presence of UPyMA units contributed significantly to the emulsion stabilizing ability of the nanoparticles (NPs) in comparison with pristine CNCs, which made them suitable to prepare high internal phase emulsions and macroporous hydrogels. Another example was reported by Wu et al., who used acrylic acid (AA) as the monomer.¹²⁴ The use of grafted poly(acrylic acid) (PAA) chains offered CNC-based hydrogels excellent water retention properties with a swelling ratio of 323 g/g in distilled water versus 33 g/g in sodium chloride (NaCl) solution.

A particular case of SI-FRP was described by Anirudhan et al., who reported the grafting of poly(methacrylic acid-co-vinylsulfonic acid) (P(MAA-co-VSA)) from the surface of magnetized CNCs.¹²⁵ First, magnetic CNCs were prepared by the chemical precipitation of magnetite (Fe₃O₄) onto the surface of CNCs. Then, the SI-FRP was carried out in the presence of KPS as initiator, the corresponding monomers and 2-(2-methylacryloyloxy)ethyl 2-methylacrylate (EGDMA) as cross-linker in water to form a three-dimensional network, which ultimately had a surface area that was five times larger than that of the parent CNCs.

Ammonium persulfate (APS) is another thermal initiator that has been used for the grafting from the surface of CNCs, in water, via SI-FRP; in this case, poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) was used as the monomer.¹²⁶ The final product contained 11.7 wt % of polymer chains, which imparted stimuli-responsive properties to the CNCs.

In addition to the radical formation via thermal homolytic decomposition of the peroxide bond, persulfate initiators can also effectively generate free radicals in the presence of a reducing agent, such as oxyacids of sulfur (e.g., sulfite, bisulfite, thiosulfate, metabisulfite and dithionate), organic acids

(e.g., ascorbic acid), metal ions (e.g., Fe²⁺ or Ag⁺) or amines (e.g., TMEDA: N,N,N',N'-tetramethylethane-1,2-diamine). This works by electron transfer from atoms or ions containing species that feature unpaired electrons and subsequent bond dissociation in the acceptor (persulfate) molecules.¹²⁷ These systems are referred to as redox initiators, redox catalysts or redox activators. They are often used at or below room temperature and are commonly employed in emulsion and aqueous polymerizations. This pathway has also been utilized for the formation of active radicals on the surface of CNCs by Zhou et al., where KPS and sodium bisulfite (SBS) were used as a redox initiator system to directly graft polyacrylamide (PAM) from the surface of CNCs dispersed in water.¹²⁸ The initiation reaction of this system is shown in Scheme 5.

<caption>Scheme 5. Radical Formation of the KPS/SBS Redox Initiator System</caption>

$$S_2O_8^{2-} + HSO_3^- \rightarrow SO_4^{2-} + SO_4^{2-} + HSO_3^- \quad (1)$$

$$SO_4^{2-} + H_2O \rightarrow OH^- + HSO_4^- \quad (2)$$

In this case, both radicals, formed through reaction 1 (Scheme 5), could activate CNCs via hydrogen abstraction at -OH groups on the surface. Upon addition of acrylamide (AM) monomer and N,N'-methylenebis(acrylamide) (NMBA) as a cross-linker, these surface radicals acted as the initiating sites for grafted PAM chains. Simultaneously, free radicals were also formed in solution promoting the polymerization of AM, which were also cross-linked by NMBA. Finally, a nanocomposite hydrogel based on CNC-g-PAM and PAM network was formed and showed well-dispersed modified-CNCs and enhanced interfacial adhesion between the two components.

The main advantage of using this type of initiator system is that the polymerization can be performed under mild conditions, as no heat is needed to promote the radical formation, which decreases the possibility of undesired side reactions while still obtaining high molecular weight polymers with a high yield. APS and TMEDA have likewise been used as a redox couple for the SI-FRP of N-isopropylacrylamide (NIPAM) from CNCs in order to form thermoresponsive hydrogels.¹²⁹ The initiation mechanism proceeds via a contact charge transfer complex and a cyclic transition state, from which two primary radicals are formed, one from the amine and another from persulfate, which both in turn initiate the polymerization process (Scheme 6).¹³⁰
<figure><img src="image_13.png" /><figcaption>Scheme 6. Radical Formation of the APS/TMEDA Redox Initiator Couple<sup>a</sup></figcaption></figure>

<figure><img src="image_14.png" /><figcaption>Scheme 7. Mechanism of Ceric Ion Reduction, Leading to the Formation of CNC-Based Radicals<sup>a</sup></figcaption></figure>

Another technique, based on ceric-ion initiated polymerization and performed in aqueous solutions of nitric acid to modify the surface of CNCs, was first reported by Kan et al.<sup>132</sup> In this approach, ceric ions (Ce<sup>4+</sup>) chelate with two adjacent -OH groups on the AGUs, possibly those at C2 and C3 positions. This step is followed by an electron transfer from the AGU to Ce<sup>4+</sup>, leading to its reduction into Ce<sup>3+</sup>, opening of the ring at C2 and C3 positions, and the formation of a radical species (Scheme 7). The ability to graft poly(4-vinylpyridine) (P4VP) from the surface of CNCs proved the effectivity of this chemistry on the surface of highly crystalline cellulose. Using similar chemistry, poly(glycidyl methacrylate) (PGMA)<sup>133</sup> and poly(methyl methacrylate) (PMMA)<sup>134</sup> have also been successfully grafted from the surface of CNCs.

In addition, Tang et al. carried out the synthesis of binary polymer brushes consisting of poly(oligoethylene glycol) methacrylate (POEGMA) and poly(methacrylic acid) (PMAA) from the surface of CNCs.<sup>135</sup> These brushes offered new properties to the final material that were effectively controlled by external stimuli such as pH and temperature. Initially, the OEGMA polymerization was performed via cerium-mediated reactions, followed by the subsequent sulfate-initiated MAA polymerization. Combining these two initiator systems allowed the authors to polymerize two vinyl monomers, with different intrinsic properties, from the surface of CNCs while assuring the accessibility to binding sites. Thus, cerium-initiated polymerization generated radicals at AGUs by ring-opening at the C2 and C3 positions and transforming their secondary alcohol, but without affecting their primary alcohols, which left the latter available for further radical-initiated polymerization through hydrogen abstraction. Indeed, it is believed that the ring opening of the AGUs at the C2-C3 position and the generation

of aldehyde groups yields a material, which undergoes thermal degradation at a lower temperature, thus reduced thermal stability can be observed compared to pristine CNCs. This was demonstrated for CNC-g-POEGMA or CNC-g-P(OEGMA-ran-MAA) ($T_d$ = 180 °C)<sup>135</sup> and CNC-g-PMMA ($T_d$ = 250 °C).<sup>134</sup>

In general, the modification of CNCs via SI-FRP comes with the formation of a substantial amount of free (unattached) homopolymers in solution, which represents a loss of monomer and a reduction in grafting efficiency, which thus is the main drawback of this method. In addition, the successful removal of such species is in some cases tedious and challenging, depending on the reactants and the final product. The purification of hydrogels, for example, request the immersion of the material into an excess of water for a period between 48 and 72 h in order to remove any water-soluble fraction. It is usually followed by a drying process (50-70 °C) in vacuum for 48 h.<sup>128,137</sup> For the removal of metals, i.e., particles released from sonication probe, the common reported procedure implies the use of glass microfiber filter paper followed by extensive dialysis and ultrafiltration.<sup>132,134</sup> If the extraction of CNC-g-polymer from the reaction media is required, it generally proceeds via subsequent centrifugation and washing steps with one or more solvents (according to the affinity of interested species), which could be followed by dialysis and drying processes.<sup>94,135</sup>

Nevertheless, inorganic peroxides, such as KPS and APS, have shown their versatility as initiator species in SI-FRP based on the large variety of polymers that have successfully been grafted from the surface of CNCs. The main advantage of using this type of initiator system is that the grafting can be performed directly from the surface of pristine CNCs, i.e., without previous modification. For cases where the polymerization should be performed at ambient or lower temperatures, a

<footnote><sup>a</sup>Scheme adapted from ref 131.</footnote>

<footnote><sup>a</sup>Scheme adapted from ref 136.</footnote>
<figure><img src="image_16.png" /><figcaption>Scheme 6. Radical Formation of the APS/TMEDA Redox Initiator Couple<sup>a</sup></figcaption></figure>

<figure><img src="image_17.png" /><figcaption>Scheme 7. Mechanism of Ceric Ion Reduction, Leading to the Formation of CNC-Based Radicals<sup>a</sup></figcaption></figure>

Another technique, based on ceric-ion initiated polymerization and performed in aqueous solutions of nitric acid to modify the surface of CNCs, was first reported by Kan et al.<sup>132</sup> In this approach, ceric ions (Ce<sup>4+</sup>) chelate with two adjacent -OH groups on the AGUs, possibly those at C2 and C3 positions. This step is followed by an electron transfer from the AGU to Ce<sup>4+</sup>, leading to its reduction into Ce<sup>3+</sup>, opening of the ring at C2 and C3 positions, and the formation of a radical species (Scheme 7). The ability to graft poly(4-vinylpyridine) (P4VP) from the surface of CNCs proved the effectivity of this chemistry on the surface of highly crystalline cellulose. Using similar chemistry, poly(glycidyl methacrylate) (PGMA)<sup>133</sup> and poly(methyl methacrylate) (PMMA)<sup>134</sup> have also been successfully grafted from the surface of CNCs.

In addition, Tang et al. carried out the synthesis of binary polymer brushes consisting of poly(oligoethylene glycol) methacrylate (POEGMA) and poly(methacrylic acid) (PMAA) from the surface of CNCs.<sup>135</sup> These brushes offered new properties to the final material that were effectively controlled by external stimuli such as pH and temperature. Initially, the OEGMA polymerization was performed via cerium-mediated reactions, followed by the subsequent sulfate-initiated MAA polymerization. Combining these two initiator systems allowed the authors to polymerize two vinyl monomers, with different intrinsic properties, from the surface of CNCs while assuring the accessibility to binding sites. Thus, cerium-initiated polymerization generated radicals at AGUs by ring-opening at the C2 and C3 positions and transforming their secondary alcohol, but without affecting their primary alcohols, which left the latter available for further radical-initiated polymerization through hydrogen abstraction. Indeed, it is believed that the ring opening of the AGUs at the C2-C3 position and the generation

of aldehyde groups yields a material, which undergoes thermal degradation at a lower temperature, thus reduced thermal stability can be observed compared to pristine CNCs. This was demonstrated for CNC-g-POEGMA or CNC-g-P(OEGMA-ran-MAA) ($T_d$ = 180 °C)<sup>135</sup> and CNC-g-PMMA ($T_d$ = 250 °C).<sup>134</sup>

In general, the modification of CNCs via SI-FRP comes with the formation of a substantial amount of free (unattached) homopolymers in solution, which represents a loss of monomer and a reduction in grafting efficiency, which thus is the main drawback of this method. In addition, the successful removal of such species is in some cases tedious and challenging, depending on the reactants and the final product. The purification of hydrogels, for example, request the immersion of the material into an excess of water for a period between 48 and 72 h in order to remove any water-soluble fraction. It is usually followed by a drying process (50-70 °C) in vacuum for 48 h.<sup>128,137</sup> For the removal of metals, i.e., particles released from sonication probe, the common reported procedure implies the use of glass microfiber filter paper followed by extensive dialysis and ultrafiltration.<sup>132,134</sup> If the extraction of CNC-g-polymer from the reaction media is required, it generally proceeds via subsequent centrifugation and washing steps with one or more solvents (according to the affinity of interested species), which could be followed by dialysis and drying processes.<sup>94,135</sup>

Nevertheless, inorganic peroxides, such as KPS and APS, have shown their versatility as initiator species in SI-FRP based on the large variety of polymers that have successfully been grafted from the surface of CNCs. The main advantage of using this type of initiator system is that the grafting can be performed directly from the surface of pristine CNCs, i.e., without previous modification. For cases where the polymerization should be performed at ambient or lower temperatures, a

<footnote><sup>a</sup>Scheme adapted from ref 131.</footnote>

<footnote><sup>a</sup>Scheme adapted from ref 136.</footnote>
<figure><img src="image_15.png" /><figcaption>Scheme 8. Overview of the Most Common SI-CRP Strategies Illustrated by SI-ATRP of Sty,¹⁴¹ S-RAFT of NIPAM,¹⁴² and SI-NMP of Methyl Acrylate (MA)¹⁴³ on the CNC Surface</figcaption></figure>

redox system involving any of these compounds can be used. In comparison to ceric ion SI-FRP, peroxy compounds have the advantage of not altering the structure of CNC surface chains, which usually results in nanomaterials with higher thermal stability, as previously discussed.

The SI-FRP methods presented above involved the formation of reactive species directly on the surface of CNCs, from which polymer chains were allowed to grow. Yang and co-workers reported a different approach, in which the CNC surface was chemically modified with reactive small molecules before polymerization.¹³⁷,¹³⁸ A chemical entity containing end vinyl groups were covalently linked to the surface of CNCs by treatment with γ-methacryloxypropyl silane. Subsequently, radicals species were formed at the end vinyl groups in the presence of KPS initiator, thus creating propagating radicals, from which AA¹³⁷ or AM¹³⁸ monomers were polymerized until termination reactions occurred by recombination, indeed promoting the formation of cross-linked networks. On the basis of the high monomer-to-surface initiator ratio¹³⁷,¹³⁸ employed and the final monomer conversion (ca. 99%),¹³⁷ the polymer grafted via this approach formed a dense shell around the CNC core due to either a high grafting density or a high molecular weight.

Espino-Perez et al. proposed the synthesis of CNC-g-poly-styrene via reaction steps consistent with principles of green chemistry.⁹⁴ The protocol involved the ozonolysis of CNCs leading to peroxide groups at their surface, and the subsequent radical polymerization in the presence of KPS and styrene (Sty) monomers within a sodium acetate buffer solution (pH = 3.5). The thermally activated sulfate radicals reduced the peroxide groups, creating free radicals, which propagated to form tethered polystyrene (PSty) chains. The Sty/AGU ratio was estimated to be 0.24, which confirmed the low grafting efficiency on CNCs through this method due to the low hydroperoxidation efficiency during the ozonolysis treatment.

Another strategy for decorating CNCs with grafted vinyl polymers was presented by Lee et al.¹³⁹ The SI-FRP took place on the surface of CNCs containing trichloroacetyl groups in the presence of molybdenumhexacarbonyl (Mo(CO)₆) as initiator and 2-methacryloyloxyethyl phosphorylcholine (MPC) and stearyl methacrylate (SMA) as monomers in ethanol to yield CNC-g-P(MPC-co-SMA), with over 90% of monomers conversion.

More recently, a new free radical strategy was presented by Wohlhauser et al. where the initiator species was covalently
<figure><img src="image_18.png" /><figcaption>Scheme 8. Overview of the Most Common SI-CRP Strategies Illustrated by SI-ATRP of Sty,¹⁴¹ S-RAFT of NIPAM,¹⁴² and SI-NMP of Methyl Acrylate (MA)¹⁴³ on the CNC Surface</figcaption></figure>

redox system involving any of these compounds can be used. In comparison to ceric ion SI-FRP, peroxy compounds have the advantage of not altering the structure of CNC surface chains, which usually results in nanomaterials with higher thermal stability, as previously discussed.

The SI-FRP methods presented above involved the formation of reactive species directly on the surface of CNCs, from which polymer chains were allowed to grow. Yang and co-workers reported a different approach, in which the CNC surface was chemically modified with reactive small molecules before polymerization.¹³⁷,¹³⁸ A chemical entity containing end vinyl groups were covalently linked to the surface of CNCs by treatment with γ-methacryloxypropyl silane. Subsequently, radicals species were formed at the end vinyl groups in the presence of KPS initiator, thus creating propagating radicals, from which AA¹³⁷ or AM¹³⁸ monomers were polymerized until termination reactions occurred by recombination, indeed promoting the formation of cross-linked networks. On the basis of the high monomer-to-surface initiator ratio¹³⁷,¹³⁸ employed and the final monomer conversion (ca. 99%),¹³⁷ the polymer grafted via this approach formed a dense shell around the CNC core due to either a high grafting density or a high molecular weight.

Espino-Perez et al. proposed the synthesis of CNC-g-poly-styrene via reaction steps consistent with principles of green chemistry.⁹⁴ The protocol involved the ozonolysis of CNCs leading to peroxide groups at their surface, and the subsequent radical polymerization in the presence of KPS and styrene (Sty) monomers within a sodium acetate buffer solution (pH = 3.5). The thermally activated sulfate radicals reduced the peroxide groups, creating free radicals, which propagated to form tethered polystyrene (PSty) chains. The Sty/AGU ratio was estimated to be 0.24, which confirmed the low grafting efficiency on CNCs through this method due to the low hydroperoxidation efficiency during the ozonolysis treatment.

Another strategy for decorating CNCs with grafted vinyl polymers was presented by Lee et al.¹³⁹ The SI-FRP took place on the surface of CNCs containing trichloroacetyl groups in the presence of molybdenumhexacarbonyl (Mo(CO)₆) as initiator and 2-methacryloyloxyethyl phosphorylcholine (MPC) and stearyl methacrylate (SMA) as monomers in ethanol to yield CNC-g-P(MPC-co-SMA), with over 90% of monomers conversion.

More recently, a new free radical strategy was presented by Wohlhauser et al. where the initiator species was covalently
attached to the surface of CNCs.⁹³ This approach guarantees the presence of a high number of reactive sites at the surface of the substrate, although it does not avoid the formation of radicals in solution. In this work, PMMA and poly(hexyl methacrylate) (PHMA) were successful grafted from CNCs through an undemanding protocol, which involved the surface modification of CNCs with benzophenone moieties as photoinitiators and subsequent surface-photoinitiated FRP of methacrylates in DMF under UV-irradiation. Benzophenone is a Norrish Type II photoinitiator that undergoes intersystem crossing from its excited singlet state to the lowest energy reactive triplet, followed by the abstraction of aliphatic hydrogen from a nearby molecule or interface, yielding a ketyl radical and a carbon centered radical.¹⁴⁰ The SI-FRP is thus initiated by the carbon centered radical and can also result in the formation of free (unattached) polymer chains (initiated from a monomer or solvent molecule). Using this method, compositions containing 80 and 90 wt % of grafted polymers were prepared for both monomers. This was achieved by using DMF as solvent and variation of the monomer conversion. In comparison to previous studies, the fraction of polymer brushes on the surface of CNCs was very high, but given the mechanism, a substantial amount of polymer (between 24 and 48 wt %, relative to the initial monomer amount) was also produced as free (unattached) polymer.

In general, the polymer grafted on the surface of CNCs via free radical polymerization from covalently immobilized reactive species depends on factors related to the propagating radical, such as surface initiator efficiency, propagation rate and the monomer-to-surface initiator ratio. Indeed, additional studies are needed in the field to better understand the influence of these parameters. Another opportunity that currently remains in this field is the investigation of SI-FRP via the attachment of other conventional radical initiators (i.e., azo-containing initiators) and subsequent comparisons with polymer brushes obtained through the above-mentioned techniques.

**3.3. Surface-Initiated Controlled Radical Polymerization.** SI-CRP is an effective way to grow polymers when a precise control over polymer architecture, composition, molecular weight, and graft density is required.²⁸ In general, CRP reactions rely on the reversible deactivation of propagating chains, such that termination reactions are minimized, thus providing first order polymerization kinetics and low dispersity. Different SI-CRP methods can be performed, which depend on the type of initiator or so-called chain transfer agent (CTA) coupled to the surface, such as Cu-mediated SI-CRP, surface reversible addition-fragmentation chain transfer (S-RAFT) or surface-initiated nitroxide-mediated polymerization (SI-NMP), among others. Scheme 8 exemplifies the three most commonly used SI-CRP techniques, i.e. surface-initiated atom transfer radical polymerization (SI-ATRP), S-RAFT, and SI-NMP. In the following sections, a brief overview of each of these techniques will be provided.

**3.3.1. Cu-Mediated SI-CRP.** Of all SI-CRP techniques, those mediated by transition metal complexes, especially Cu species, as in SI-ATRP, have been the most widely employed to graft polymers from CNCs. The control in ATRP arises from the existence of a dynamic equilibrium between dormant and propagating species, thus lowering radical concentration and minimizing termination reactions.¹⁴⁴ The reversible deactivation occurs through the exchange of a halogen atom (Br or Cl) between a metal complex (based on Cu in most cases) and the propagating species. As the presence of residual metal in the final polymers may be a problem for some applications,

techniques were then developed to decrease the required amount of catalytic system to ppm by (re)generating Cu(I) *in situ* activators by electron transfer (A(R)GET) ATRP.¹⁴⁵⁻¹⁴⁷ CRP in the presence of Cu(0) was also reported with a debate on the model of the electron transfer mechanism, i.e. single-electron transfer living radical polymerization (SET LRP) and supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP).¹⁴⁸⁻¹⁵⁰ As the present Perspective aims at reporting the different structures obtained from CNCs and their related properties rather than debating the mechanism, techniques involving Cu species are grouped under the generic term “Cu-mediated SI-CRP.”

Conducting a Cu-mediated SI-CRP from CNC requires the preliminary introduction of initiating sites on their surface. This is usually achieved by reacting the numerous –OH functionalities on the CNC surface with 2-bromoisobutyryl bromide (BiBB) in a solvent (THF or DMF), usually in the presence of a base (triethylamine) and a catalyst (DMAP).¹⁴¹,¹⁵¹⁻¹⁶¹ The extent of this modification can be tailored through the reaction duration and the amount of BiBB introduced.¹⁶²,¹⁶³ Through a combination of active initiators and inactive “dummy” initiators, the overall grafting density can be controlled.²⁸ Over the years, strategies were developed to improve the CNCs dispersibility during the modification by either performing (1) solvent exchanges (rather than freeze-drying), which permitted avoiding a redispersion step of dried CNCs,¹⁶³,¹⁶⁴ or (2) a chemical vapor deposition of BiBB on a CNC aerogel to partially hydrophobize the CNCs prior to a subsequent liquid phase reaction with BiBB.¹⁶⁵⁻¹⁶⁹ Higher modification degrees (up to 15 wt % Br determined by elemental analysis) were then reached with lower BiBB excess.¹⁶⁵ Although less widely used, the incorporation of the initiating sites was also reported by reacting the –OH functions with α-bromoisobutyric acid activated with carbonyl diimidazole.¹⁷⁰,¹⁷¹ In another study, the use of 2-bromopropionic acid was proposed for the introduction of active sites on the CNCs surface with a yield of 46% and 2.86 wt % of Br content determined by elemental analysis.⁹⁵ The protocol involve a pretreatment of pristine CNCs with HCl, followed by one-step reaction, which combines Fischer esterification and acid hydrolysis.

In all the previous studies, the –OH functions were statistically and homogeneously modified on the entire nano-crystal surface. Recently, Zoppe et al. reported an approach to introduce initiating sites specifically on the reducing end groups of CNCs through their oxidation and reaction with ethylenediamine and subsequently 2-bromoisobutanoic acid N-hydroxysuccinimide ester.¹⁷² This approach is discussed in further detail in section 3.4.

The CNC macroinitiators have been used in the Cu-mediated SI-CRP of various styrenic-, (meth)acrylic, or acrylamide-based monomers (Figure 5) with catalytic systems and solvents optimized for the various monomers employed (see Table S1). The polymerization control can be elucidated through the saponification of the grafted polymers (via either acidic or alkaline conditions) and further analysis of the degrafted polymer in solution; however the degrafting reactions require an extended reaction time (2 days to 3 weeks) and can potentially alter the polymer structure.¹⁴¹,¹⁵²⁻¹⁵⁴,¹⁶³,¹⁶⁴,¹⁷⁰,¹⁷² The versatility of Cu-mediated SI-CRP techniques and their high tolerance toward many functional groups has given access to CNCs grafted with a large variety of polymeric chains ranging from neutral PSty,¹¹⁵,¹⁴¹,¹⁵⁷,¹⁶² poly(alkyl ((meth)acrylate),¹³⁸,¹⁶⁹,¹⁷⁰ to cationic poly(amino(alkyl) methacrylate/methacrylamide)¹⁵⁵,¹⁷² or
attached to the surface of CNCs.⁹³ This approach guarantees the presence of a high number of reactive sites at the surface of the substrate, although it does not avoid the formation of radicals in solution. In this work, PMMA and poly(hexyl methacrylate) (PHMA) were successful grafted from CNCs through an undemanding protocol, which involved the surface modification of CNCs with benzophenone moieties as photoinitiators and subsequent surface-photoinitiated FRP of methacrylates in DMF under UV-irradiation. Benzophenone is a Norrish Type II photoinitiator that undergoes intersystem crossing from its excited singlet state to the lowest energy reactive triplet, followed by the abstraction of aliphatic hydrogen from a nearby molecule or interface, yielding a ketyl radical and a carbon centered radical.¹⁴⁰ The SI-FRP is thus initiated by the carbon centered radical and can also result in the formation of free (unattached) polymer chains (initiated from a monomer or solvent molecule). Using this method, compositions containing 80 and 90 wt % of grafted polymers were prepared for both monomers. This was achieved by using DMF as solvent and variation of the monomer conversion. In comparison to previous studies, the fraction of polymer brushes on the surface of CNCs was very high, but given the mechanism, a substantial amount of polymer (between 24 and 48 wt %, relative to the initial monomer amount) was also produced as free (unattached) polymer.

In general, the polymer grafted on the surface of CNCs via free radical polymerization from covalently immobilized reactive species depends on factors related to the propagating radical, such as surface initiator efficiency, propagation rate and the monomer-to-surface initiator ratio. Indeed, additional studies are needed in the field to better understand the influence of these parameters. Another opportunity that currently remains in this field is the investigation of SI-FRP via the attachment of other conventional radical initiators (i.e., azo-containing initiators) and subsequent comparisons with polymer brushes obtained through the above-mentioned techniques.

**3.3. Surface-Initiated Controlled Radical Polymerization.** SI-CRP is an effective way to grow polymers when a precise control over polymer architecture, composition, molecular weight, and graft density is required.²⁸ In general, CRP reactions rely on the reversible deactivation of propagating chains, such that termination reactions are minimized, thus providing first order polymerization kinetics and low dispersity. Different SI-CRP methods can be performed, which depend on the type of initiator or so-called chain transfer agent (CTA) coupled to the surface, such as Cu-mediated SI-CRP, surface reversible addition-fragmentation chain transfer (S-RAFT) or surface-initiated nitroxide-mediated polymerization (SI-NMP), among others. Scheme 8 exemplifies the three most commonly used SI-CRP techniques, i.e. surface-initiated atom transfer radical polymerization (SI-ATRP), S-RAFT, and SI-NMP. In the following sections, a brief overview of each of these techniques will be provided.

**3.3.1. Cu-Mediated SI-CRP.** Of all SI-CRP techniques, those mediated by transition metal complexes, especially Cu species, as in SI-ATRP, have been the most widely employed to graft polymers from CNCs. The control in ATRP arises from the existence of a dynamic equilibrium between dormant and propagating species, thus lowering radical concentration and minimizing termination reactions.¹⁴⁴ The reversible deactivation occurs through the exchange of a halogen atom (Br or Cl) between a metal complex (based on Cu in most cases) and the propagating species. As the presence of residual metal in the final polymers may be a problem for some applications,

techniques were then developed to decrease the required amount of catalytic system to ppm by (re)generating Cu(I) *in situ* activators by electron transfer (A(R)GET) ATRP.¹⁴⁵⁻¹⁴⁷ CRP in the presence of Cu(0) was also reported with a debate on the model of the electron transfer mechanism, i.e. single-electron transfer living radical polymerization (SET LRP) and supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP).¹⁴⁸⁻¹⁵⁰ As the present Perspective aims at reporting the different structures obtained from CNCs and their related properties rather than debating the mechanism, techniques involving Cu species are grouped under the generic term “Cu-mediated SI-CRP.”

Conducting a Cu-mediated SI-CRP from CNC requires the preliminary introduction of initiating sites on their surface. This is usually achieved by reacting the numerous –OH functionalities on the CNC surface with 2-bromoisobutyryl bromide (BiBB) in a solvent (THF or DMF), usually in the presence of a base (triethylamine) and a catalyst (DMAP).¹⁴¹,¹⁵¹⁻¹⁶¹ The extent of this modification can be tailored through the reaction duration and the amount of BiBB introduced.¹⁶²,¹⁶³ Through a combination of active initiators and inactive “dummy” initiators, the overall grafting density can be controlled.²⁸ Over the years, strategies were developed to improve the CNCs dispersibility during the modification by either performing (1) solvent exchanges (rather than freeze-drying), which permitted avoiding a redispersion step of dried CNCs,¹⁶³,¹⁶⁴ or (2) a chemical vapor deposition of BiBB on a CNC aerogel to partially hydrophobize the CNCs prior to a subsequent liquid phase reaction with BiBB.¹⁶⁵⁻¹⁶⁹ Higher modification degrees (up to 15 wt % Br determined by elemental analysis) were then reached with lower BiBB excess.¹⁶⁵ Although less widely used, the incorporation of the initiating sites was also reported by reacting the –OH functions with α-bromoisobutyric acid activated with carbonyl diimidazole.¹⁷⁰,¹⁷¹ In another study, the use of 2-bromopropionic acid was proposed for the introduction of active sites on the CNCs surface with a yield of 46% and 2.86 wt % of Br content determined by elemental analysis.⁹⁵ The protocol involve a pretreatment of pristine CNCs with HCl, followed by one-step reaction, which combines Fischer esterification and acid hydrolysis.

In all the previous studies, the –OH functions were statistically and homogeneously modified on the entire nano-crystal surface. Recently, Zoppe et al. reported an approach to introduce initiating sites specifically on the reducing end groups of CNCs through their oxidation and reaction with ethylenediamine and subsequently 2-bromoisobutanoic acid N-hydroxysuccinimide ester.¹⁷² This approach is discussed in further detail in section 3.4.

The CNC macroinitiators have been used in the Cu-mediated SI-CRP of various styrenic-, (meth)acrylic, or acrylamide-based monomers (Figure 5) with catalytic systems and solvents optimized for the various monomers employed (see Table S1). The polymerization control can be elucidated through the saponification of the grafted polymers (via either acidic or alkaline conditions) and further analysis of the degrafted polymer in solution; however the degrafting reactions require an extended reaction time (2 days to 3 weeks) and can potentially alter the polymer structure.¹⁴¹,¹⁵²⁻¹⁵⁴,¹⁶³,¹⁶⁴,¹⁷⁰,¹⁷² The versatility of Cu-mediated SI-CRP techniques and their high tolerance toward many functional groups has given access to CNCs grafted with a large variety of polymeric chains ranging from neutral PSty,¹¹⁵,¹⁴¹,¹⁵⁷,¹⁶² poly(alkyl ((meth)acrylate),¹³⁸,¹⁶⁹,¹⁷⁰ to cationic poly(amino(alkyl) methacrylate/methacrylamide)¹⁵⁵,¹⁷² or
<figure><img src="image_16.png" /><figcaption>**Figure 5.** Monomers used in Cu-mediated SI-CRP from CNC macroinitiators; Sty,<sup>115,141,157,162</sup> 4-SS, sodium 4-vinylbenzenesulfonate;<sup>172</sup> MA;<sup>158,170</sup> BA, butyl acrylate;<sup>169</sup> tBA, *tert*-butyl acrylate;<sup>165,168</sup> MMA, methyl methacrylate;<sup>169</sup> AEM, 2-aminoethyl methacrylate;<sup>155</sup> METAC, [2-(methacryloyl-oxy)ethyl]trimethylammonium chloride;<sup>172</sup> OEGMEMA, oligoethylene glycol methyl ether methacrylate;<sup>159,160</sup> NIPAM;<sup>153,154,163,164,172</sup> DMAM, N,N-dimethylacrylamide;<sup>173</sup> AEMA, 2-aminoethyl methacrylamide;<sup>155</sup> DMAEMA, dimethylaminoethyl methacrylate;<sup>152,166,167,171</sup> DEAEMA, diethylaminoethyl methacrylate;<sup>171</sup> EANI, 4-ethoxy-9-allyl-1,8-naphtalimide;<sup>154</sup> SBAM, soybean amide methacrylate;<sup>156</sup> NVC, N-vinylcaprolactam;<sup>161</sup> MMAZO, 6-[4-(4-methoxyphenylazo)phenoxy] hexyl methacrylate;<sup>151</sup> NpMA, naphtyl methacrylate.<sup>166</sup></figcaption></figure>

anionic polystyrenesulfonate<sup>172</sup> and PAA (through the hydrolysis of poly(*tert*-butyl acrylate) (PtBA)).<sup>165,168</sup> Thermosensitive polymers, such as PNIPAM,<sup>153,154,163,164,172</sup> PDMAEMA,<sup>152,166,167,171</sup> poly(N-vinylcaprolactam) (PNVC),<sup>161</sup> or poly-[(oligoethylene glycol) methyl ether acrylate] (POEGMEMA)<sup>159,160</sup> have also been grafted with this approach.

The use of a sacrificial initiator (i.e., free initiator species added to the reaction medium, usually EBiB, ethyl α-bromoisobutyrate) is common as it provides a simple means to monitor the polymerization process, allows targeting the DP, and also contributes to a better control by increasing the concentration of Cu(II) deactivator species in the reaction.<sup>154,157,162,165,167–169</sup> A good correlation between molecular weight of the free PSty chains (initiated from the sacrificial initiator) and the molecular weight of PSty grafts was demonstrated by incorporating *o*-nitrobenzyl photosensitive moieties between the CNC surface and the surface-grafted initiator, degrafting the PSty grafts under mild conditions (UV-irradiation) and comparing their molecular weight to the free chains (number-average molecular weight ($M_n$) = 8.5 kDa, dispersity ($D = M_w/M_n$) = 1.08 versus $M_n$ = 10.1 kDa, $D$ = 1.07 for degrafted and free polymers, respectively).<sup>115</sup> In contrast, Zoppe et al. showed high dispersity of surface-grown PNIPAM, however this was likely due to insufficient amounts of CuX<sub>2</sub> deactivator species and complexation with NIPAM monomers, in addition to having been conducted in highly polar media.<sup>163</sup> Using a similar SI-AGET-ATRP approach with ascorbic acid as reducing agent, Wu et al. found that free PNIPAM chains had a bimodal molecular weight distribution, whereas cleaved PNIPAM chains were unimodal and had a lower dispersity.<sup>154</sup> In addition, a significantly higher $M_n$ was observed for the free PNIPAM chains.

Monte Carlo simulations of surface-initiated polymerization from neutral planar substrates have shown that bulk polymers tend to grow at faster rates and with lower dispersity, however this is highly dependent on the grafting density of initiating sites.<sup>174</sup> Along with the importance of CuX/CuX<sub>2</sub> ratio,<sup>28,29</sup> it is clear that the monomer type/concentration, solvent and bulk vs solution synthesis all play a role in the control of Cu-mediated

SI-CRP.<sup>28</sup> Thus, the assumption that the polymerization kinetics of surface-initiated polymers is similar to that of free polymers is not always valid.

In general, Cu-mediated SI-CRP conducted in higher polarity solvents, such as water, tend to yield polymers with higher dispersity, as a result of high rates of activation.<sup>175</sup> Such high dispersity implies higher rates of termination and the loss of active polymer chain ends, thus low overall control. The kinetics of Cu-mediated SI-CRP conducted at high monomer concentrations in less polar solvents or in bulk, e.g., Sty, tend to resemble those of solution Cu-mediated CRP.<sup>115</sup> Such aspects are important considerations when more complex architectures are required, such as block copolymer brushes from CNCs, for example, in which good control and high chain-end fidelity are critical for success.

The impact of the CNC surface charges on SI-ATRP control was also investigated through the initiation of a DMAM polymerization from CNCs with various charge densities.<sup>173</sup> It was demonstrated that electrostatic interactions lead to an enrichment of catalytic species on the surface and increased the initiator efficiency, but at the expense of a higher dispersity. A qualitative model of catalyst enrichment for SI-ATRP from CNCs is shown in Figure 6. Since Cu(II) halide species readily dissociate in aqueous environments, divalent Cu<sup>2+</sup>/ligand complexes can strongly interact with sulfate half-ester groups on the surfaces of the CNCs, which in turn has a negative impact on deactivation of growing polymer chains. Therefore, low charge density CNCs are advantageous if low dispersity of grafted chains is required in the final hybrid materials; however, a lower initiator efficiency can be expected.

It was also recently demonstrated that a controlled Cu-mediated SI-CRP of MA can be photoinduced from modified CNCs in a commercial UV nail box.<sup>158</sup> The CNCs were previously modified with BiBB, then the polymerization of the monomer proceed under UV irradiation at 360 nm during 90 min in the presence of a sacrificial initiator. The analysis of the free PMA synthesized simultaneously in solution demonstrated first order kinetics and, thus, good control. The use of this technique
<figure><img src="image_19.png" /><figcaption>**Figure 5.** Monomers used in Cu-mediated SI-CRP from CNC macroinitiators; Sty;<sup>115,141,157,162</sup> 4-SS, sodium 4-vinylbenzenesulfonate;<sup>172</sup> MA;<sup>158,170</sup> BA, butyl acrylate;<sup>169</sup> tBA, *tert*-butyl acrylate;<sup>165,168</sup> MMA, methyl methacrylate;<sup>169</sup> AEM, 2-aminoethyl methacrylate;<sup>155</sup> METAC, [2-(methacryloyl-oxy)ethyl]trimethylammonium chloride;<sup>172</sup> OEGMEMA, oligoethylene glycol methyl ether methacrylate;<sup>159,160</sup> NIPAM;<sup>153,154,163,164,172</sup> DMAM, N,N-dimethylacrylamide;<sup>173</sup> AEMA, 2-aminoethyl methacrylamide;<sup>155</sup> DMAEMA, dimethylaminoethyl methacrylate;<sup>152,166,167,171</sup> DEAEMA, diethylaminoethyl methacrylate;<sup>171</sup> EANI, 4-ethoxy-9-allyl-1,8-naphtalimide;<sup>154</sup> SBAM, soybean amide methacrylate;<sup>156</sup> NVC, N-vinylcaprolactam;<sup>161</sup> MMAZO, 6-[4-(4-methoxyphenylazo)phenoxy] hexyl methacrylate;<sup>151</sup> NpMA, naphtyl methacrylate.<sup>166</sup></figcaption></figure>

anionic polystyrenesulfonate<sup>172</sup> and PAA (through the hydrolysis of poly(*tert*-butyl acrylate) (PtBA)).<sup>165,168</sup> Thermosensitive polymers, such as PNIPAM,<sup>153,154,163,164,172</sup> PDMAEMA,<sup>152,166,167,171</sup> poly(N-vinylcaprolactam) (PNVC),<sup>161</sup> or poly-[(oligoethylene glycol) methyl ether acrylate] (POEGMEMA)<sup>159,160</sup> have also been grafted with this approach.

The use of a sacrificial initiator (i.e., free initiator species added to the reaction medium, usually EBiB, ethyl α-bromoisobutyrate) is common as it provides a simple means to monitor the polymerization process, allows targeting the DP, and also contributes to a better control by increasing the concentration of Cu(II) deactivator species in the reaction.<sup>154,157,162,165,167–169</sup> A good correlation between molecular weight of the free PSty chains (initiated from the sacrificial initiator) and the molecular weight of PSty grafts was demonstrated by incorporating o-nitrobenzyl photosensitive moieties between the CNC surface and the surface-grafted initiator, degrafting the PSty grafts under mild conditions (UV-irradiation) and comparing their molecular weight to the free chains (number-average molecular weight ($M_n$) = 8.5 kDa, dispersity ($D = M_w/M_n$) = 1.08 versus $M_n$ = 10.1 kDa, $D = 1.07$ for degrafted and free polymers, respectively).<sup>115</sup> In contrast, Zoppe et al. showed high dispersity of surface-grown PNIPAM, however this was likely due to insufficient amounts of CuX<sub>2</sub> deactivator species and complexation with NIPAM monomers, in addition to having been conducted in highly polar media.<sup>163</sup> Using a similar SI-AGET-ATRP approach with ascorbic acid as reducing agent, Wu et al. found that free PNIPAM chains had a bimodal molecular weight distribution, whereas cleaved PNIPAM chains were unimodal and had a lower dispersity.<sup>154</sup> In addition, a significantly higher $M_n$ was observed for the free PNIPAM chains.

Monte Carlo simulations of surface-initiated polymerization from neutral planar substrates have shown that bulk polymers tend to grow at faster rates and with lower dispersity, however this is highly dependent on the grafting density of initiating sites.<sup>174</sup> Along with the importance of CuX/CuX<sub>2</sub> ratio,<sup>28,29</sup> it is clear that the monomer type/concentration, solvent and bulk vs solution synthesis all play a role in the control of Cu-mediated

SI-CRP.<sup>28</sup> Thus, the assumption that the polymerization kinetics of surface-initiated polymers is similar to that of free polymers is not always valid.

In general, Cu-mediated SI-CRP conducted in higher polarity solvents, such as water, tend to yield polymers with higher dispersity, as a result of high rates of activation.<sup>175</sup> Such high dispersity implies higher rates of termination and the loss of active polymer chain ends, thus low overall control. The kinetics of Cu-mediated SI-CRP conducted at high monomer concentrations in less polar solvents or in bulk, e.g., Sty, tend to resemble those of solution Cu-mediated CRP.<sup>115</sup> Such aspects are important considerations when more complex architectures are required, such as block copolymer brushes from CNCs, for example, in which good control and high chain-end fidelity are critical for success.

The impact of the CNC surface charges on SI-ATRP control was also investigated through the initiation of a DMAM polymerization from CNCs with various charge densities.<sup>173</sup> It was demonstrated that electrostatic interactions lead to an enrichment of catalytic species on the surface and increased the initiator efficiency, but at the expense of a higher dispersity. A qualitative model of catalyst enrichment for SI-ATRP from CNCs is shown in Figure 6. Since Cu(II) halide species readily dissociate in aqueous environments, divalent Cu<sup>2+</sup>/ligand complexes can strongly interact with sulfate half-ester groups on the surfaces of the CNCs, which in turn has a negative impact on deactivation of growing polymer chains. Therefore, low charge density CNCs are advantageous if low dispersity of grafted chains is required in the final hybrid materials; however, a lower initiator efficiency can be expected.

It was also recently demonstrated that a controlled Cu-mediated SI-CRP of MA can be photoinduced from modified CNCs in a commercial UV nail box.<sup>158</sup> The CNCs were previously modified with BiBB, then the polymerization of the monomer proceed under UV irradiation at 360 nm during 90 min in the presence of a sacrificial initiator. The analysis of the free PMA synthesized simultaneously in solution demonstrated first order kinetics and, thus, good control. The use of this technique
<figure><img src="image_17.png" /><figcaption>**Figure 6.** Qualitative model describing the composition of the interfacial region around CNCs prior to polymer brush growth from CNCs with high (CNC-1) and low (CNC-2) density of surface sulfate groups. (small red circle = initiator, L = ligand, $κ^{-1}$ = Debye length). Reprinted with permission from ref 173. Copyright 2016 American Chemical Society.</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>**Scheme 9.** (A) R-Group vs Z-Group CTA Immobilization Strategies, (B) S-RAFT of VAc from CNCs Using the R-Group Approach,¹⁷⁹ and (C) S-RAFT of Acrylamide from CNCs Using Free macroCTA and Surface Free Radicals Generated by CAN⁹⁶</figcaption></figure>

allowed high grafting density in short reaction times and a straightforward purification due to the significant reduction in the amounts of Cu and ligand employed in comparison with a traditional Cu-mediated CRP. The authors observed an increase of the glass transition temperature ($T_g$) of CNC-g-PMA compared with the free PMA, indicative of reduced chain mobility when tethered to the CNC surface. The development of such

user-friendly SI-CRP techniques opens up a wide range of synthetic possibilities for grafting polymers from CNCs.

Even though the above-mentioned techniques has been widely used in the field of grafting polymers from CNCs, the removal of Cu residues after the polymerization is still challenging and needs more investigation. Several purification methods are used in solution polymerization but they are difficult to
<figure><img src="image_20.png" /><figcaption>Figure 6. Qualitative model describing the composition of the interfacial region around CNCs prior to polymer brush growth from CNCs with high (CNC-1) and low (CNC-2) density of surface sulfate groups. (small red circle = initiator, L = ligand, $κ^{-1}$ = Debye length). Reprinted with permission from ref 173. Copyright 2016 American Chemical Society.</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>Scheme 9. (A) R-Group vs Z-Group CTA Immobilization Strategies, (B) S-RAFT of VAc from CNCs Using the R-Group Approach,¹⁷⁹ and (C) S-RAFT of Acrylamide from CNCs Using Free macroCTA and Surface Free Radicals Generated by CAN⁹⁶</figcaption></figure>

allowed high grafting density in short reaction times and a straightforward purification due to the significant reduction in the amounts of Cu and ligand employed in comparison with a traditional Cu-mediated CRP. The authors observed an increase of the glass transition temperature ($T_g$) of CNC-g-PMA compared with the free PMA, indicative of reduced chain mobility when tethered to the CNC surface. The development of such

user-friendly SI-CRP techniques opens up a wide range of synthetic possibilities for grafting polymers from CNCs.

Even though the above-mentioned techniques has been widely used in the field of grafting polymers from CNCs, the removal of Cu residues after the polymerization is still challenging and needs more investigation. Several purification methods are used in solution polymerization but they are difficult to
employ when the reaction involves CNCs, e.g., purification over silica or neutral alumina columns.¹⁴⁴ The (re)generation of Cu(I) in solution partially solved this problem, since much lower amounts of Cu are used in the reaction. Nevertheless, in case of biomedical applications, the removal of trace amounts of Cu appears to be crucial.

Dialysis has been used to remove Cu after polymerization reactions.¹⁷⁶ Prior to dialysis, Rosilo et al. performed several centrifugations of CNC-g-poly(N,N-dimethylaminoethyl methacrylate) in different organic solvents, i.e., MeOH, DMF, and dioxane.¹⁶⁷ In both cases, the approaches used may not be sufficient to remove the adsorbed Cu on the surface of the grafted CNCs. In another study, Morandi et al. proposed two subsequent Soxhlet extractions on freeze-dried samples in dichloromethane and ethanol for the removal of Cu traces. The blue color, related to the Cu adsorbed on the surface of the CNCs, was removed by this treatment. Nevertheless, no quantitative analysis of remaining traces of Cu was performed after this process. Another method to remove Cu could involves stirring the grafted CNCs with an ion-exchange resin.¹⁷⁷ Or, as discussed in section 3.2, the particles could be removed by filtering the modified CNCs through glass microfiber filter paper.¹³²

### 3.3.2. Surface-RAFT Polymerization.
Reversible addition-fragmentation chain transfer polymerization (RAFT) involves a reversible chain transfer process and is carried out in the presence of a CTA, in addition to a free radical initiator. Such CTAs are often based on dithiocarbamates, dithiocarbonates, or dithioesters and contain so-called R- and Z-groups. The function of the CTA is to mediate addition, fragmentation and chain transfer reactions, resulting in an overall controlled molecular weight and dispersity. During RAFT polymerization, radicals either reversibly react with the CTA or monomers. Consequently, S-RAFT polymerization is conducted by the prior immobilization of either free radical initiators or CTAs to the substrate surface. If the CTA is attached to the substrate surface, it can be tethered so as to present either the R- or Z-group, the former of which being the most commonly utilized. Scheme 9 shows (A) the two different CTA immobilization strategies and examples of S-RAFT from CNCs using (B) the R-group approach and (C) surface free radicals and free CTA.

One of the first studies utilizing S-RAFT to modify CNCs was reported by Zeinali et al., in which PNIPAM and random copolymers of PNIPAM and PAA (P(PNIPAM-ran-AA)) were grafted via the immobilization of the CTA with the R-group approach.¹⁴² The CTA, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT), was grafted to CNC -OH groups via DMAP-catalyzed esterification in the presence of N,N'-dicyclohexylcarbodiimide (DCC) in chloroform. A sulfur content of 2.1 wt %, determined by elemental analysis, confirmed the successful attachment of the CTA. S-RAFT polymerizations of NIPAM and NIPAM-ran-AA were conducted with 2,2'-azobis(2-methylpropionitrile) (AIBN) in dioxane and in the presence of free CTA, i.e., DDMAT. The authors obtained statistical copolymer grafts using different monomer feed ratios, in which the CNC hybrids were characterized by FTIR, dynamic light scattering (DLS) and thermal analysis. From the results of DSC experiments, the authors concluded that as the ratio of AA was increased, while the heat capacity variation ($\Delta C_p$) decreased. In comparison to free (co)polymers, those grafted from CNCs displayed higher $T_g$ due to surface chain confinement.

Similar block copolymers were grafted from CNCs coated with a cross-linked poly(2-hydroxyethyl methacrylate) (PHEMA) shell by immobilization of the CTA, S-(thiobenzoylthioglycolic)

acid.¹⁷⁸ CNCs were first coated with PHEMA cross-linked with ethylene glycol dimethacrylate (EGDMA), using a free radical approach with AIBN in solution, to increase the -OH groups content on the surface. From X-ray photoelectron spectroscopy (XPS) analysis of CTA-modified CNC/PHEMA particles, the surface S content was determined to be 1.8%. In this case, the authors likely obtained a higher grafting density of PNIPAM-block-PAA chains due to grafting from a cross-linked PHEMA network at the surface of CNCs. In other work, PMMA was grafted from CNCs using 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPADB) functionalized CNCs.¹¹⁸ Here the authors determined a sulfur content of 2.9 wt % by elemental analysis and estimated that 1 out of 6 AGUs was modified with CPADB, which is one of the highest values reported so far for CTA-functionalized CNCs. The authors further utilized ¹H NMR spectroscopy in DMSO-d₆ in the presence of an ionic liquid to observe CPADB-functionalized CNCs.

Boujemaoui et al. investigated the grafting of vinyl acetate (VAc) via S-RAFT and the interchange of xanthates (MADIX) polymerization.¹⁷⁹ The xanthate-based CTA, 2-((ethoxycarbonothioyl)thio)propanoic acid, was attached to CNCs either by Steglich esterification or a two-step approach, the latter of which resulted in a lower grafting yield of poly(vinyl acetate) (PVAc). The kinetics of the S-RAFT process was monitored using sacrificial CTA and demonstrated good control over the molecular weight and dispersity. However, for all the aforementioned studies, little is known regarding the molecular weight, dispersity and grafting density of the tethered polymers.

S-RAFT of various acrylamides from CNCs has also been mediated by free macromolecular CTAs and conducted utilizing free radical initiators, such as ceric(IV) ammonium nitrate (CAN) (Scheme 9C).⁹⁶,¹⁸⁰ In these cases, macromolecular CTAs with molecular weight between 1.4 and 2 kDa were used to mediate S-RAFT polymerization of AM⁹⁶,¹⁸⁰ and DMAM¹⁸⁰ from CNCs, in which surface radicals were generated as shown in Scheme 7. PAM chains were cleaved from the surface of CNCs and determined to have $M_n$ = 14 kDa and $D$ = 1.28, the latter demonstrating involvement of the macromolecular CTA.⁹⁶ In addition, increased S contents were observed for the polymer-modified CNCs, which indicated that at least some of the grafted PAM chains were end-capped with macromolecular CTA moieties.

In a unique approach, poly[poly(ethylene glycol)ethyl ether methacrylate] (PPEGEEMA) and PDMAEMA were grafted from a difunctional CNC macroinitiator via a combination of S-RAFT and SI-ATRP.¹⁷⁶ The difunctional CNC macroinitiator was obtained by first transforming the -OH groups on the CNC surface into reactive amines, and then, transforming them into bromoisobutyryl functionalities and azo initiator sites via consecutive reactions with $\alpha$-bromoisobutyric acid (BIBA) and 4,4'-azobis(4-cyanovaleric acid) (ACVA), respectively. The subsequent step involved the growth of PPEGEEMA brushes from the surface of modified CNCs via S-RAFT polymerization, followed by the grafting of PDMAEMA via SI-ATRP. The polymerizations were sequentially performed in this way to avoid potential destruction of the azo initiator sites by the SI-ATRP processes. Free PPEGEEMA formed in solution via RAFT had a $M_n$ of 4.7 kDa and $D$ of 1.2. After SI-ATRP, PDMAEMA chains were successfully cleaved from the CNCs by acid hydrolysis, yielding $M_n$ in the range of 4.2-7.7 kDa and $D$ of 1.2-1.3, depending on the SI-ATRP reaction time between 5 and 30 min. However, under such conditions (64 wt % of sulfuric acid, 3 h, 45 °C), the reported size-exclusion chromatography (SEC)
employ when the reaction involves CNCs, e.g., purification over silica or neutral alumina columns.¹⁴⁴ The (re)generation of Cu(I) in solution partially solved this problem, since much lower amounts of Cu are used in the reaction. Nevertheless, in case of biomedical applications, the removal of trace amounts of Cu appears to be crucial.

Dialysis has been used to remove Cu after polymerization reactions.¹⁷⁶ Prior to dialysis, Rosilo et al. performed several centrifugations of CNC-g-poly(N,N-dimethylaminoethyl methacrylate) in different organic solvents, i.e., MeOH, DMF, and dioxane.¹⁶⁷ In both cases, the approaches used may not be sufficient to remove the adsorbed Cu on the surface of the grafted CNCs. In another study, Morandi et al. proposed two subsequent Soxhlet extractions on freeze-dried samples in dichloromethane and ethanol for the removal of Cu traces. The blue color, related to the Cu adsorbed on the surface of the CNCs, was removed by this treatment. Nevertheless, no quantitative analysis of remaining traces of Cu was performed after this process. Another method to remove Cu could involves stirring the grafted CNCs with an ion-exchange resin.¹⁷⁷ Or, as discussed in section 3.2, the particles could be removed by filtering the modified CNCs through glass microfiber filter paper.¹³²

### 3.3.2. Surface-RAFT Polymerization.
Reversible addition-fragmentation chain transfer polymerization (RAFT) involves a reversible chain transfer process and is carried out in the presence of a CTA, in addition to a free radical initiator. Such CTAs are often based on dithiocarbamates, dithiocarbonates, or dithioesters and contain so-called R- and Z-groups. The function of the CTA is to mediate addition, fragmentation and chain transfer reactions, resulting in an overall controlled molecular weight and dispersity. During RAFT polymerization, radicals either reversibly react with the CTA or monomers. Consequently, S-RAFT polymerization is conducted by the prior immobilization of either free radical initiators or CTAs to the substrate surface. If the CTA is attached to the substrate surface, it can be tethered so as to present either the R- or Z-group, the former of which being the most commonly utilized. Scheme 9 shows (A) the two different CTA immobilization strategies and examples of S-RAFT from CNCs using (B) the R-group approach and (C) surface free radicals and free CTA.

One of the first studies utilizing S-RAFT to modify CNCs was reported by Zeinali et al., in which PNIPAM and random copolymers of PNIPAM and PAA (P(PNIPAM-ran-AA)) were grafted via the immobilization of the CTA with the R-group approach.¹⁴² The CTA, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT), was grafted to CNC -OH groups via DMAP-catalyzed esterification in the presence of N,N'-dicyclohexylcarbodiimide (DCC) in chloroform. A sulfur content of 2.1 wt %, determined by elemental analysis, confirmed the successful attachment of the CTA. S-RAFT polymerizations of NIPAM and NIPAM-ran-AA were conducted with 2,2'-azobis(2-methylpropionitrile) (AIBN) in dioxane and in the presence of free CTA, i.e., DDMAT. The authors obtained statistical copolymer grafts using different monomer feed ratios, in which the CNC hybrids were characterized by FTIR, dynamic light scattering (DLS) and thermal analysis. From the results of DSC experiments, the authors concluded that as the ratio of AA was increased, while the heat capacity variation ($\Delta C_p$) decreased. In comparison to free (co)polymers, those grafted from CNCs displayed higher $T_g$ due to surface chain confinement.

Similar block copolymers were grafted from CNCs coated with a cross-linked poly(2-hydroxyethyl methacrylate) (PHEMA) shell by immobilization of the CTA, S-(thiobenzoylthioglycolic)

acid.¹⁷⁸ CNCs were first coated with PHEMA cross-linked with ethylene glycol dimethacrylate (EGDMA), using a free radical approach with AIBN in solution, to increase the -OH groups content on the surface. From X-ray photoelectron spectroscopy (XPS) analysis of CTA-modified CNC/PHEMA particles, the surface S content was determined to be 1.8%. In this case, the authors likely obtained a higher grafting density of PNIPAM-block-PAA chains due to grafting from a cross-linked PHEMA network at the surface of CNCs. In other work, PMMA was grafted from CNCs using 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPADB) functionalized CNCs.¹¹⁸ Here the authors determined a sulfur content of 2.9 wt % by elemental analysis and estimated that 1 out of 6 AGUs was modified with CPADB, which is one of the highest values reported so far for CTA-functionalized CNCs. The authors further utilized ¹H NMR spectroscopy in DMSO-d₆ in the presence of an ionic liquid to observe CPADB-functionalized CNCs.

Boujemaoui et al. investigated the grafting of vinyl acetate (VAc) via S-RAFT and the interchange of xanthates (MADIX) polymerization.¹⁷⁹ The xanthate-based CTA, 2-((ethoxycarbonothioyl)thio)propanoic acid, was attached to CNCs either by Steglich esterification or a two-step approach, the latter of which resulted in a lower grafting yield of poly(vinyl acetate) (PVAc). The kinetics of the S-RAFT process was monitored using sacrificial CTA and demonstrated good control over the molecular weight and dispersity. However, for all the aforementioned studies, little is known regarding the molecular weight, dispersity and grafting density of the tethered polymers.

S-RAFT of various acrylamides from CNCs has also been mediated by free macromolecular CTAs and conducted utilizing free radical initiators, such as ceric(IV) ammonium nitrate (CAN) (Scheme 9C).⁹⁶,¹⁸⁰ In these cases, macromolecular CTAs with molecular weight between 1.4 and 2 kDa were used to mediate S-RAFT polymerization of AM⁹⁶,¹⁸⁰ and DMAM¹⁸⁰ from CNCs, in which surface radicals were generated as shown in Scheme 7. PAM chains were cleaved from the surface of CNCs and determined to have $M_n$ = 14 kDa and $D = 1.28$, the latter demonstrating involvement of the macromolecular CTA.⁹⁶ In addition, increased S contents were observed for the polymer-modified CNCs, which indicated that at least some of the grafted PAM chains were end-capped with macromolecular CTA moieties.

In a unique approach, poly[poly(ethylene glycol)ethyl ether methacrylate] (PPEGEEMA) and PDMAEMA were grafted from a difunctional CNC macroinitiator via a combination of S-RAFT and SI-ATRP.¹⁷⁶ The difunctional CNC macroinitiator was obtained by first transforming the -OH groups on the CNC surface into reactive amines, and then, transforming them into bromoisobutyryl functionalities and azo initiator sites via consecutive reactions with $\alpha$-bromoisobutyric acid (BIBA) and 4,4'-azobis(4-cyanovaleric acid) (ACVA), respectively. The subsequent step involved the growth of PPEGEEMA brushes from the surface of modified CNCs via S-RAFT polymerization, followed by the grafting of PDMAEMA via SI-ATRP. The polymerizations were sequentially performed in this way to avoid potential destruction of the azo initiator sites by the SI-ATRP processes. Free PPEGEEMA formed in solution via RAFT had a $M_n$ of 4.7 kDa and $D$ of 1.2. After SI-ATRP, PDMAEMA chains were successfully cleaved from the CNCs by acid hydrolysis, yielding $M_n$ in the range of 4.2-7.7 kDa and $D$ of 1.2-1.3, depending on the SI-ATRP reaction time between 5 and 30 min. However, under such conditions (64 wt % of sulfuric acid, 3 h, 45 °C), the reported size-exclusion chromatography (SEC)
results likely represented a mixture of cleaved PDMAEMA and PPEGEEMA or their corresponding hydrolysates, e.g. PMAA. There is still much to learn about S-RAFT from CNCs, given the complexity of the RAFT process and the lack of knowledge on the kinetics of such polymerizations conducted from CNCs.

**3.3.3. Surface-Initiated NMP.** Similar to Cu-mediated SI-CRP, surface-initiated nitroxide-mediated polymerization (SI-NMP) mechanistically depends on the reversible activation–deactivation of propagating polymer chains by a deactivating species, in this case a nitroxide radical. In general, alkoxyamines, such as TEMPO and N-tert-butyl-N-[1-diethylphosphono(2,2-dimethylpropyl)] nitroxide (DEPN), are commonly used for SI-NMP. Roeder et al. were the first to use SI-NMP to graft polymers from CNCs.<sup>143</sup> The authors first functionalized CNCs with 4-(diethoxyphosphinyl)-2,2,5,5-tetramethyl-3-azahexane-N-oxyl, a.k.a. BlocBuilder, and subsequently grafted PMA and PMMA from CNCs in the presence of sacrificial BlocBuilder initiators (see Scheme 8). The grafting yields obtained were 75–80 wt %. The authors were unable to cleave the grafted PMA or PMMA from the CNC surfaces, despite several attempts. However, SI-NMP conducted in the presence of sacrificial BlocBuilder allowed SEC analysis of free polymers. While free PMA showed M<sub>n</sub> values from 3.3 to 43.7 kDa and D of 1.35–1.4, the free PMMA had a M<sub>n</sub> in the range of 30.8–140 kDa and similar dispersity, despite similar monomer conversion. These results suggested that higher sacrificial initiator efficiency was obtained for PMA.

The same group later employed the same approach to graft a variety of methacrylamides, such as DMAEMA, DEAEMA and N-[3-(dimethylamino)propyl] methacrylamide (DMAPMAM), from CNCs.<sup>181</sup> In addition to elemental analysis and thermal analysis, these CNC hybrids were characterized by cross-polarization magic-angle spinning (CP-MAS) <sup>13</sup>C NMR spectroscopy. The latter technique revealed that the composition depended on the reaction time, however molecular weight and dispersity of the grafted polymers were not reported. The highest grafting yield was obtained for SI-NMP of DEAEMA from CNCs, i.e., 65 wt %. Overall, little is known regarding the kinetics of SI-NMP from CNCs in comparison to solution NMP, which leaves plenty of research opportunities for this field.

**3.4. Polymer Grafting from the Reducing End Groups of CNCs.** Individual cellulose chains contain chemically distinct end groups, commonly referred to as the nonreducing end and the reducing end. The directionality of cellulose chains is a direct result of the mechanism of the cellulose biosynthesis.<sup>58</sup> This reducing end can be reversibly converted between an aldehyde and a cyclic hemiacetal under a variety of conditions. Thus, site-specific, or “patchy” CNCs can be obtained by modifying this reducing end through aldehyde-selective reaction pathways. CNCs with end-tethered polymers chains present an interesting alternative to uniformly modified CNCs for certain applications, in which their self-assembly can be exploited toward structural hierarchies with unique properties, including both assemblies on surfaces and in nanocomposite materials.<sup>8</sup> Depending on the molecular weight of end-tethered polymers, one can see such anisotropic colloidal building blocks as somewhat analogous to rod–coil block copolymers.<sup>182</sup>

Different procedures have been established in order to attach a small molecule or initiator to the reducing end groups of CNCs. Arcot et al. reported a method to introduce thiol groups on the reducing ends of the CNCs by reductive amination in an aqueous medium.<sup>183</sup> The authors synthesized thiolated cellulose nanocrystals (CNC-SH) by using 6-amino-1-hexanethiol

hydrochloride (NH<sub>2</sub>(CH<sub>2</sub>)<sub>6</sub>SH·HCl), a reducing agent (Cn, sodium cyanoborohydride; Ac, sodium triacetoxyborohydride; or Pc, 2-picoline-borane complex) and an aqueous CNC dispersion at different pH (5, 7 or 9.2) values. The CNC-SH were then functionalized with silver nanoparticles (AgNPs) in order to characterize the successful reducing end group modification, qualitatively using transmission electron microscopy (TEM) and quantitatively using XPS. The authors determined that the highest yield of functionalization was obtained when using basic conditions.

The same group later introduced a second thiolation procedure with shorter reaction times, milder conditions, and higher colloidal stability of the modified CNCs.<sup>184</sup> Usuda and coworkers had previously demonstrated the selective oxidation of reducing ends to carboxylates with sodium chlorite (NaClO<sub>2</sub>), which gives access to a wider variety of potential modification chemistries.<sup>185</sup> Thus, the reducing ends were first reacted with NaClO<sub>2</sub> to generate carboxylate functionalized CNC reducing ends (CNC-COOH). After this oxidation step, a mild activation step of the carboxyl group was carried out mediated by N-hydroxysuccinimide (NHS) and N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC), which was followed by a nucleophilic substitution with NH<sub>2</sub>(CH<sub>2</sub>)<sub>6</sub>SH·HCl, resulting in CNC-SH.

Gruber et al. proposed a different reducing end group functionalization strategy via hydrazone linkages.<sup>186</sup> The authors used 4-hydrazino-benzoic acid or radical initiators 4,4'-azobis(4-cyanopentanoic dihydrazide) and 4-carboxyphenylazomethylmalonodinitrile to form the hydrazone linkage, which resulted in a phenylcarboxylate terminated CNC with a yield of 44.6%. Similar yields with the conversion of 4-hydrazinobenzoic acid were obtained for the other initiators. The authors hypothesized that the low yield was due to the poor accessibility of the reducing end groups. If one considers individualized CNCs to consist of 18-chains,<sup>58</sup> this would imply a maximum of 18 reducing end groups at one end of the cellulose I crystal. Given the rod-like shape of CNCs, the reducing end-groups are likely distributed heterogeneously. This may lead to varying degrees of reducing end group accessibility for chemical reactions, however this remains to be explored.

Gruber and coworkers grafted PAM by SI-FRP from different azo initiators discussed in section 3.2 and shown in Table 1.<sup>186</sup> The molecular weight of the polymers were determined by SEC measurements after degrafting the PAM from the CNCs. The authors observed high molecular weights for PAM, but only a small amount of the initiators reacted to yield the polymer-grafted CNCs. Initiator efficiencies for surface-initiated polymerizations are reported to be very low, e.g., 5–30%.<sup>28</sup>

Recently, Zoppe et al. reported a route to functionalize the end groups with an SI-ATRP initiator containing α-bromoisobutyryl groups.<sup>172</sup> The reducing ends were first oxidized with NaClO<sub>2</sub> and further modified with ethylenediamine (EDA), following the work of Arcot et al.<sup>184</sup> Subsequently, primary amine groups were reacted with 2-bromoisobutanoic acid N-hydroxysuccinimide ester (NHS-BiB) to yield the ATRP initiator-functionalized reducing end groups. Although the authors were unable to quantify the success of the initiator modification reactions with conventional techniques, such as FTIR, XPS and elemental analysis, they subsequently grafted a variety of polymers from the functionalized reducing end groups of CNCs via SI-ATRP. The monomers investigated included NIPAM, METAC, and 4-SS. Thermogravimetric analysis (TGA), FTIR, and XPS were used to qualitatively analyze the
results likely represented a mixture of cleaved PDMAEMA and PPEGEEMA or their corresponding hydrolysates, e.g. PMAA. There is still much to learn about S-RAFT from CNCs, given the complexity of the RAFT process and the lack of knowledge on the kinetics of such polymerizations conducted from CNCs.

**3.3.3. Surface-Initiated NMP.** Similar to Cu-mediated SI-CRP, surface-initiated nitroxide-mediated polymerization (SI-NMP) mechanistically depends on the reversible activation–deactivation of propagating polymer chains by a deactivating species, in this case a nitroxide radical. In general, alkoxyamines, such as TEMPO and N-tert-butyl-N-[1-diethylphosphono(2,2-dimethylpropyl)] nitroxide (DEPN), are commonly used for SI-NMP. Roeder et al. were the first to use SI-NMP to graft polymers from CNCs.<sup>143</sup> The authors first functionalized CNCs with 4-(diethoxyphosphinyl)-2,2,5,5-tetramethyl-3-azahexane-N-oxyl, a.k.a. BlocBuilder, and subsequently grafted PMA and PMMA from CNCs in the presence of sacrificial BlocBuilder initiators (see Scheme 8). The grafting yields obtained were 75–80 wt %. The authors were unable to cleave the grafted PMA or PMMA from the CNC surfaces, despite several attempts. However, SI-NMP conducted in the presence of sacrificial BlocBuilder allowed SEC analysis of free polymers. While free PMA showed M<sub>n</sub> values from 3.3 to 43.7 kDa and D of 1.35–1.4, the free PMMA had a M<sub>n</sub> in the range of 30.8–140 kDa and similar dispersity, despite similar monomer conversion. These results suggested that higher sacrificial initiator efficiency was obtained for PMA.

The same group later employed the same approach to graft a variety of methacrylamides, such as DMAEMA, DEAEMA and N-[3-(dimethylamino)propyl] methacrylamide (DMAPMAM), from CNCs.<sup>181</sup> In addition to elemental analysis and thermal analysis, these CNC hybrids were characterized by cross-polarization magic-angle spinning (CP-MAS) <sup>13</sup>C NMR spectroscopy. The latter technique revealed that the composition depended on the reaction time, however molecular weight and dispersity of the grafted polymers were not reported. The highest grafting yield was obtained for SI-NMP of DEAEMA from CNCs, i.e., 65 wt %. Overall, little is known regarding the kinetics of SI-NMP from CNCs in comparison to solution NMP, which leaves plenty of research opportunities for this field.

**3.4. Polymer Grafting from the Reducing End Groups of CNCs.** Individual cellulose chains contain chemically distinct end groups, commonly referred to as the nonreducing end and the reducing end. The directionality of cellulose chains is a direct result of the mechanism of the cellulose biosynthesis.<sup>58</sup> This reducing end can be reversibly converted between an aldehyde and a cyclic hemiacetal under a variety of conditions. Thus, site-specific, or “patchy” CNCs can be obtained by modifying this reducing end through aldehyde-selective reaction pathways. CNCs with end-tethered polymers chains present an interesting alternative to uniformly modified CNCs for certain applications, in which their self-assembly can be exploited toward structural hierarchies with unique properties, including both assemblies on surfaces and in nanocomposite materials.<sup>8</sup> Depending on the molecular weight of end-tethered polymers, one can see such anisotropic colloidal building blocks as somewhat analogous to rod–coil block copolymers.<sup>182</sup>

Different procedures have been established in order to attach a small molecule or initiator to the reducing end groups of CNCs. Arcot et al. reported a method to introduce thiol groups on the reducing ends of the CNCs by reductive amination in an aqueous medium.<sup>183</sup> The authors synthesized thiolated cellulose nanocrystals (CNC-SH) by using 6-amino-1-hexanethiol

hydrochloride (NH<sub>2</sub>(CH<sub>2</sub>)<sub>6</sub>SH·HCl), a reducing agent (Cn, sodium cyanoborohydride; Ac, sodium triacetoxyborohydride; or Pc, 2-picoline-borane complex) and an aqueous CNC dispersion at different pH (5, 7 or 9.2) values. The CNC-SH were then functionalized with silver nanoparticles (AgNPs) in order to characterize the successful reducing end group modification, qualitatively using transmission electron microscopy (TEM) and quantitatively using XPS. The authors determined that the highest yield of functionalization was obtained when using basic conditions.

The same group later introduced a second thiolation procedure with shorter reaction times, milder conditions, and higher colloidal stability of the modified CNCs.<sup>184</sup> Usuda and coworkers had previously demonstrated the selective oxidation of reducing ends to carboxylates with sodium chlorite (NaClO<sub>2</sub>), which gives access to a wider variety of potential modification chemistries.<sup>185</sup> Thus, the reducing ends were first reacted with NaClO<sub>2</sub> to generate carboxylate functionalized CNC reducing ends (CNC-COOH). After this oxidation step, a mild activation step of the carboxyl group was carried out mediated by N-hydroxysuccinimide (NHS) and N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC), which was followed by a nucleophilic substitution with NH<sub>2</sub>(CH<sub>2</sub>)<sub>6</sub>SH·HCl, resulting in CNC-SH.

Gruber et al. proposed a different reducing end group functionalization strategy via hydrazone linkages.<sup>186</sup> The authors used 4-hydrazino-benzoic acid or radical initiators 4,4'-azobis(4-cyanopentanoic dihydrazide) and 4-carboxyphenylazomethylmalonodinitrile to form the hydrazone linkage, which resulted in a phenylcarboxylate terminated CNC with a yield of 44.6%. Similar yields with the conversion of 4-hydrazinobenzoic acid were obtained for the other initiators. The authors hypothesized that the low yield was due to the poor accessibility of the reducing end groups. If one considers individualized CNCs to consist of 18-chains,<sup>58</sup> this would imply a maximum of 18 reducing end groups at one end of the cellulose I crystal. Given the rod-like shape of CNCs, the reducing end-groups are likely distributed heterogeneously. This may lead to varying degrees of reducing end group accessibility for chemical reactions, however this remains to be explored.

Gruber and coworkers grafted PAM by SI-FRP from different azo initiators discussed in section 3.2 and shown in Table 1.<sup>186</sup> The molecular weight of the polymers were determined by SEC measurements after degrafting the PAM from the CNCs. The authors observed high molecular weights for PAM, but only a small amount of the initiators reacted to yield the polymer-grafted CNCs. Initiator efficiencies for surface-initiated polymerizations are reported to be very low, e.g., 5–30%.<sup>28</sup>

Recently, Zoppe et al. reported a route to functionalize the end groups with an SI-ATRP initiator containing α-bromoisobutyryl groups.<sup>172</sup> The reducing ends were first oxidized with NaClO<sub>2</sub> and further modified with ethylenediamine (EDA), following the work of Arcot et al.<sup>184</sup> Subsequently, primary amine groups were reacted with 2-bromoisobutanoic acid N-hydroxysuccinimide ester (NHS-BiB) to yield the ATRP initiator-functionalized reducing end groups. Although the authors were unable to quantify the success of the initiator modification reactions with conventional techniques, such as FTIR, XPS and elemental analysis, they subsequently grafted a variety of polymers from the functionalized reducing end groups of CNCs via SI-ATRP. The monomers investigated included NIPAM, METAC, and 4-SS. Thermogravimetric analysis (TGA), FTIR, and XPS were used to qualitatively analyze the
<table><caption>Table 1. Polymerization of Different Monomers from the CNC Reducing Ends<sup>172,186</sup></caption><thead><tr><th>Initiator</th><th>Technique</th><th>Link</th><th>Monomer</th><th>Structure</th></tr></thead><tbody><tr><td>4,4'-Azobis(4-cyanopentanoic dihydrazide)</td><td>SI-FRP</td><td>Hydrazone</td><td>AM</td><td>Cell-OH-OH-OH-OH-CN-OH-NH-CO-OH-CN-OH<br>NH<sub>2</sub></td></tr><tr><td>p-Carboxyphenylazomethylmalonodinitrile</td><td>SI-FRP</td><td>Hydrazone</td><td>AM</td><td>Cell-OH-OH-OH-OH-CO-OH-CO-OH-CN-OH-NH-CO-OH-CN-OH<br>NH<sub>2</sub></td></tr><tr><td rowspan="3">NHS-BiB</td><td rowspan="3">SI-ATRP</td><td rowspan="3">Diacetamide</td><td>NIPAM</td><td>Cell-OH-OH-OH-OH-CN-OH-NH-CO-OH-CN-OH-NH-CO-OH-CN-OH-Br</td></tr><tr><td>METAC</td><td>Cell-OH-OH-OH-OH-CN-OH-NH-CO-OH-CN-OH-Br<br>(H<sub>3</sub>C)<sub>3</sub>N-Cl</td></tr><tr><td>4-SS</td><td>Cell-OH-OH-OH-OH-CN-OH-NH-CO-OH-CN-OH-Br<br>Na-O-S-O-OH-CN-OH-Br</td></tr></tbody></table>

successful grafting of the CNCs. In order to directly visualize the selective modification of CNC reducing ends with grafted polymers, patchy CNC/PMETAC hybrids were labeled with gold nanoparticles (AuNPs) and observed by cryo-TEM (Figure 7).

<figure><img src="image_22.png" /><figcaption>Figure 7. Cryo-TEM micrograph of patchy CNC/PMETAC hybrids labeled with AuNPs. Reprinted with permission from ref. 172. Copyright 2017 American Chemical Society.</figcaption></figure>

This image supports the point that the authors successfully achieved the preferential modification of the CNC ends, but nevertheless, optimization of the reaction conditions for both the end group functionalization and the AuNPs adsorption is

necessary in future work, since it was clear that not all CNCs were successfully functionalized. Moreover, better characterization techniques are needed to quantify the number of initiators on the CNC reducing end groups.

## 4. EMERGING APPLICATIONS AND PROPERTIES OF POLYMER-MODIFIED CNCS VIA "GRAFTING FROM"

The rapidly growing academic and commercial interest in CNCs and hybrid nanomaterials thereof has driven their suggested use in unprecedented applications.<sup>3,5,7,8,24,26,92</sup> These include Picking emulsions, nanotemplates for functional materials, supercapacitors, biosensors, drug delivery, and enantioselective catalysis. Here, we will focus on some of the emerging applications of polymer-modified CNCs, in which the grafting from approach, where high grafting density and molecular weight, is a priori advantageous over other polymer grafting strategies. In the following sections, applications in hybrid colloidal liquid crystals (LC), stimuli-responsive nanohybrids, nanocomposites and hydrogels as well as their unique properties will be discussed.

### 4.1. Self-Assembly, Hybrid Colloidal Liquid Crystals, and Optical Properties.
CNCs have the intriguing ability to form chiral nematic LC phases, which depend on several factors, notably aspect ratio, surface charge density, ionic strength, external fields and solvent conditions.<sup>31c</sup> Above a critical concentration, CNCs undergo a phase transition from an isotropic phase to a chiral nematic (cholesteric) liquid-crystalline phase (Figure 8).<sup>187</sup> The rods align preferentially with their long axes along a director. Each director of every pseudolayer (defined as an infinitesimally thin layer taken perpendicularly to the helicoidal axis) is slightly rotated around the helicoidal axis for each subsequent layer. The vertical distance required to complete a 360° rotation of the director is defined as the pitch.<sup>188,189</sup> When the suspension is left to dry on a substrate, the chiral nematic structure that forms upon increasing sample concentration is maintained after complete evaporation of the solvent, resulting in a film whose pitch
<figure><img src="image_19.png" /><figcaption>**Figure 8.** Schematic representation of the self-assembly of a CNC suspension upon evaporation to form a structurally organized array. (A) Phase diagram showing the transition from isotropic to cholesteric phase upon increase of CNC concentration. (B) Atom force microscopy image of individual CNCs. (C) Polarized optical microscopy (POM) image of a typical fingerprint pattern of the cholesteric phase. Reprinted with permission from ref 31. Copyright 2017, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>**Figure 9.** POM images showing (A) the fingerprint texture of pure CNC in water (3.1 wt %), and the fingerprint of different polymer-modified CNCs in their zone of anisotropic phase, i.e., (B) CNC-g-PMMAZO in chlorobenzene (5.1 wt %), (C) CNC-g-PSty in DMF (5.7 wt %), and (D) CNC-g-PDMAEMA in water (4.7 wt %, after 3 days) scale bar 10 µm for A, C and D and 100 µm for B. Adapted with permission from ref 141 (A, C) and ref 151 (B), copyright 2008 Elsevier Ltd., and ref 152 (D), copyright 2009, Springer Science+Business Media B.V.</figcaption></figure>

dimension decreases to the submicrometer scale.³¹ By exerting control over the drying conditions, the film can be homogeneous in thickness while retaining the long-range order of the CNCs.¹⁹⁰ Thus, the film behaves as a photonic crystal, and only light that is circularly polarized with the same handedness as the colloidal helices is reflected, in this case left-handed circularly polarized light.¹⁸⁸ These attractive properties of the self-assembled CNCs give a wide variety of interesting applications, such as devices for optical encryption, sensors, mesoporous chiral nanotemplates, and security coatings.¹⁸⁸,¹⁸⁹,¹⁹¹,¹⁹²

Grafting polymers from the surface of CNCs, without a doubt, will affect their assembly behavior. Self-assembly of polymer-tethered CNCs is of great interest since more complex assemblies can be targeted through entropy-driven processes. Furthermore, their self-assembly can be potentially obtained in a variety of solvents, since the dispersibility of the CNCs can be tuned by surface modification. Such grafted-CNCs could give rise to a variety of CNC-based functional materials and templates based on colloidal LC assemblies. Nevertheless, self-assembly of polymer-grafted CNCs has not been widely explored to date, since the grafts likely alter the packing density of the CNCs, where they may behave more like spherical objects upon reaching a certain graft molecular weight and grafting density.¹⁹³ However, by using grafting to approaches, Jean et al. showed that polyetheramine-modified CNCs self-assembled into chiral nematic phases, albeit at a lower critical concentration in comparison to unmodified CNCs.¹⁹⁴

Zhang and co-workers investigated the thermotropic and lyotropic LC phase behavior of CNC-g-PMMAZO,¹⁵¹ CNC-g-PSty,¹⁴¹

and CNC-g-PDMAEMA¹⁵² (Figure 9). The CNC-g-PMMAZO showed a smectic to nematic transition at 95 °C and a nematic to isotropic transition at 135 °C in the absence of a solvent.¹⁵¹ Furthermore, for these grafted CNCs, the POM image reveals a lyotropic LC phase in chlorobenzene above a concentration of 5.1 wt %. The authors reported a chiral nematic liquid crystalline behavior for the CNC-g-PSty, in both the lyotropic (DMF) and thermotropic states.¹⁴¹ In the latter case, chiral nematic phases were observed upon cooling CNC-g-PSty to 163 °C, highlighting the potential of generating thermotropic LC phases based on CNCs grafted with nonmesogenic polymers. The authors later grafted thermosensitive PDMAEMA from CNCs.¹⁵² They observed the fingerprint pattern for the CNC-g-PDMAEMA in the anisotropic phase, indicating chiral organization in aqueous media. The spacing of the fingerprint lines in aqueous suspensions could be tuned by changing the temperature. Below the lower critical solution temperature (LCST) of PDMAEMA, the polymer brushes existed in an extended conformation and therefore increased the spacing of the fingerprint, the opposite of which occurred above the LCST. Such systems are of great potential in developing colloidal analogues of stimuli-responsive molecular mesogens. The ability to externally manipulate the LC phases of CNCs in a reversible manner opens a number of interesting possibilities, including templating chiral 3D nanostructures,¹⁹² optical sensors,³¹ nanocomposite liquid crystalline elastomers (LCEs),¹⁹⁵ and multiresponsive supracolloidal systems.¹⁹⁶

**4.2. Stimuli-Responsive Nanohybrids.** Biopolymers in living organisms are ubiquitously involved in dynamic systems
<figure><img src="image_23.png" /><figcaption>**Figure 8.** Schematic representation of the self-assembly of a CNC suspension upon evaporation to form a structurally organized array. (A) Phase diagram showing the transition from isotropic to cholesteric phase upon increase of CNC concentration. (B) Atom force microscopy image of individual CNCs. (C) Polarized optical microscopy (POM) image of a typical fingerprint pattern of the cholesteric phase. Reprinted with permission from ref 31. Copyright 2017, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>**Figure 9.** POM images showing (A) the fingerprint texture of pure CNC in water (3.1 wt %), and the fingerprint of different polymer-modified CNCs in their zone of anisotropic phase, i.e., (B) CNC-g-PMMAZO in chlorobenzene (5.1 wt %), (C) CNC-g-PSty in DMF (5.7 wt %), and (D) CNC-g-PDMAEMA in water (4.7 wt %, after 3 days) scale bar 10 µm for A, C and D and 100 µm for B. Adapted with permission from ref 141 (A, C) and ref 151 (B), copyright 2008 Elsevier Ltd., and ref 152 (D), copyright 2009, Springer Science+Business Media B.V.</figcaption></figure>

dimension decreases to the submicrometer scale.³¹ By exerting control over the drying conditions, the film can be homogeneous in thickness while retaining the long-range order of the CNCs.¹⁹⁰ Thus, the film behaves as a photonic crystal, and only light that is circularly polarized with the same handedness as the colloidal helices is reflected, in this case left-handed circularly polarized light.¹⁸⁸ These attractive properties of the self-assembled CNCs give a wide variety of interesting applications, such as devices for optical encryption, sensors, mesoporous chiral nanotemplates, and security coatings.¹⁸⁸,¹⁸⁹,¹⁹¹,¹⁹²

Grafting polymers from the surface of CNCs, without a doubt, will affect their assembly behavior. Self-assembly of polymer-tethered CNCs is of great interest since more complex assemblies can be targeted through entropy-driven processes. Furthermore, their self-assembly can be potentially obtained in a variety of solvents, since the dispersibility of the CNCs can be tuned by surface modification. Such grafted-CNCs could give rise to a variety of CNC-based functional materials and templates based on colloidal LC assemblies. Nevertheless, self-assembly of polymer-grafted CNCs has not been widely explored to date, since the grafts likely alter the packing density of the CNCs, where they may behave more like spherical objects upon reaching a certain graft molecular weight and grafting density.¹⁹³ However, by using grafting to approaches, Jean et al. showed that polyetheramine-modified CNCs self-assembled into chiral nematic phases, albeit at a lower critical concentration in comparison to unmodified CNCs.¹⁹⁴

Zhang and co-workers investigated the thermotropic and lyotropic LC phase behavior of CNC-g-PMMAZO,¹⁵¹ CNC-g-PSty,¹⁴¹

and CNC-g-PDMAEMA¹⁵² (Figure 9). The CNC-g-PMMAZO showed a smectic to nematic transition at 95 °C and a nematic to isotropic transition at 135 °C in the absence of a solvent.¹⁵¹ Furthermore, for these grafted CNCs, the POM image reveals a lyotropic LC phase in chlorobenzene above a concentration of 5.1 wt %. The authors reported a chiral nematic liquid crystalline behavior for the CNC-g-PSty, in both the lyotropic (DMF) and thermotropic states.¹⁴¹ In the latter case, chiral nematic phases were observed upon cooling CNC-g-PSty to 163 °C, highlighting the potential of generating thermotropic LC phases based on CNCs grafted with nonmesogenic polymers. The authors later grafted thermosensitive PDMAEMA from CNCs.¹⁵² They observed the fingerprint pattern for the CNC-g-PDMAEMA in the anisotropic phase, indicating chiral organization in aqueous media. The spacing of the fingerprint lines in aqueous suspensions could be tuned by changing the temperature. Below the lower critical solution temperature (LCST) of PDMAEMA, the polymer brushes existed in an extended conformation and therefore increased the spacing of the fingerprint, the opposite of which occurred above the LCST. Such systems are of great potential in developing colloidal analogues of stimuli-responsive molecular mesogens. The ability to externally manipulate the LC phases of CNCs in a reversible manner opens a number of interesting possibilities, including templating chiral 3D nanostructures,¹⁹² optical sensors,³¹ nanocomposite liquid crystalline elastomers (LCEs),¹⁹⁵ and multiresponsive supracolloidal systems.¹⁹⁶

**4.2. Stimuli-Responsive Nanohybrids.** Biopolymers in living organisms are ubiquitously involved in dynamic systems
<figure><img src="image_21.png" /><figcaption>**Figure 10.** Schematic representation of stimuli-responsive hybrids based on polymer-grafted CNCs. (A) Transitions in solution of stimuli-responsive polymer modified CNCs from well dispersed to aggregates or vice versa as response to an external stimulus, such as pH, temperature or carbon dioxide (CO₂) atmosphere. (B) Thermally triggered triple shape memory behavior is achieved with poly(soybean amide methacrylate) (PSBAM) grafted CNCs dispersed in a PSBAM matrix in the presence of 4,4'-(1,4-phenylene)-bis(1,2,4-triazoline-3,5-dione) (bis-TAD) as cross-linker, where H-bonding between PSBAM chains is also at play.</figcaption></figure>

that promote structural changes in response to the conditions in their surrounding environment.¹⁹⁷ These properties are based on highly cooperative interactions that provide a significant driving force for the response caused by small external changes. Similar adaptive behavior can be imparted to synthetic polymers by incorporating functional groups in their backbone capable of responding to diverse external chemical (pH, ionic factor or chemical agents) or physical stimuli (temperature, electronic or magnetic fields, light or mechanical stress) (Figure 10). These are known as stimuli-responsive or smart polymers. By grafting such polymers from the surface of CNCs, hybrid nanomaterials bearing reversible interfacial properties can be fabricated.

**pH-Responsive Hybrids.** Polymers that undergo chemical and morphological changes as a result of pH variations are known as pH-responsive polymers. They usually contain functional groups that can be protonated, resulting in a transition from a hydrophilic to a hydrophobic state or vice versa, which may induce precipitation in aqueous solutions. CNC-g-P4VP was reported to display a completely reversible pH-responsive behavior in water.¹³² P4VP is a well-known polyelectrolyte with an acid dissociation constant ($pK_a$) of around 5, and is thus present in a hydrophilic protonated state at a pH of lower than 5, but it can be rendered hydrophobic at higher pH values due to the deprotonation of the pyridyl groups. Accordingly, suspensions of CNC-g-P4VP in water show good colloidal stability in acidic environments but flocculate above a pH of 5, due to the change from hydrophilic to hydrophobic character of the grafts.

In a different study, CNC-g-PAA also showed equivalent properties due to the nature of its brushes.¹⁷⁸ PAA is a weak polyelectrolyte, which undergoes pH-dependent conformation transition according to the ionization state of its carboxylic groups; i.e., the chains adopt a partial globular conformation in their protonated form (at low pH) and change to a coiled conformation when deprotonated at high pH.¹⁹⁸ The CNC-g-PAA suspensions showed lower UV-vis absorbance at pH values higher than 7 as consequence of the ionizations of the carboxylic

groups present in the grafts, which promotes the complete solvation of polymer chains.¹⁷⁸

pH variations of a colloid can also be promoted by its exposition to acidic or basic gases, such as CO₂ and N₂, respectively. In addition, it is known that polymers containing tertiary amines are easily protonated in the presence of water and CO₂, which involves the formation of carbonic acid and an increase in the hydrophilicity character of the polymer. The combination of these two concepts was applied to CNCs by decoration with PDMAEMA, PDEAEMA and PDMAPMAM either via S-NMP¹⁸¹ or SI-ATRP.¹⁷¹ The CO₂-responsiveness of these systems was evaluated by determining the ζ-potential versus pH in alternating CO₂/N₂ exposure cycles (Figure 11). In all cases, high ζ-potential values were observed for stable dispersion when grafts were protonated under CO₂ atmosphere (low pH), while low ζ-potential values were reported when the dispersion flocculated in the presence of N₂ (high pH). From the three hybrids studied, CNC-g-PDEAEMA clearly demonstrated the pH-switchability of the system due to its sole solubility in water under its protonated form, i.e., in the presence of CO₂.

**Temperature-Responsive Hybrids.** Thermosensitive polymers respond to a change in their solvation state upon heating or cooling. According to the way the measurement is performed, the reported value should be cited as cloud point, upper (UCST) or lower critical solution temperature (LCST). The cloud point is an experimentally measured value at which the first appearance of turbidity or cloudiness are observed due to the phase separation of the components of a mixture as a response of a stimulus (i.e., temperature).¹⁹⁹ When plotting these cloud-point temperatures (CPT) vs the overall polymer concentration, the cloud-point curve is obtained, where the minimum point is known as the precipitation threshold (or LCST).²⁰⁰ In this sense, the term LCST only refers to the lowest temperature at which phase separation can happen.

Decorating CNCs with thermosensitive polymers can lead to hybrid materials with temperature-responsive features. CNC-g-PNIPAM has demonstrated thermal behavior due to the ability
<figure><img src="image_25.png" /><figcaption>**Figure 10.** Schematic representation of stimuli-responsive hybrids based on polymer-grafted CNCs. (A) Transitions in solution of stimuli-responsive polymer modified CNCs from well dispersed to aggregates or vice versa as response to an external stimulus, such as pH, temperature or carbon dioxide (CO₂) atmosphere. (B) Thermally triggered triple shape memory behavior is achieved with poly(soybean amide methacrylate) (PSBAM) grafted CNCs dispersed in a PSBAM matrix in the presence of 4,4'-(1,4-phenylene)-bis(1,2,4-triazoline-3,5-dione) (bis-TAD) as cross-linker, where H-bonding between PSBAM chains is also at play.</figcaption></figure>

that promote structural changes in response to the conditions in their surrounding environment.¹⁹⁷ These properties are based on highly cooperative interactions that provide a significant driving force for the response caused by small external changes. Similar adaptive behavior can be imparted to synthetic polymers by incorporating functional groups in their backbone capable of responding to diverse external chemical (pH, ionic factor or chemical agents) or physical stimuli (temperature, electronic or magnetic fields, light or mechanical stress) (Figure 10). These are known as stimuli-responsive or smart polymers. By grafting such polymers from the surface of CNCs, hybrid nanomaterials bearing reversible interfacial properties can be fabricated.

**pH-Responsive Hybrids.** Polymers that undergo chemical and morphological changes as a result of pH variations are known as pH-responsive polymers. They usually contain functional groups that can be protonated, resulting in a transition from a hydrophilic to a hydrophobic state or vice versa, which may induce precipitation in aqueous solutions. CNC-g-P4VP was reported to display a completely reversible pH-responsive behavior in water.¹³² P4VP is a well-known polyelectrolyte with an acid dissociation constant ($pK_a$) of around 5, and is thus present in a hydrophilic protonated state at a pH of lower than 5, but it can be rendered hydrophobic at higher pH values due to the deprotonation of the pyridyl groups. Accordingly, suspensions of CNC-g-P4VP in water show good colloidal stability in acidic environments but flocculate above a pH of 5, due to the change from hydrophilic to hydrophobic character of the grafts.

In a different study, CNC-g-PAA also showed equivalent properties due to the nature of its brushes.¹⁷⁸ PAA is a weak polyelectrolyte, which undergoes pH-dependent conformation transition according to the ionization state of its carboxylic groups; i.e., the chains adopt a partial globular conformation in their protonated form (at low pH) and change to a coiled conformation when deprotonated at high pH.¹⁹⁸ The CNC-g-PAA suspensions showed lower UV-vis absorbance at pH values higher than 7 as consequence of the ionizations of the carboxylic

groups present in the grafts, which promotes the complete solvation of polymer chains.¹⁷⁸

pH variations of a colloid can also be promoted by its exposition to acidic or basic gases, such as CO₂ and N₂, respectively. In addition, it is known that polymers containing tertiary amines are easily protonated in the presence of water and CO₂, which involves the formation of carbonic acid and an increase in the hydrophilicity character of the polymer. The combination of these two concepts was applied to CNCs by decoration with PDMAEMA, PDEAEMA and PDMAPMAM either via S-NMP¹⁸¹ or SI-ATRP.¹⁷¹ The CO₂-responsiveness of these systems was evaluated by determining the ζ-potential versus pH in alternating CO₂/N₂ exposure cycles (Figure 11). In all cases, high ζ-potential values were observed for stable dispersion when grafts were protonated under CO₂ atmosphere (low pH), while low ζ-potential values were reported when the dispersion flocculated in the presence of N₂ (high pH). From the three hybrids studied, CNC-g-PDEAEMA clearly demonstrated the pH-switchability of the system due to its sole solubility in water under its protonated form, i.e., in the presence of CO₂.

**Temperature-Responsive Hybrids.** Thermosensitive polymers respond to a change in their solvation state upon heating or cooling. According to the way the measurement is performed, the reported value should be cited as cloud point, upper (UCST) or lower critical solution temperature (LCST). The cloud point is an experimentally measured value at which the first appearance of turbidity or cloudiness are observed due to the phase separation of the components of a mixture as a response of a stimulus (i.e., temperature).¹⁹⁹ When plotting these cloud-point temperatures (CPT) vs the overall polymer concentration, the cloud-point curve is obtained, where the minimum point is known as the precipitation threshold (or LCST).²⁰⁰ In this sense, the term LCST only refers to the lowest temperature at which phase separation can happen.

Decorating CNCs with thermosensitive polymers can lead to hybrid materials with temperature-responsive features. CNC-g-PNIPAM has demonstrated thermal behavior due to the ability
<figure><img src="image_22.png" /><figcaption>**Figure 11.** ζ-Potential and pH at room temperature of aqueous dispersions (1 mg·mL⁻¹) of (A) CNC-g-PDMAEMA, (B) CNC-g-PDEAEMA and (C) CNC-g-PDMAPMAM in alternating CO₂/N₂ exposure cycles. Adapted from ref 181. Copyright 2017, Royal Society of Chemistry.</figcaption></figure>

of PNIPAM to undergo a coil-to-globule transition at about 32 °C.<sup>164,178,201</sup> Zoppe et al. reported for the first time the thermoresponsive behavior of such materials as a function of grafting density and degree of polymerization of PNIPAM, which was prepared by SI-ATRP.<sup>201</sup> In this study, suspensions of CNC-g-PNIPAM were shown to respond to temperature changes between 30 and 32 °C (CPT) by a significant increase in their viscosity (Figure 12A). The CPT of the hybrid materials was shown to approach the LCST of the free polymer as the grafting density was increased. In addition to the nature of the grafts, the CPT was also influenced by the differences in the steric repulsive forces between adjacent polymer-modified CNCs. The latter was also shown to affect the size of aggregates at temperatures higher than the LCST of free polymer. In the same study, the CPT of these materials also was shown to decrease upon increasing the ionic strength (i.e., by varying the concentration of NaCl), as is the case of free PNIPAM in aqueous solution (Figure 12B).

In another study, CNC-g-PNIPAM also demonstrated a sol–gel phase transition at about 34.3 °C, which may be exploited for embolization of hepatocellular carcinoma.<sup>164</sup> The weight-average molecular weight (M<sub>w</sub>) of these grafts was more than 10 times lower than the one reported by Zoppe et al.<sup>201</sup>

<figure><img src="image_23.png" /><figcaption>**Figure 12.** Thermoresponsive behavior of CNC-g-PNIPAM. (A) Viscosity of CNC-g-PNIPAM with various graft densities (i.e., VHD, very high; HD, high; MD, medium; and LD, low densities) at different temperature normalized by the viscosity of the corresponding dispersion at 25 °C. (B) LCST of aqueous dispersions of medium density CNC-g-PNIPAM at various NaCl concentrations. Adapted from ref 201. Copyright 2011, American Chemical Society.</figcaption></figure>

This indicates that the molecular weight of the grafts has a direct influence on the thermal behavior of the hybrid nanoparticles. Furthermore, Haqani et al. also reported the thermoresponsiveness of CNC-g-PNIPAM with a CPT around 29 °C.<sup>178</sup> Such differences in CPTs are often associated with differences in molecular weight, which is known for PNIPAM chains in solution,<sup>202</sup> and the grafting density of PNIPAM brushes.

CNC-g-PNVC was also reported to display a thermally induced phase transition above 36 °C from a stable colloidal aqueous suspension to collapsed PNVC brushes due to a change of the hydrophilicity of the grafts.<sup>161</sup> CNC-g-POEGMEMA<sup>189</sup> and CNC-g-poly(ethylene glycol)methyl acrylate<sup>160</sup> have exhibited tunable thermal behavior (between 23.8 and 63.8 °C and between 34 and 66 °C, respectively); the respective cloud points could readily be tailored by adjusting the comonomer ratio in the grafted polymers. In all cases, the transition was confirmed by changes in the viscoelastic properties of the materials.

A thermoresponsive enhanced fluorescence hybrid based on CNC-g-P(NIPAM-co-EANI) was recently synthesized. This
<figure><img src="image_26.png" /><figcaption>**Figure 11.** ζ-Potential and pH at room temperature of aqueous dispersions (1 mg·mL⁻¹) of (A) CNC-g-PDMAEMA, (B) CNC-g-PDEAEMA and (C) CNC-g-PDMAPMAM in alternating CO₂/N₂ exposure cycles. Adapted from ref 181. Copyright 2017, Royal Society of Chemistry.</figcaption></figure>

of PNIPAM to undergo a coil-to-globule transition at about 32 °C.<sup>164,178,201</sup> Zoppe et al. reported for the first time the thermoresponsive behavior of such materials as a function of grafting density and degree of polymerization of PNIPAM, which was prepared by SI-ATRP.<sup>201</sup> In this study, suspensions of CNC-g-PNIPAM were shown to respond to temperature changes between 30 and 32 °C (CPT) by a significant increase in their viscosity (Figure 12A). The CPT of the hybrid materials was shown to approach the LCST of the free polymer as the grafting density was increased. In addition to the nature of the grafts, the CPT was also influenced by the differences in the steric repulsive forces between adjacent polymer-modified CNCs. The latter was also shown to affect the size of aggregates at temperatures higher than the LCST of free polymer. In the same study, the CPT of these materials also was shown to decrease upon increasing the ionic strength (i.e., by varying the concentration of NaCl), as is the case of free PNIPAM in aqueous solution (Figure 12B).

In another study, CNC-g-PNIPAM also demonstrated a sol–gel phase transition at about 34.3 °C, which may be exploited for embolization of hepatocellular carcinoma.<sup>164</sup> The weight-average molecular weight (M<sub>w</sub>) of these grafts was more than 10 times lower than the one reported by Zoppe et al.<sup>201</sup>

<figure><img src="image_27.png" /><figcaption>**Figure 12.** Thermoresponsive behavior of CNC-g-PNIPAM. (A) Viscosity of CNC-g-PNIPAM with various graft densities (i.e., VHD, very high; HD, high; MD, medium; and LD, low densities) at different temperature normalized by the viscosity of the corresponding dispersion at 25 °C. (B) LCST of aqueous dispersions of medium density CNC-g-PNIPAM at various NaCl concentrations. Adapted from ref 201. Copyright 2011, American Chemical Society.</figcaption></figure>

This indicates that the molecular weight of the grafts has a direct influence on the thermal behavior of the hybrid nanoparticles. Furthermore, Haqani et al. also reported the thermoresponsiveness of CNC-g-PNIPAM with a CPT around 29 °C.<sup>178</sup> Such differences in CPTs are often associated with differences in molecular weight, which is known for PNIPAM chains in solution,<sup>202</sup> and the grafting density of PNIPAM brushes.

CNC-g-PNVC was also reported to display a thermally induced phase transition above 36 °C from a stable colloidal aqueous suspension to collapsed PNVC brushes due to a change of the hydrophilicity of the grafts.<sup>161</sup> CNC-g-POEGMEMA<sup>189</sup> and CNC-g-poly(ethylene glycol)methyl acrylate<sup>160</sup> have exhibited tunable thermal behavior (between 23.8 and 63.8 °C and between 34 and 66 °C, respectively); the respective cloud points could readily be tailored by adjusting the comonomer ratio in the grafted polymers. In all cases, the transition was confirmed by changes in the viscoelastic properties of the materials.

A thermoresponsive enhanced fluorescence hybrid based on CNC-g-P(NIPAM-co-EANI) was recently synthesized. This
nanomaterial reacted to temperature variations while display-
ing a simultaneous increase in its fluorescence intensity.¹⁵⁴
The latter was attributed to the thermoresponsiveness of
PNIPAM brushes, which altered the distance between the dye
groups.

**Dual pH- and Temperature-Responsive Hybrids.** Contributions of polymeric chains bearing different stimuli-responsive properties can result in copolymers with multiple response functions. In the domain of polymer-grafted CNCs, the first example of hybrids reporting such dual-responsiveness was CNC-g-P(NIPAM-ran-AA) containing different amounts of AA, which responded to pH and temperature variations.¹⁴² The CPT of this system was tuned by three factors: polymer tethering to CNCs, AA content, and pH (Figure 13). The CPT increased

<figure><img src="image_24.png" /><figcaption>**Figure 13.** LCST diagram of 0.2 wt % aqueous suspensions of P(NIPAM-ran-AA) as free copolymer (red) or grafted to CNCs (blue) bearing different AA content at low and high pH values. Reprinted with permission of ref 142. Copyright 2014, Royal Society of Chemistry.</figcaption></figure>

from 38 to 45 °C at high pH (ca. 7–8) while it decreased from
30 to 18 °C at lower pH values (ca. 3–4), when the AA content
of the grafts was increased from 5 to 30 mol %. The reduction
observed in the CPT was mainly attributed to the hydrophobic
behavior of AA in protic media. In addition, a comparison
between free and CNC-grafted copolymers demonstrated that
tethering copolymers to CNCs resulted in an increase up to 6 °C
in the CPT, with respect to the LCST of free copolymer.

Using the same monomers, albeit with block rather than
random distribution of comonomers and varying the inner
and outer block, Haqani et al. also reported thermoresponsive
systems. A dual-sensitive characteristic was only observed when
the PAA was at the outer block, i.e. CNC-g-PNIPAM-block-
PAA.¹⁷⁸ Furthermore, Tang et al. developed a hybrid, containing
POEGMA and PMAA grafted-CNCs, in order to control the
stability of particle-stabilized emulsions manipulated by pH and
temperature.¹³⁵ PMAA brushes formed hydrogen bonds with
POEGMA at low pH values (<4.5), leading to the formation of
aggregates, while the presence of electrostatic repulsions at higher
pH disrupted hydrogen bonding to yield stable dispersions.
Additionally, POEGMA chains exhibited temperature-dependent

conformational changes that influenced the stability of the
emulsion.

*Shape-Memory Hybrids.* Shape-memory polymers (SMPs) are those capable of being deformed and returned to their original state through a specific stimulus, for example, via thermal transitions or activation–deactivation of supramolecular interactions, often in elastomeric systems. Wang et al. proposed a shape-memory nanocomposite derived from biomass, i.e., soybean oil and CNC.¹⁵⁶ The system was composed of CNC-g-PSBAM (synthesized via SI-ATRP with a theoretical M~n~ = 22 kDa) and bis-TAD using PSBAM as a matrix (Figure 10B). The network of the hybrid was formed by using two types of cross-links within the material: one covalently linked by bis-TAD and another dynamic physically cross-linked via hydrogen bonding with the plant oil functional groups (and also the bis-TAD moiety). Here, the covalently bonded network was responsible for the permanent shape of the material, while the physical network provided multiresponsive and multi shape-memory properties. The material showed triple-shape-memory with two recovery steps performed at staged temperatures of 40 and 80 °C, respectively. Additionally, water and THF induced the shape-recovery of the material within 30 and 5 min of immersion, respectively.

The properties exhibited by the above-mentioned stimuli-
responsive nanohybrids are mainly attributed to the type of
polymer grafted on the CNC surface. Thus, the main function of
the CNCs is to provide either mechanical reinforcement¹⁵⁶ to
the material or enhance their solubility¹⁴² in polar solvents due
to the large amount of –OH groups at the surface of CNCs.
Most of these investigations have been focused on the synthesis
of stimuli-responsive materials and their behavior upon applica-
tion of different stimuli, but little is known about the minimum
amount of grafted polymer required to observe such stimuli-
responsive behavior. Furthermore, it is in many cases unclear
how the grafting density of the polymer influences the respon-
siveness of the material.

**4.3. Nanocomposites.** Nanocomposites are materials composed of two or more components, often a matrix and a filler, in the solid or gel-like state, the filler being on the nanoscale. There are different processing routes reported in the literature for the production of cellulose nanocomposites, such as solution casting, melt extrusion, compression molding, injection molding, ball milling, or even precipitation methods.⁴,⁵,⁷,²⁰³ The outstanding mechanical properties and the high specific area suggest that CNCs would provide good reinforcement to other polymeric materials. The reinforcement potential of CNCs was first reported by Favier et al., who incorporated tunicate-derived CNCs into a copolymer of Sty and BA (P(Sty-co-BA)).²⁰⁴,²⁰⁵ The reinforcement effect of CNCs is often explained by the formation of a rigid percolating network in which the filler nanoparticles interact via hydrogen bonding.⁵ The experimental evidence and statistical models stipulate that the matrix, the filler characteristics, such as aspect ratio, dispersion and volume fraction, and their interactions (filler–matrix and filler–filler) all influence the mechanical properties of the nanocomposites.³

Even though unmodified CNCs have been reported to offer
reinforcement in some materials,²⁰⁴⁻²⁰⁶ the hydrophilicity of
the CNC surface makes their dispersion difficult in polymer
matrices, especially those that are nonpolar or hydrophobic.
In addition, the numerous –OH groups at their surface promotes
the agglomeration of CNCs via hydrogen bonding limiting the
extent of mechanical reinforcement. In that regard, surface modi-
fication of CNCs is a viable approach to create good interfacial