*3.1. Synthesis and Characterization of Cationic CNCs*

CNCs with cationic polymer brushes were synthesized by grafting poly (METAC) or poly(AEM) via SI-SET-LRP from the surface of pristine CNCs (Scheme 1). The first step required the covalent attachment of an initiator on the surface of CNCs using a well-known esterification method of the hydroxyl groups of CNCs with an acyl bromide [23,26,27]. CNC-initiator (CNC-BriB) were prepared at two different initiator loading capacity with respect to anhydroglucose units (AGU) in CNCs ([Br]/[AGU]), 5:3 for CNC-BriB-1 and 5:12 for CNC-BriB-2). The next key step involved a grafting from approach to introduce cationic poly(METAC) or poly(AEM) brushes by SI-SET-LRP on CNCs under mild reaction conditions (Scheme 1B). SET-LRP is a robust and versatile polymerization technique for materials synthesis [28] and had been previously used to graft thermoresponsive and methacrylamide-based polymers from the surface of CNCs [23,26,27,29]. A series of cationic CNCs was synthesized by varying the ratio between the immobilized initiator and the monomers (Table 1). After the polymerization, the resulting cationic CNCs were extensively purified by several centrifugations and dialysis over a week to ensure the removal of unreacted materials or any trace impurities.

**Scheme 1.** (**A**) chemical structure and schematic representation of pristine CNCs; (**B**) synthetic strategy for the fabrication of a series of cationic CNCs.

The success of the grafting of cationic brushes on the surface of CNCs was confirmed by FTIR spectroscopy, DLS, zeta potential and elemental analysis. Pristine CNCs displayed typical IR peaks characteristic of cellulosic functional groups at 3000–3600 cm<sup>−</sup>1, 1645 cm−<sup>1</sup> and 900–1150 cm−<sup>1</sup> corresponding to the -OH, -OH bending of water and -C-O-C vibrations, respectively [30,31] (Figure 1, spectrum A; Supplementary Figure S2). The spectra of CNCs after the polymerization showed a notable change with the appearance of new IR peaks at 1730 cm−<sup>1</sup> and 1728 cm−<sup>1</sup> for CNC-METAC-1A (Figure 1, spectrum B) and CNC-AEM-2A (Figure 1, spectrum C), respectively. These peaks were attributed to the typical carbonyl stretching vibration (C = O) of ester moieties confirming the presence of poly (METAC) and poly (AEM) on the surface of CNCs [27].

**Figure 1.** FTIR spectra of cationic CNCs: (A) pristine CNC, (B) CNC-METAC-1A, (C) CNC-AEM-2A.

Dynamic light scattering (DLS) and zeta potential measurements were conducted to explore the surface properties and stability of CNC suspensions before and after polymer grafting. Although the DLS technique is used for spherical particles, the measurements have been commonly employed to compare the relative sizes of rod-like CNCs before and after a chemical modification as well as the state of dispersion of the particles [16,21]. However, particle size measurements by DLS are not absolute and should not be considered as the exact dimensions of pristine and modified CNCs. The apparent hydrodynamic diameter of CNC particles was obtained by DLS in water at a 0.05 wt% concentration. Pristine CNCs showed an apparent particle size of ~101 nm which is in close agreement with the data obtained from AFM analysis (Figure 2D). An increase in apparent particle size was observed for all cationic CNCs (Table 2, Supplementary Figure S3) which indicated the presence of the polymer brushes on the surface of CNCs and as expected, the cationic CNCs would be highly hydrated. Furthermore, DLS analysis indicated no apparent aggregation of CNC particles at low concentrations. The nature of the surface charges on pristine and cationic CNCs was examined by measuring the zeta potential of the particles in water at 0.25 wt% concentration. Pristine CNCs had a negative zeta potential (-34.8 mV) due to the presence of anionic sulfate half-ester groups. On the other hand, cationic CNCs displayed positive zeta potential values in the range of +31.8 to +45.0 mV (Table 2). This further confirmed a successful polymerization reaction and the grafted cationic polymer brushes shielded the anionic sulfate half-ester groups. An increase in zeta potential (becomes more positive) was observed with an increase in monomer concentration ([monomer/AGU]: 50:3 v/s 60:3, Table 1). For instance, CNC-METAC-1B which had a higher monomer concentration compared to CNC-METAC-1A showed a more positive zeta potential value (+44.9 mV v/s +31.8 mV). The same trend was observed for CNC-METAC-2B (+38.2 mV) compared to CNC-METAC-2A (+32.0 mV). Overall, the cationic nature and colloidal stability of CNC-METAC and CNC-AEM materials are crucial for consistent results in biological studies.

The atomic composition of CNC samples before and after polymer grafting was measured by elemental analysis (Table 3). As expected, a small percentage of sulfur was detected for pristine CNCs due to the presence of the sulfate half-ester groups. While pristine CNCs displayed the absence of nitrogen, all the prepared cationic CNCs indicated the appearance of nitrogen deriving from the cationic monomers (METAC or AEM). This further supported the success of the polymerization reaction. The cationic CNC with a fixed initiator content and increased monomer concentration (CNC-METAC-1B v/s CNC-METAC-1A, Table 1) had a higher nitrogen content (~7.68%).

**Figure 2.** Morphological analyses of pristine and cationic CNCs by TEM and AFM: Representative TEM images of (**A**) pristine CNCs; (**B**) CNC-METAC-1A; (**C**) CNC-METAC-1B and AFM phase images of (**D**) pristine CNCs, (**E**) CNC- METAC-2A; (**F**) CNC-METAC-2B. TEM and AFM images of other cationic CNCs are depicted in Figures S4 and S5.



**Table 3.** Elemental analysis of pristine and cationic CNCs.


The morphological features of the cationic CNCs were analyzed by both transmission electron microscopy (TEM) and atomic force microscopy (AFM) to verify whether the characteristic rod-like crystalline structure of pristine CNCs (Figure 2A,D) was maintained after the surface modification reactions. Figure 2B–F, Figures S4 and S5 indicated that the rod-like morphology of the cationic CNCs was retained and the samples did not degrade into more simple carbohydrates [32]. Accurate size measurement of the rod-like particles by TEM was difficult as the edges of the grafted CNCs were less defined as well as a tendency to aggregate during air-drying or staining [33]. AFM analysis of pristine CNCs depicted a good distribution of the nanorods possessing an average length of 104 ± 68 nm and diameter of 4.0 ± 0.5 nm with a resultant aspect ratio of 26 (Figure 2D). The size of the cationic CNCs was found to be in the range of 112–164 nm in average length and 4.9–5.3 nm in cross-section. Moreover, AFM phase images of the cationic CNCs (Figure 2D,E) showed a halo that was indicative of the presence of grafted polymer brushes [26]. Aggregation of nanoparticles was observed for CNC-METAC-2B (Figure 2F) as it was prepared with a high monomer concentration (compared to CNC-METAC-2A (Figure 2E)), and hence a high degree of polymerization was expected [34].
