*2.6. Statistical analysis*

Statistical significance was determined using an analysis of variance (ANOVA) and Tukey's test (OriginPro, version 9.0.0, OriginLab Corporation, Northampton, MA, USA). Statistical significance was established at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. PMPC*/*BNC nanocomposites: preparation and characterization*

The one-pot *in-situ* free radical polymerization of MPC, inside the swollen BNC network and using MBA as cross-linker, was used to produce two PMPC/BNC nanocomposites (Figure 1) with distinct compositions (Table 1). The cross-linker was chosen based on former studies [23,24] and utilized with the goal of preserving the water-soluble zwitterionic homopolymer inside the BNC porous network during washing and utilization. The resulting nanocomposites contain 21 ± 3 wt.% and 46 ± 13 wt.% of BNC ( *W*BNC/*W*total), and concomitantly 79 ± 3 wt.% and 54 ± 13 wt.% of PMPC (*W*PMPC/*W*total), which correspond to nanocomposite materials containing 479 ± 118 mg and 859 ± 90 mg of PMPC *per* cm<sup>3</sup> of membrane, respectively, as listed in Table 1. The thickness of the membranes increased from 92 ± 21 μm for neat BNC to 133 ± 65 μm for PMPC/BNC\_1 and 226 ± 35 μm for PMPC/BNC\_2 (Table 1) due to the inclusion of the cross-linked PMPC into the three-dimensional structure of BNC. The membranes are macroscopically homogeneous with no discernible irregularities on either side of the materials surfaces, indicating a good dispersion of the cross-linked PMPC polymer inside the BNC network. After the incorporation of PMPC into the BNC network, the

transparency of the nanocomposites significantly increased, as displayed in Figure 1B and confirmed by transmittance values in the visible range (400–700 nm) of 58.1–65.6% for PMPC/BNC\_1 and 60.5–68.2% for PMPC/BNC\_2 (Figure 1C). In the ultraviolet region (200–400 nm), the transmittance remained below 5% until 265 nm for PMPC/BNC\_1 and 250 nm for PMPC/BNC\_2, and then steadily increased to 58.0% and 60.5% at 400 nm for PMPC/BNC\_1 and PMPC/BNC\_2, respectively. Furthermore, PMPC/BNC\_2 presents higher values of transmittance and concomitantly lower absorbance values, which points to a transmittance augmen<sup>t</sup> with higher content of cross-linked PMPC (Figure 1C). An analogous trend was observed for other BNC-based nanocomposites containing for example polycaprolactone [25], poly(methacroylcholine chloride) [23] and polyaniline [26].

**Figure 1.** (**A**) Radical polymerization of MPC in the presence of MBA as cross-linker, yielding cross-linked PMPC, (**B**) photographs of neat BNC and nanocomposites PMPC/BNC\_1 and PMPC/BNC\_2, and (**C**) the corresponding UV-visible transmission spectra.


**Table 1.** List of the studied membranes with the respective weight compositions and thicknesses.

a The composition was calculated by considering the weight of the nanocomposite membrane (*W*total), BNC (*W*BNC) and PMPC cross-linked polymer (*W*PMPC = *W*total – *W*BNC); b Ratio between the mass of the cross-linked PMPC (*W*PMPC) and the volume of the nanocomposite membrane (*V*total); all values are the mean of at least three replicates with the respective standard deviations.

The infrared spectra of neat BNC, cross-linked PMPC, and nanocomposites PMPC/BNC\_1 and PMPC/BNC\_2 are shown in Figure 2. The ATR-FTIR spectra of the PMPC/BNC membranes present the absorption bands of cellulose at 3340 cm<sup>−</sup><sup>1</sup> (O–H stretching), 2900 cm<sup>−</sup><sup>1</sup> (C–H stretching), 1310 cm<sup>−</sup><sup>1</sup> (O–H in plane bending) and 1030 cm<sup>−</sup><sup>1</sup> (C–O stretching) [27], jointly with those of the cross-linked PMPC at 1715 cm<sup>−</sup><sup>1</sup> (C=O stretching), 1479 cm<sup>−</sup><sup>1</sup> (N+(CH3)3 bending), 1228 cm<sup>−</sup><sup>1</sup> (P=O stretching), 1056 cm<sup>−</sup><sup>1</sup> (P–O–C stretching) and 953 cm<sup>−</sup><sup>1</sup> (N+(CH3)3 stretching) [28,29]. The presence of these absorption bands and the absence of one at about 1640 cm<sup>−</sup><sup>1</sup> corresponding to the C=C stretching of the methacrylic group of the starting monomer corroborated the occurrence of the *in-situ* free radical polymerization of MPC inside the BNC network. Furthermore, the relative intensity of the bands assigned to the cross-linked PMPC is in accordance with the *W*PMPC/*W*total ratio measured for each nanocomposite (Table 1).

**Figure 2.** ATR-FTIR spectra of cross-linked PMPC, neat BNC, and nanocomposites PMPC/BNC\_1 and PMPC/BNC\_2.

The solid-state 13C CP/MAS NMR spectra (Figure 3) of the membranes show the typical carbon resonances of cellulose at δ 65.2 ppm (C6), 71.6–74.5 ppm (C2,3,5), 88.9 ppm (C4) and 105.1 ppm (C1) [27], in combination with those of cross-linked PMPC at δ 18.4 ppm ( *C* H3 of polymer backbone), 44.9 ppm (quaternary C of polymer backbone), 54.3 ppm ( *C* H2 of polymer backbone and N<sup>+</sup>(*C* H3)3), 59.7 ppm (O *C* H2CH2N+(CH3)3), 65.5 ppm (O *C* H2*C* H2O and CH2*C* H2N+(CH3)3) and 176.6 ppm ( *C*=O). In addition, the truancy of the resonances allocated to the C=C double bond of the methacrylic group of the monomer [30] and cross-linker, supports their complete consumption during the polymerization and/or removal during the washing steps, as previously established by ATR-FTIR analysis.

The XRD patterns of the nanocomposites were compared with those of the individual components, namely cross-linked PMPC and BNC, to obtain an indication of the nanomaterials' crystallinity. Figure 4 shows the amorphous character of the cross-linked PMPC with a very broad band centered at 2θ ≈ 18◦, and the crystalline nature of BNC with a di ffraction pattern characteristic of cellulose I (native cellulose) composed of highly-ordered and least-ordered regions. The nanocomposites display a di ffractogram with three peaks corresponding to the (101) plane at *2*θ ≈ 14.7◦, (101) plane at *2*θ ≈ 16.8◦ and (002) plane at *2*θ ≈ 22.8◦ [27], which are representative of the cellulosic substrate. The addition of the cross-linked PMPC is evident through the reduction of the peaks of the (101) and (101) planes, most likely linked to the augmen<sup>t</sup> of disordered cellulose domains due to the presence of the amorphous polymer. A comparable trend was reported for other BNC-based nanocomposites containing for instance poly(bis[2-(methacryloyloxy)ethyl] phosphate) [31] and poly(4-styrene sulfonic acid) [32].

**Figure 3.** 13C CP/MAS NMR spectra of cross-linked PMPC, neat BNC and nanocomposite PMPC/BNC\_2.

**Figure 4.** X-ray di ffractograms of cross-linked PMPC, neat BNC, and nanocomposites PMPC/BNC\_1 and PMPC/BNC\_2.

SEM micrographs of the cross-section of neat BNC and nanocomposites PMPC/BNC\_1 and PMPC/BNC\_2 are compiled in Figure 5A. It is clearly visible that the lamellar microstructure of neat BNC disappeared in the nanocomposites due to the filling of the lamellar spaces by the cross-linked PMPC, particularly in the case of PMPC/BNC\_2 with 859 ± 90 mg of PMPC *per* cm<sup>3</sup> of membrane. The SEM/EDS analysis reiterates the presence of PMPC and BNC through the detection of carbon (C), nitrogen (N), oxygen (O) and phosphorous (P) peaks at 0.27, 0.39, 0.51 and 2.01 keV, respectively, as illustrated in Figure 5B for PMPC/BNC\_2. Moreover, the SEM/EDS mapping (Figure 5C) confirmed the uniform distribution of nitrogen and phosphorous of the cross-linked polymer within the BNC nanofibrous network, since both elements are only present in the zwitterionic PMPC.

**Figure 5.** (**A**) SEM micrographs of the cross-section of neat BNC, and nanocomposites PMPC/BNC\_1 and PMPC/BNC\_2; EDS spectrum (**B**) and micrograph (**C**) for nitrogen and phosphorous elemental mapping of nanocomposite PMPC/BNC\_2.
