Cellulose nanocrystals from sugarcane bagasse and its graft with GMA: Synthesis, characterization, and biocompatibility assessment

Materials

SCB (agro-waste mixtures of different types of sugarcane) was provided from Egyptian Sugar and Integrated Industries Company. Sodium chlorite (NaClO₂), glacial acetic acid, sodium hydroxide, and sulfuric acid (85%) were obtained from El-Nasr Co. for Intermediate Chemicals, Egypt. Cellulose dialysis membrane is a semi-permeable membrane, and cellulose acetate, GMA, nitric acid, and IV nitrate (CAN) were purchased from Sigma–Aldrich, Germany. Acetone, THF, methanol, and nitric acid were purchased from Fluka, Germany.

Methods of isolation of cellulose from SCB

Five grams of dried SCB were suspended in 125 ml distilled water for bleaching in a 250 ml conical flask, then 0.5 g of sodium chlorite was added to the bagasse colloidal and glacial acetic acid was added drop by drop to adjust pH < 4.0. The mixture was heated in a water bath at 70°C for 1 hour. The bleaching process was repeated at hourly intervals. Four hours of extraction was sufficient for the product to become white. The contents of the flask were filtered and the bleached cellulose fibers were washed with distilled water until the mixture neutralized. The bleached SCB was air-dried. Three grams of dry bleached bagasse was torn into small pieces in a beaker and kept in a water bath at 20°C. Afterward, 50 ml of 17.5% (w/w) of NaOH was added with continuous stirring for 45 minutes. Then, 100 ml of distilled water was added to the mixture and it was harshly stirred for further 30 minutes. The produced cellulose suspension was filtered and washed with 500 ml distilled water then with 100 ml of 10% acetic acid. The washing with distilled water was repeated until the cellulose became neutral. The pure cellulose was air-dried and preserved in a brown bag.

Synthesis of CNCs

The hydrolysis method was employed for preparing the nanocellulose as explained by Ferreira et al. (2018). One gram of cellulose only was added to 40 ml of 65% (w/w) sulfuric acid. The suspension was warmed to 50°C and vigorously stirred for 40 minutes. For stopping the reaction, 100 ml of distilled water was added, and then centrifuged at 6,000 rpm for 15 minutes. The mixture was washed with distilled water and centrifuged five times until pH was >5. The nanocellulose suspension was dialyzed against distilled water for 5 days and then stored at 4°C. Dried nanocellulose was obtained by lyophilization for 48 hours.

Scheme 1. Reaction mechanisms of grafting of CNCs with GMA using cerium-initiated copolymerization. [Click here to view]

Instrumental characterization of CNCs and CNCs-g-GMA copolymer

Grafting efficiency

The gravimetric calculation was used in order to assess the grafting process of CNCs-g-GMA, where CNCs-g-GMA was weighed and the results were employed to determine the graft yield using the given equation (2)

$\mathrm{Graft}\mathrm{yield}=\frac{\mathit{Wg}}{\mathit{Wc}}\times 100\%.$ (2)

where W_g is the obtained mass of CNCs-g-GMA and W_c is the initial mass of CNCs.

Antimicrobial properties of CNCs and CNCs-g-GMA copolymer

In this work, different human pathogens, such as Escherichia coli (−ve), Klebsiella pneumonia (−ve), Staphylococcus aureus (+ve), Salmonella typhimurium (+ve), and Candida albicans, were tested to detect the antimicrobial activities for the synthesized CNCs (1, 2, and 4 gl⁻¹) and the grafted CNCs-g-GMA copolymer (1, 2, and 4 gl⁻¹) separately. Furthermore, the inhibitory effects of different dilutions of CNCs-g-GMA (3.5, 3, 2.5, and 1.5 gl⁻¹) against the tested human pathogens were checked and mathematically estimated using turbidity measurements according to Koch’s method (Goy et al., 2016). First, these pathogens were cultured using nutrient broth (2 g/l) at 37°C ± 2°C for 24 hours to prepare the stock cultures. Subsequently, these cultures were diluted with sterilized nutrient broth to get an optical density of 0.08 at 600 nm. Afterward, the CNCs and CNCs-g-GMA solutions were added separately to these cultures except the control (untreated sample) which contained the culture only. These solutions were then incubated at 200 rpm, at 37°C ± 2°C for 18 hours. After the incubation period, the microbial growth was estimated via spectrophotometer at 600 nm to calculate the antimicrobial effectiveness. Since the reduction of the microbial growth quantified as a function of the tested polymer, the antimicrobial efficiency can be mathematically determined employing the maximum absorbance measurements in each case (turbidity data). Three replicas were measured for each sample and the final data were exposed to statistical evaluation by one-way analysis alteration (p ≤ 0.05) utilizing Minitab 17 software. In this case, the antimicrobial efficiency can be established as a relationship between the maximum microbial growth (stationary phase) with and without the tested toxins which coded as G_P and G_C, respectively via Eq. 3 (Da Silva et al., 2010).

$\begin{array}{c}\mathit{Ac}\to {\left(\frac{{\mathit{dG}}_{\mathit{c}}}{\mathit{dt}}\right)}_{\mathit{max}}^{\mathit{Control}}=0\mathit{and}\mathit{Ac}\to {\left(\frac{{\mathit{dG}}_{\mathit{p}}}{\mathit{dt}}\right)}_{\mathit{max}}^{\mathit{Polymer}}=0\hfill \\ \mathit{Af}\mathrm{(\%)}=\left[\frac{\mathit{Ac}-\mathit{Ap}}{\mathit{Ac}}\right]\times 100.\hfill \end{array}$ (3)

where A_C and A_P are the maximum absorbances at 600 nm for the control and treated cultures, respectively, and A_f is considered as the calculated inhibitory proportional factor.

Cytocompatibility of CNCs and CNCs-g-GMA on the viability of normal cells

The effect of CNCs and CNCs-g-GMA on the cell viability of HDF (fibroblast cells originated from human dermal tissue) and WI38 (fibroblast cells obtained from human lung tissue) normal cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) cell viability procedure as previously assessed by Mosmann (1983) and El-Fakharany et al. (2020). In brief, HDF and WI38 cells (1.0 × 10³) were scattered in a 96-well flat bottom tissue culture sterile microplates and cultured in the DMEM media (Lonza, USA) enhanced with 10% fetal bovine serum at 37°C for 24 hours in a 5% CO₂ incubator. Then, both CNCs and CNCs-g-GMA samples were added to the cells at concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/ml and incubated at 37°C in 5% CO₂ incubator for 1, 2, and 3 days. The cells were rinsed three times with fresh media after incubation to remove the samples and dead cells. Then, about 200 μl of MTT solution at a concentration of 0.5 mg/ml was inserted into each well and developed for 3 hours at 37°C in the 5% CO₂ incubator. The formed formazan crystals were dissolved in 200 μl/well of DMSO and the optical density was measured at 630 nm and subtracted from measuring at 570 nm using reader a microplate ELISA reader. All tests were carried out in triplicates and the relative cell viability (%) evaluated to control wells containing cells without treatment was analyzed by applying the formula: (X) test/ (Y) control × 100%.

Particle size measurement of CNCs

The dispersed CNCs/water mixture solution was employed to verify the actual particle size of CNCs, as shown in Figure S1 (supplementary materials), using the duplicated particle size analyzer. The dispersed CNCs show a narrow and sharp peak at around 156–370 nm diameter. This result refers to the prepared CNCs which belong to the nanoscale with a mean particle size of ~260 nm.

Grafting yield (%) optimization of CNCs-g-GMA copolymer

Different degrees of grafting yields of CNCs-g-GMA were obtained with variations in grafting reaction conditions, e.g., GMA content, CAN initiator concentration, grafting time, and grafting temperature. Table 1 displays the obtained graft yield of CNCs-g-GMA as a function of grafting conditions.

As shown in Figure 1a, the grafting yield (%) of the resultant CNCs-g-GMA increases clearly to be 115%, 180%, and 170% for GMA ratios of 20, 40, and 80 ml mol/g of CNC, respectively. This is owing to the increasing portion of GMA results in increasing the vinyl groups in CNCs-g-GMA, which facilitates the copolymerization reaction and accompanies the increase in the grafting yield % (Sun et al., 2018). Thus, 40 ml mol was chosen as the optimum GMA ratio, due to its own high grafting yield (%) (i.e., 180%).In the same manner, the influence of the CAN initiator concentrations in the range of 1, 2, and 4 ml mol were studied based on the resultant graft yield of CNCs-g-GMA copolymer (Table 1 and Fig. 1b). It is noted that the grafting yield increases to 120%, 180%, and 150% with CAN initiator concentrations of 1, 2, and 4 ml mol, respectively. It could be ascribed to the high concentration of CAN initiator >2 ml mol which might terminate the reaction of the grafting process (Fig. 1b) (Tosh and Routray, 2014). Thus, the CAN initiator concentration of 2 ml mol was selected as an optimum condition for a further grafting reaction.On the other hand, the influence of the grafting time was in the range of 30, 60, and 120 minutes on the resultant grafting yield of CNCs-g-GMA copolymer, while the other grafting conditions were kept constant (Table 1 and Fig. 1c). It is clear that the grafting yield of the CNCs-g-GMA copolymer is dramatically increased from 120% to 180% after prolonging the grafting time from 30 to 60 minutes. However, the grafting yield decreased clearly to 135% as the grafting time increased to 120 minutes. Thus, 60 minutes as grafting time was chosen as the highest grafting yield of CNCs-g-GMA copolymer.

Table 1. Grafting yield optimization of CNCs-g-GMA copolymer. [Click here to view]

Figure 1. Grafting yield (%) of CNCs–GMA copolymer. Effect of (a) GMA monomer concentration, (b) CAN initiator concentration, (c) grafting time, and (d) grafting temperature on grafting yield (%) of CNC-g-GMA copolymer. [Click here to view]

Figure 2. FTIR spectra of CNCs-g-GMA copolymer. (a) IR spectra of CNCs, GMA, and CNCs-g-GMA, and (b) as function of GMA monomer concentrations (20, 40, and 80 mmol/g of CNCs). [Click here to view]

Likely, increasing the grafting temperature from 25°C to 40°C resulted in a significant increase of grafting yield from 110% to 180%, respectively (Fig. 1d). Notably, the grafting yield decreased to 115%, when the temperature of the grafting reaction was increased to 60°C. The reason might be referred to the decomposition of the CAN initiator at a high grafting temperature (i.e., 60°C) (Fig. S2, supplementary).

FTIR spectra of CNCs and CNCs-g-GMA copolymers

The prepared CNCs, GMA, and CNCs-g-GMA copolymers were characterized by the FTIR, as shown in Figure 2a. As can be seen, both CNCs and CNCs-g-GMA spectra provided the absorption peaks at ѵ 3,451 cm⁻¹ and 2,899 cm⁻¹, respectively, and were assigned to the O-H and C-H stretching vibrations, respectively. However, the peak C-O vibrations contained in the polysaccharide rings of cellulose is observed at ѵ 1,377 cm⁻¹, while the vibration of C-O-C in pyranose ring is indicated by the absorption peak at 1,055 cm⁻¹(Mandal and Chakrabarty, 2011; Purwanti et al., 2015). CNCs-g-GMA spectrum also showed a new and strong absorption band at ѵ 1,720 cm⁻¹ as a characteristic ester carbonyl group, as found in the GMA spectrum. Characteristic C–H stretching peaks (symmetric and asymmetric) were also detected in the wavenumber range of ѵ 2,960–2,850 cm⁻¹. In addition, a medium strength absorption peak at ѵ 840 cm⁻¹ characterized for an epoxy group was detected in the spectra of GMA and CNCs-g-GMA (Hase, 1995).Interestingly, the low grafting yield (%) values exhibited significantly low intensities at ѵ 1,720 cm⁻¹ as observed in CNCs-g-GMA copolymer with the low concentration of GMA (20 ml mol/g), and it resulted in a very low grafting yield (%) accompanying low intensity at ѵ 1,720 cm⁻¹ (Fig. 2b).

Unexpectedly, the intensity of the feature absorption peak of ester carbonyl group at 1,720 cm⁻¹ was detected in a low intensity for low and high CAN initiator concentrations (1 and 4 ml mol) during the grafting process, while the methacrylation peak intensity was detected in a significantly high intensity with the middle CAN initiator concentration (2 ml mol) during the grafting process (Fig. 3a). Thus, the middle CAN initiator concentration (2 ml mol) was chosen as the optimized CAN initiator concentration for further grafting attempts. The intensity of ester carbonyl group of CNCs-g-GMA copolymer at ѵ 1,720 cm⁻¹ was raised notably with prolonging the grafting reaction time from 30 to 120 minutes at 60°C (Fig. 3b).

X-ray diffraction of CNCs and CNCs-g-GMA copolymer

The C_I of CNCs was analyzed by the XRD pattern as shown in Figure 4. Three diffraction peaks at 2θ = 12.2°, 20.1°, and 22° were detected with the CNCs. The C_I for CNCs was estimated employing equation (1), where I₂₀₀ at 2θ = 22° and I_a at 2θ = 17.5° (Wada et al., 2004). This method was progressed by Segal et al. (1959), where the C_I was evaluated from the ratio of the height of the 002 peak (I₀₀₂) and the height of the minimum between the 002 and 101 peaks. The diffractograms are shown in Figure 4. The C_I for isolated CNCs from SCB is calculated ca. 77.33% as previously measured by Park et al. (2010).

Figure 3. FTIR spectra of CNCs-g-GMA copolymer as function of (a) CAN initiator concentration (1, 2, and 4 mmol/g of CNC) and (b) grafting reaction time (30, 60, and 120 minutes) during grafting process. [Click here to view]

Figure 4. XRD patterns of resultant CNCs (left) and CNCs-g-GMA copolymer (right). [Click here to view]

The crystallinity of the prepared CNC-g-GMA is shown in Figure 4. The XRD pattern exhibits the loss of the crystal structure of CNC after the grafting reaction, corresponding to the formation of CNCs-g-GMA, which resulted in changing the crystal structure of CNCs and reducing peaks intensity in the CNC-g-GMA. However, the pattern of CNCs-GMA displayed a significant increase in hydrophobic properties of CNC-g-GMA compared to CNCs (Giljean et al., 2011), where the C_I of CNC-g-GMA could not be measured due to losing the intensity of crystal peak at 2θ~22°.

Figure 5. SEM photographs of CNCs and CNCs-g-GMA copolymer. Images were taken under original magnifications (1,000x and 3,000x) under 30 Kv. [Click here to view]

Surface morphology of CNCs and CNCs-g-GMA copolymer

The surface morphology of CNCs and CNCs-g-GMA was investigated by SEM with 1,000x and 3,000x original magnifications under 30 kV. SEM micrographs are shown in Figure 5. The SEM images revealed that hydrolysis of cellulose produced a reduction in its raw cellulose fiber diameter (Sèbe et al., 2012). Moreover, cellulose particles after hydrolysis with regard to CNCs-g-GMA showed a more irregular shape and more asymmetrical edges than CNCs. CNCs images involved fibrils that were significantly lesser than 40 nm in diameter, but thicker fibril-aggregated bundles were also observed. Comparison of these images demonstrates that the nanoscale fibrils structure is preserved during the grafting process in case CNCs-g-GMA, while the aggregation of CNCs appeared to be significantly reduced after grafting, owing to the methacrylation with regard to CNCs-g-GMA copolymer. Also, the fibrils of the CNCs-g-GMA copolymer were somewhat thicker than those of CNCs; it might be attributed to the large amounts of grafted GMA onto CNCs nanofibrils (Habibi, 2014).

TGA thermographs of CNCs and CNCs-g-GMA copolymer

TGA analysis of CNCs and CNCs-g-GMA is shown in Figure 6. As can be seen, CNCs started to thermo-decompose close to 238°C–327°C as initial decomposition stage, showing a drop in weight loss (%) to complete decomposition at almost 400°C. However, grafted CNCs exhibited a slight increase in initial decomposition stage close to 264°C–342°C, with a complete decomposition at 448°C. It means that the grafting process of CNCs might improve the thermal stability of CNCs to ca. 26°C.

Evaluation of antimicrobial efficiency of CNCs and CNCs-g-GMA

The antimicrobial effects of CNCs and CNCs-g-GMA were represented graphically using the optical density of tested pathogens, as shown in Figure 7. Since the growth of all tested human pathogens was affected by all CNCs and CNCs-g-GMA treatments, the treatment coded as F (4 gl⁻¹ of CNCs-g-GMA) showed the highest antimicrobial activity and the growth of S. aureus (+ve) was reduced from 2.44 nm to 1.36 nm, while S. typhimurium (−ve) decreased from 2.39 nm to 1.35 nm (Fig. 7a). Furthermore, using turbidity data, this figure indicates that the fungal growth was also reduced using 4 gl⁻¹ of CNCs-g-GMA treatment (F) from 2.71 nm to 1.62 nm, as shown in the case of tested C. albicans. Other than the growth of all tested fungi, pathogens could be reduced slightly by using 1, 2, and 4 gl⁻¹ of CNC treatments coded as A, B, and C, respectively. Accordingly, the growth of the human pathogens can be reduced perfectly by using 4 gl⁻¹ of grafted CNCs-g-GMA, compared to untreated CNCs (Fig. 7a).

Figure 7b shows the calculated inhibitory proportional factor (%) which indicated the reduction in the microbial populations concerning the tested control, especially in case of S. aureus (+ve), S. typhimurium (−ve), and C, albicans to 44.30%, 43.09%, and 40.29%, respectively, as a function of tested treatment coded as F (4 gl⁻¹of CNCs-g-GMA). With regard to the rest of the treatment, E. coli (−ve) has the highest inhibitory factor which recorded 13.62%, 17.25%, 21.24%, 28.34%, and 29.38% using A (1 gl⁻¹ CNCs), B (2 gl⁻¹ CNCs), C (4 gl⁻¹ CNCs), D (1 gl⁻¹of CNCs-g-GMA), and E (2 gl⁻¹of CNCs-g-GMA) treatments, respectively. However, the lowest inhibitory factors were recorded as 5.5% with K. pneumonia, followed by 7.12% of C. albicans and 7.8% of S. aureus via the treatment coded as A (1 gl⁻¹ CNCs).

Figure 6. TGA thermograph results of CNCs and CNCs-g-GMA copolymer. [Click here to view]

Furthermore, the derivative relationship for different dilutions of the tested CNCs-g-GMA copolymer (3.5, 3, 2.5, and 1.5 gl⁻¹) and the microbial growth were calculated and are graphically represented in Figure 7c. The highest antimicrobial activity recorded by calculating the inhibitory proportional factor was 79.5% against S. aureus, followed by S. typhimurium (71.4%), and K. pneumonia (69.5%) using 2.5 gl⁻¹ of CNCs-g-GMA copolymer (F2). In addition, 1.5 gl⁻¹ of CNCs-g-GMA copolymer (F1) reduced the C. albicans and E. coli growth by 69.4, and 66.07%, respectively. The lowest inhibitory proportional factor was recorded by using 3.5 gl⁻¹ of CNCs-g-GMA copolymer (F4) against all tested pathogens, especially C. albicans recorded at 16.34%.

In light of these findings, synthesized CNCs and its graft (i.e., CNCs-g-GMA) concentrations inhibited the growth of human pathogens whether Gram +ve, Gram −ve, or fungal cells. Conversely, the maximum activity was achieved when 2.5 g l⁻¹ of CNCs-g-GMA copolymer is added since the microbial population (S. aureus, S. typhimurium, and K. pneumonia) reduced as close to 70% and 80%. The mode of action of CNCs-g-GMA copolymer might be due to its remarkable physicochemical properties that disrupted the microbial membranes by the effect of cytoplasmic membrane potentials, net surface charge, or permeability, which led to the leakage of the cellular compounds and eventually cell death (Jiang et al., 2012; Liang et al., 2014), since CNCs possess a special surface chemistry and excellent physical properties which make them appealing for antimicrobial applications (Panchal et al., 2019).

Figure 7. (a) Microbial growth absorbance at 600 nm as a function of different treatments; A, 1 gl−1 CNCs; B, 2 gl−1 of CNCs; C, 4 gl−1 CNCs; D, 1 gl−1of CNCs-g-GMA; E, 2 gl−1of CNCs-g-GMA; and F, 4 gl−1of CNCs-g-GMA against different human pathogens using turbidity method after 18 hours. (b) Inhibitory proportional factor (%) according to Eq.(3) which indicated the reduction in the microbial growth in function of different CNCs, and CNCs-g-GMA treatments coded as A, 1 gl−1 CNCs; B, 2 gl−1 CNCs; C, 4 gl−1 CNCs; D, 1 gl−1of CNCs-g-GMA; E, 2 gl−1of CNCs-g-GMA; and F, 4 gl−1of CNCs-g-GMA. (c) Affection of the tested concentrations of CNCs-g-GMA copolymer coded as F1: 1.5 gl−1 of CNCs-g-GMA; F2: 2.5 gl−1 of CNCs-g-GMA; F3: 3 gl−1 of CNCs-g-GMA; and F4: 3.5 gl−1 of CNCs-g-GMA against different human pathogens indicated via calculating the inhibitory proportional factor (%). [Click here to view]

Cytocompatibility of CNC and CNCs-g-GMA copolymer

The cytotoxicity of CNCs and CNCs-g-GMA with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/ml) was assessed in vitro on human normal HDF and WI38 cell lines after 1 and 3 days of incubation (Fig. 8). As shown in Figure 8, the results revealed that CNCs has high cell viability (%) and proliferation reached to 100% with all tested concentrations (0.5–3.0 mg/ml) on both days 1 and 3 of incubation. While CNCs-g-GMA copolymer with low-tested concentrations (0.5–1.5 mg/ml) shows high cell viability ranged 90%–100%, high-tested concentrations (2.0–3.0 mg/ml) exhibited a moderated and clinically accepted cell viability (%) range of 60%–80%, regardless of the type of cell lines. This observation could be attributed to CNCs-g-GMA, which might release and contain unreacted or excess of free GMA and which has cytotoxic effect on tested cell lines; thus, this cytotoxic behavior was noted only tested in high concentrations of CNCs-g-GMA in the range of 2.0–3.0 mg/ml. Accordingly, synthesized CNCs and CNCs-g-GMA possess high cytocompatibility which make them good candidates as biomaterials for biomedical applications.

Figure 8. Influence of CNCs and CNCs-g-GMA at different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/ml) on human normal HDF and WI 38 cell lines proliferation after 1 and 3 days of incubation. [Click here to view]