Cellulose nanocrystals/silver nanoparticles: in-situ preparation and application in PVA films

Materials

Bleached hardwood dissolving pulp was purchased from Shandong Babeibo Pulp Co., Ltd (Shandong, China). Silver nitrate (AgNO₃, purity >99%), PVA, glucose (C₆H₁₂O₆), sodium hydroxide (NaOH, reagent grade ≥98%), sulfuric acid (H₂SO₄), sodium chloride (NaCl), trypton peptone, phyton peptone, agar-agar and anhydrous ethanol were purchased from Tianjin Jiangtian Chemical Technology Co., Ltd (Tianjin, China). Deionized water was used for all experiments. Escherichia coli and Staphylococcus aureus were obtained from the Tianjin Key Laboratory of Pulp & Paper, Tianjin University of Science and Technology (Tianjin, China).

Preparation of CNC

CNCs were prepared by treating the dissolving pulp with sulfuric acid according to a previously published procedure (Bharimalla et al. 2015). Hardwood dissolving pulp was dispersed into 64% (wt) sulfuric acid at 45°C and stirred for 50 min. Afterward, the reaction solution was washed with deionized water, centrifuged repeatedly and then dialyzed against deionized water until neutrality. The resulting CNC samples were kept in a refrigerator until use.

Preparation of silver-loaded nanocellulose

First, 0.17 g of AgNO₃ was dissolved in 10.00 ml of deionized water, and 0.40 g of NaOH was dissolved in 10 ml of distilled water and were stored in the dark at room temperature. AgNO₃ was added to 50 ml of CNC in a water bath at 80°C. The NaOH solution was added dropwise to this solution with constant stirring until the pH was adjusted to 8. Then, 2.00 g of C₆H₁₂O₆ was added to the mixture solution over a period of 4 h. The resulting solution was then sequentially centrifuged with absolute ethanol and distilled water, and then sonicated for 0.5 h.

Preparation of CNC-PVA-AgNP nanocomposite films

Different concentrations of silver-loaded nanocellulose were used to prepare CNC-PVA-AgNP films. First, 10 g of PVA and 90 ml of water were mixed at 80°C for 4 h with continuous stirring. Silver-loaded nanocellulose and PVA solutions (10 wt%) were mixed in a 1:1 (w/w) ratio with constant stirring at room temperature for at least 1 h to ensure complete homogeneity of the mixed solution. The mixture was stirred at 80°C for 3 h, and then 20 ml of CNC-PVA-AgNP solution at 25°C was poured into petri dishes and stored at room temperature for 24 h to allow solvents to evaporate. After the solvents had evaporated, films were then peeled off the petri dish and placed in the oven at 50°C for 3 h to ensure complete evaporation of water. Finally, the dried films were stored in sealed bags until use. Samples of silver-loaded nanocellulose formulated with different concentrations of silver nitrate were mixed with PVA solutions to prepare composite films that are denoted as: CNC-PVA-AgNPs-0.05, CNC-PVA-AgNPs-0.1, CNC-PVA-AgNPs-0.2, CNC-PVA-AgNPs-0.3, CNC-PVA-AgNPs-0.4 and CNC-PVA-AgNPs-0.5. The numbers 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 denote the respective molar concentration of AgNO₃.

Material characterization

The morphology of AgNPs was revealed using an ultraviolet-visible (UV-vis) spectrophotometer (Shimadzu, UV-2550 PC, Japan) and by transmission electron microscopy (TEM; JEOL, JEOL-2010, Japan) operating at 200 kV. Wide-angle X-ray diffraction (WAXD; Shimadzu, XRD-6100, Japan) measurements using Ni-filtered Cu Kα radiation (λ=0.15405 nm) at room temperature were used to analyze crystalline structures of films. Tensile tests of the films were performed using an electromechanical universal testing machine (MTS, CMT4503, China) according to the ISO 527–3-2018 standard method (ISO 527-2, Plastic), and tests were carried out at 20°C and 65% relative humidity (RH). At each treatment level, films were cut into 150 mm×25 mm samples, with an original scale distance of 150 mm. Five replicates were tested, and the results are presented as the average of the tested samples. A crosshead speed of 50 mm min⁻¹ was used to determine the tensile deformation. The moisture absorption properties of CNC-PVA-AgNP nanocomposite films were measured as follows based on a previous procedure (Peleg 1988; Zhang et al. 2018): pure PVA films and CNC-PVA-AgNP nanocomposite films (20 mm×20 mm) were divided into eight groups, which were dried in an air oven at 105°C for 4 h. Then, the mass of the eight groups was obtained using a balance with an accuracy to four decimal places (±mg) and recorded as Md. Next, the eight groups were placed into separate closed containers with saturated salt solutions at 25°C and measured by a hygrometer. The RH of the saturated salt solutions at 25°C were CH₃COOK: 23%, K₂CO₃: 42%, NaBr: 57%, KI: 63%, NaCl: 75%, KBr: 80%, KCl: 86% and KNO₃: 92%. Afterward, the films were re-weighed, and this mass was recorded as Mh. To accurately gather data, three replicates were performed, and the average of the data is reported.

Moisture absorption (Ma)=Mh−MdMd.

where

Ma is the moisture absorption expressed as g H₂O g⁻¹ solids;

Mh is the weight of the films after being placed into closed containers with saturated salt solutions;

Md is the weight of the films before being placed into closed containers with saturated salt solutions.

Antibacterial activity

The antibacterial property of a substance is determined by its ability to inhibit bacterial growth on a surface. Escherichia coli (ATCC 25922) and S. aureus (ATCC 29213) were used as model bacteria to evaluate the antibacterial properties of the materials prepared in this study, and the standardized agar disk diffusion plate test method was used in accordance with ISO20645:2004.

The nutrient broth for bacterial growth was selected and poured into each sterillzed petri dish to allow the agar to congeal on an aseptic table. The plates were incubated for 24 h at 37°C immediately after placing the test specimens with 6 mm circular diameter CNC-PVA-AgNP films on the agar. The bacterial growth was then checked. The diameter of the inhibition zone was measured after the culture dish was removed from the bacterial incubator. The width of the inhibition zone (i.e. the bacteria-free zone near the edge of the specimen) was calculated using the following formula:

H=D−d2

where

H is the inhibition zone in mm;

D is the total diameter of the specimen and inhibition zone in mm;

d is the diameter of the specimen in mm.

TEM analysis

TEM images of AgNPs, CNCs and silver-loaded nanocellulose are shown in Figure 1. The AgNPs are shown in Figure 1a and they appear to be spherically shaped with an average diameter of 25 nm. Figure 1b shows that the CNC has a rod-like structure with a diameter of about 10 nm. Figure 1c shows that spherical AgNPs are dispersed homogeneously on the surface of CNCs, and the AgNPs show no apparent aggregation due to the loading and dispersing function of CNCs. The surface of the nano argent crystal nucleus is bonded to the O atom in the CNC during the entire reaction process. The long chain that extends to the four sides can effectively prevent agglomeration of the AgNPs, so that the formed silver atoms can be evenly adsorbed onto the nucleation surface, allowing spherical AgNPs to be obtained.

Figure 1:

TEM images of (a) AgNPs, (b) CNC and (c) silver-loaded nanocellulose.

UV spectrophotometer analysis

As the shape and position of a spectral absorption peak is related to the morphology and particle size of the metal nanoparticles, spectral analysis is an effective method to study these properties in metal nanoparticles. In Figure 2, there is a strong absorption peak between 300 and 600 nm with a maximum around 425–450 nm, which is the typical plasmon resonance band of AgNPs (Sharma et al. 2009). This peak and its location indicate that the Ag⁺ ions in the silver nitrate solution were successfully reduced to AgNPs. Meanwhile, the absorption peak indicates the presence of many spherical or nearly spherical AgNPs in the solutions. At low AgNO₃ concentrations (a and b in Figure 2), the absorption band appears at lower wavelengths. As the AgNO₃ content increases, the absorption peak intensity increases, which indicates that the concentration of the AgNPs increases. When the concentration of silver nitrate was increased from 0.4 mol l⁻¹ to 0.5 mol l⁻¹, the peak value slowly increased. The colors of the solutions gradually deepened as the concentration of AgNO₃ increased.

Figure 2:

UV-visible absorption spectra of silver-loaded nanocellulose solutions prepared with different AgNO₃ concentrations.

WAXD analysis

As can be seen from Figure 3, the AgNPs were well crystallized and showed sharp peaks at 2θ angles of 38.2°, 44.4°, 64.5° and 78.7°. These were assigned to the Ag(111), Ag(200), Ag(220) and Ag(311) planes of the face-centered cubic Ag and confirm that the AgNP crystals were successfully prepared (Subarani et al. 2013). The diffraction peak of this curve was quite sharp, which indicates that the silver has good crystallization performance. The figure also shows the characteristic diffraction peaks of CNCs, which has three distinct diffraction peaks at 2θ values of 16.5°, 22.5° and 34.7°, which correspond to the (101), (002) and (040) crystals, respectively (Zhang et al. 2014). The surface diffraction peaks indicate that the respective crystal forms of the CNCs and the AgNPs did not change.

Figure 3:

WAXD spectra of CNC-PVA-AgNPs-0.1 films.

Mechanical property analysis

Figure 4 shows the stress and strain at the rupture of CNC-PVA-AgNP nanocomposite films. As the concentration of AgNO₃ solutions increased, the tensile strength of the CNC-PVA-AgNP nanocomposite films increased significantly, while the elongation at break significantly decreased. The results show that adding silver-loaded nanocellulose can improve the mechanical properties of PVA, but it also reduces its flexibility. The tensile strength of this material is mainly affected by the stiffness of the CNC and the strength of its intermolecular hydrogen bonds (Lee et al. 1999). The large numbers of hydroxyls on the surface of the CNC can easily form hydrogen bonds with hydroxyls of PVA molecules. This increases the intermolecular forces, which reduces the mobility of the polymer chain. With the increase in RH, these hydroxyl groups easily form hydrogen bonds with water molecules in a humid environment, resulting in changes in the aggregation structure of PVA, which reduces the mechanical properties of PVA.

Figure 4:

Stress and strain at the rupture of CNC-PVA-AgNP films prepared with different AgNO₃ concentrations.

Moisture absorption analysis

Figure 5 depicts the weight gain of CNC-PVA-AgNPs-0.1 films and pure PVA films at different RH conditions. There is an obvious trend that the introduction of nanomaterials into PVA decreases moisture absorption when the RH increases beyond 45%. When the RH is less than 50%, the moisture content curve shows that the moisture content increases slowly, but when the RH is higher, a rapid increase in moisture content is observed. The PVA film has good mechanical properties and acts as a gas barrier in its dry state, but when it is in a humid or wet environment, it will swell and possibly even dissolve in water. This is primarily because of the many hydroxyl groups present in the PVA side chains making it hydrophilic. Thin CNC-PVA-AgNP films absorbing less moisture than pure PVA films make them possible to be used in moist circumstances (Mbhele et al. 2003; Khanna et al. 2005).

Figure 5:

Moisture absorption of PVA and CNC-PVA-AgNPs-0.1 films.

Antibacterial activity of CNC-PVA-AgNP nanocomposite films

The films showed strong antimicrobial activity against both E. coli and S. aureus. Figure 6 and Table 1 show that as the concentration of silver particles continuously increases, the inhibition zone width also increases. As the concentration of AgNO₃ increased from 0.1 mol l⁻¹ to 0.4 mol l⁻¹, the inhibition zone gradually became larger. This indicated that there were more AgNPs produced at higher AgNO₃ concentrations, and that the antibacterial effects were more obvious. The best antibacterial effects were observed at AgNO₃ concentrations of 0.3 mol l⁻¹ and 0.4 mol l⁻¹, which had nearly identical inhibition zone sizes. The maximum inhibition zone widths of CNC-PVA-AgNP films against E. coli and S. aureus were 5±0.5 cm and 6±0.5 cm, respectively.

Figure 6:

Antibacterial activity of CNC-PVA-AgNP solutions prepared with different AgNO₃ concentrations (1, 0.1 mol l⁻¹; 2, 0.2 mol l⁻¹; 3, 0.3 mol l⁻¹; 4, 0.4 mol l⁻¹) on Escherichia coli (a) and Staphylococcus aureus (b).

Table 1:

The width of the inhibition zone against E. coli and S. aureus.

Films	The width of the inhibition zone (H, mm)
	E. coli	S. aureus
CNC-PVA-AgNPs-0.1	4±0.5	5±0.1
CNC-PVA-AgNPs-0.2	5±0.1	5±0.3
CNC-PVA-AgNPs-0.3	5±0.5	5±0.4
CNC-PVA-AgNPs-0.4	6±0.2	6±0.3

1. AgNPs, Silver nanoparticles; CNC, cellulose nanocrystal; PVA, polyvinyl alcohol.