Ag-lignin hybrid nanoparticles for high-performance solar absorption in photothermal antibacterial chitosan films

Summary There is an urgent need for antimicrobial films based on sustainable resources and production methods. In this study, we present a bio-based nanocomposite film composed of chitosan (∼60 wt %), lignin nanoparticles (LNPs, ∼40 wt %), a small amount of glutaraldehyde (1.5 wt %), and a trace level of silver nanoparticles (AgNPs, 0.072 wt %). The uniform dispersion with LNPs prevented aggregation of metallic silver, resulting in small (diameter 3.3 nm) AgNPs. The nanocomposite film absorbs 89% of radiation across the entire solar spectrum and exhibits a remarkable photothermally triggered antibacterial effect, which is further enhanced by the dark color of lignin. Under simulated solar light illumination, the nanocomposite films demonstrated a significant reduction in viable Escherichia coli count compared to control scenarios. The potential applications of these nanocomposites extend to sunlight-activated antimicrobial films and coatings, addressing the growing demand for sustainable and effective antimicrobial materials.


RESULTS
Our approach to prepare composited antibacterial film and demonstration of the photothermal antibacterial application is conveyed in Figure 1.Firstly, the prepared LNPs were used as a reducing agent and stabilizer to reduce Ag + ions to AgNPs with the assistance of UV light, and the product was named Ag@LNP.Then the dispersion of Ag@LNP was composited with chitosan in the presence of glutaraldehyde as a crosslinker and casted into films.Chitosan was chosen as a polymer matrix because of its biodegradability 39 and electrostatic attraction with lignin. 29,40,41rom the TEM images of the prepared Ag@LNP, we observed spherical LNPs surrounded by AgNPs, while AgNPs appeared to be covered by a thin layer of lignin (Figure 2A).By measuring the particle size from the TEM images and fitting with a Gaussian distribution, we found that the mean size of LNPs was 96 nm and that of AgNPs was 3.3 nm (Figures 2B and 2C).The size of AgNP was within the domain that is desired for better antibacterial performance. 6,42Such small silver nanoparticles (<10 nm) readily release ions from their oxidized surfaces that dominate their antibacterial performance in contrast to larger particles (>10 nm) that release ions slowly. 7,13Therefore, the size of the silver nanoparticles in the prepared Ag@LNP is suitable for antibacterial application.LNPs demonstrate the ability to produce small AgNPs and prevent their aggregation, which is central to achieving high-quality nanocomposite films through the dispersion casting method.Table S1 summarizes different methods for forming AgNP.Among them, the UV method that we used does not require alkaline pH or heating conditions and preserves the morphology of LNP.
The interaction of LNPs and silver ions under UV irradiation can be revealed by monitoring changes in size distribution and zeta potential of the colloidal system.Dynamic light scattering showed that UV irradiation had no significant effect on the size distribution of LNPs that had a Z-average hydrodynamic diameter of 210 nm (Figure 2D).In addition, electrophoretic mobility results implied that the hydrodynamic shear surface of LNPs did not change significantly after 30 min of UV irradiation, with the zeta potential remaining at À17 mV (Figure 2E).In contrast, when silver nitrate was added to a dispersion of LNPs and then subjected to 30-min UV light irradiation, Ag@LNPs with an increased particle size of 250 nm were obtained.In addition, the zeta potential of Ag@LNP increased to À8 mV, indicating that uncharged silver particles were bound to the surface layer of LNPs.The increased zeta potential explains why Ag@LNP tended to agglomerate over extended storage durations, as shown in Figure S1, with mean particle size increasing from 257 nm to 540 nm after two weeks.
To overcome the long-term colloidal instability of AgNPs and address the challenge of retrieving silver nanoparticles from solid-waste incineration plants after their intended use, 32 we opted to immobilize the AgNPs within nanocomposite films.The Ag@LNP dispersion was used to prepare a composited cross-linked chitosan-Ag@LNP (CC-Ag@LNP) film that was dark green (Figure 3A) owing to the presence of AgNPs, as revealed by SEM-EDS images and elemental mapping spectra.Silver, as a heavy element, appeared bright in the SEM image of CC-Ag@LNP compared to lighter elements such as carbon or oxygen.Elemental mapping showed that silver was uniformly distributed on the surface of the film (Figures 3B and 3C).The presence of silver in CC-Ag@LNP can be further evidenced by X-ray diffraction (XRD) pattern (Figure 3D).Peaks (2W) at 38.2 , 44.5 , 64.7 , 77.5 , and 81.7 correspond to (111), ( 200), (220), (311), and (222) planes of silver, respectively. 30,35,36,43The pattern is consistent with the standard Ag crystalline structure (JCPDF No. 04-0783) provided by the National Institute of Standards and Technology. 36The interactions of chitosan, glutaraldehyde, and LNPs in the composited suspension and films was revealed by recording the FTIR spectra and measuring zeta potential (Figure 4).Chitosan dissolves in water as a cationic polyelectrolyte at pH below its pKa 6.5. 44Owing to its primary amine groups, the zeta potential was + 37 mV for chitosan solution at pH 4.5 (Figure 4B).When chitosan was cross-linked with glutaraldehyde, the zeta potential decreased, suggesting reactivity of the primary amines via imine chemistry.As shown in Figure 4A, the FTIR spectrum of CC-LNP showed a peak at 1,548 cm À1 (amide II 45 ), which was absent from the spectrum of either chitosan or lignin.The interaction mechanisms between CC and LNPs are probably quite complex and involve electrostatic interactions at long-range and a series of short-distance forces such as hydrogen bonding and van der Waals forces.To further study the nature of interactions between LNP and CC, electrophoretic mobility measurements were carried out for different ratios of lignin/CC.The zeta potential decreased from +32 to +27 mV, when 20% LNP was added to CC (Figure 4B).This is likely because protonated primary amine groups of chitosan interacted with LNPs through double-layer and/or hydrogen bond interactions.It further decreased to +25 mV and remained at this plateau even with 50% LNP content, indicating that the LNPs were effectively covered by chitosan.
[24]30,31 Here, we studied the tensile strength of the chitosan-based films in dry and wet states.As shown in Figure 5A, the tensile strength of CC was 104.4 MPa and that of CC-LNP was 49.9 MPa, which is higher than other lignin-composited films. 29,30The tensile strength of CC-Ag@LNP (lignin content 40 wt%) was 69.9 MPa, which was higher than CC-LNP (lignin content 40 wt%).The Young's modulus and toughness showed the same trend (Figure 5B).Nevertheless, the strains of CC-LNP and CC-Ag@LNP were very similar, 3.3% and 4.3%, respectively (Figure 5C).Interestingly, the films performed differently under wet condition.As shown in Figure 5D, the mechanical strength of CC-LNP (0.67 MPa) was three times that of CC (0.24 MPa) after immersing in water for 2 h.The tensile strength of CC-Ag@LNP was 0.62 MPa, which was only slightly lower than that of CC-LNP.Young's modulus showed the same trend with tensile strength, that is, CC-LNP showed highest value of 3.0 MPa, CC was lowest (1.9 MPa), and CC-Ag@LNP (2.3 MPa) was in between the two.Under wet condition, LNPs and Ag@LNPs rendered the nanocomposite films (lignin content 40 wt%) stronger compared to the cross-linked chitosan alone, with toughness and strain at break increasing from CC (0.02 MJ/m 3 , 14.6%) to CC-LNP (0.07 MJ/m 3 , 23.2%) to CC-Ag@LNP (0.11 MJ/m 3 , 34.2%).Overall, the incorporation of LNPs in the cross-linked chitosan films resulted in lower dry strength, but improved wet strength, suggesting the possibility for use under moist conditions, for example, in contact with bacterial suspension.
We further investigated the stability of the films under wet condition by measuring the swelling degree and water contact angles.The swelling degree of CC, CC-LNP, and CC-Ag@LNP films were significantly lower than that of pure chitosan film (Figures 5G and 5H), which was consistent with the literature. 46Among the films, CC-Ag@LNP had the lowest swelling degree, which might explain its highest toughness (0.11 MJ/m 3 ) and strain (34.2%).Comparing the thickness changes after immersion in water for 2 h, the changes of CC-LNP and CC-Ag@LNP films were notably smaller than that of CC, which might be the reason that their tensile strength and Young's modulus were higher than that of  S1.
CC alone.By comparing their wettability, we observed that CC-LNP showed lowest water contact angles both at the beginning of contact with water and after 20 s of contact (Figure 5I), implying that LNPs increased the hydrophilicity of the composited films.The hydrophilic nature of LNPs is well documented in the literature. 47The existence of LNPs decreased the swelling degree of chitosan-LNP composited films (Figure S2).When the lignin content increased from 0 to 100%, the swelling degree significantly decreased from 60 to 4 times of swelling relative to the original weight.
To achieve stimuli-responsive triggered bactericidal properties, we first investigated the photothermal performance of the films.Infrared (IR) camera images demonstrated the photothermal properties of the films.After irradiation for 15 min by simulated solar light, CC-Ag@LNP showed highest temperature increment (Figure 6A).More accurate temperature changes during the irradiation were recorded by a temperature data logger (Figure 6B).All films reached a stable temperature after 110 s, with CC-Ag@LNP showing the highest equilibrium temperature of 52 C, followed by CC-LNP at 42 C, and CC at 31 C.
Absorption of light is crucial for photothermal heating.We measured the optical spectra of the three kinds of films to analyze their absorption (A) across the whole solar spectrum.We define here that A = 100% À T À R, where T is the transmittance and R is the reflectance of the  films.The measured transmittance (T%) and reflectance (R%) spectra of the films are shown in Figure S3, and the calculated absorption (Abs.%)spectra are shown in Figure 6C.CC-Ag@LNP had the lowest transmittance and relatively low reflectance; therefore it absorbed 89% of the radiation across 200À2500 nm, i.e., the majority of the solar radiance, which is a priority requirement for high-efficiency photothermal materials. 48In the UV range (200-400 nm), CC-LNP provided the same absorption with CC-Ag@LNP, because their transmittance properties were equally low.In the visible range (400À800 nm), CC had the highest transmittance, CC-Ag@LNP was opaque, and CC-LNP was in between, which can also be predicted from their appearance (Figure 6A, digital photos).In the NIR range (800À2500 nm), both CC and CC-LNP exhibited similar transmittance values.However, CC-LNP demonstrated a lower reflectance compared to CC, resulting in a higher absorbance for CC-LNP than CC.
After understanding the photothermal and optical properties of CC-Ag@LNP film, we tested the photothermal antibacterial properties of the prepared films using E. coli (for details, see experimental section and Figure S4).Survival of E. coli was followed and compared to the control suspension of E. coli suspended in water (Figure 7A).There was a slight reduction of similar magnitude in the colony forming units (CFU) due to contact with CC or CC-LNP films, but no significant difference, indicating that LNPs were not the active material.However, in the case of CC-Ag@LNP, the agar remained clean compared to the control samples, even when the dilution factor of the CC-Ag@LNP sample was 100-fold lower than that of the control sample.The survival of E. coli was calculated in CFU units and showed in Figure 7B.Analysis of variance was used to compare differences between two groups (Table S2).Clearly, for the control sample, the irradiation alone had no effect on the change of CFU, implying that the presence of the photothermal material is responsible for the effective sterilization.Table S1 summarizes the sterilization effect of different antibacterial materials which contain AgNP.It seems that materials with photothermal properties can kill bacteria effectively in a short time.In the present study, the CC-Ag@LNP with a low amount of silver (0.072 wt %) reduced cell viability by more than 99.9%.Such a good sterilization performance of CC-Ag@LNP may be stemming from the large surface area of AgNPs 6 and superior broad-range absorbance of solar light as discussed previously.

DISCUSSION
We reported antibacterial films that can be activated photothermally by utilizing lignin nanoparticles and silver with cross-linked chitosan.The interactions between silver ions and lignin resulted in formation of finely dispersed AgNPs ($3.3 nm) on the surfaces of colloidal lignin particles under mild reducing conditions (pH 3.8, room temperature).In addition to their superior antimicrobial effect against E. coli the composited films (CC-Ag@LNP) showed higher mechanical strength under wet condition compared to the cross-linked chitosan films.Such ligninbased nanocomposite films can be photothermally heated to 51 C in less than 2 min.The superior photothermal performance of the   S1 and S2.

Figure 1 .
Figure 1.Preparation of Ag@LNP and CC-Ag@LNP film and demonstration of phtothermal antibacterial application

Figure 2 .
Figure 2. Morphology of Ag@LNP and stability of the dispersion (A) Representative TEM images of Ag@LNP.(B) Diameter distribution of LNPs measured from TEM images, including that shown in (A).A total of 64 particles were measured.(C) Diameter distribution of AgNP measured from TEM images, including that shown in (A).A total of 27 particles were measured.(D) Diameter distribution of LNPs, LNPs-UV, and Ag@LNPs from DLS measurement.(E) Zeta (z) potential of the aqueous dispersion of LNPs (pH 3.8), LNPs-UV (pH 3.8), and Ag@LNP (pH 3.6).Error bars represent standard deviation based on the entire population (n = 3).See also Figure S1 and TableS1.

Figure 3 .
Figure 3. Characterization of silver in the nanocomposite films (A) Schematic illustration and digital photo of CC-Ag@LNP film.(B) SEM-EDS image of CC-Ag@LNP film and elemental mapping of carbon, oxygen, and silver.(C) EDS spectrum of the SEM image.(D) X-ray diffraction (XRD) pattern of CC-Ag@LNP film.

Figure 4 .
Figure 4. Interactions in the nanocomposite dispersion and films (A) FTIR spectra of LNP, chitosan, and CC-LNP.(B) Zeta (z) potential of the aqueous dispersion of chitosan, cross-linked chitosan (CC), and CC-LNP composites.Error bars represent standard deviation based on the entire population (n = 3).

Figure 5 .
Figure 5. Mechanical properties and water stability of the nanocomposite films with a constant lignin content of 40 wt%.(A-C) Mechanical properties of dry films.(D-F) Mechanical properties of films immersed in water after 2 h.(G) Swelling degree of chitosan, CC, CC-LNP, and CC-Ag@LNP films immersed in water after 2 h.(H) Thickness of dry and wet CC, CC-LNP, and CC-Ag@LNP films.(I) Extraction of contact angle from obtained images of water drop on the given substrate (CC, CC-LNP, and CC-Ag@LNP films).Error bars represent standard deviation based on the entire population (n = 3).See also Figure S2.

Figure 6 .
Figure 6.Photothermal and optical properties of the cross-linked chitosan and nanocomposite films with a constant lignin content of 40 wt% (A) Infrared images of irradiated CC (left), CC-LNP (middle), and CC-Ag@LNP (right) films after 15 min of exposure to simulated solar irradiation (0.1 W/cm 2 ) with the corresponding digital images.Color map shows the temperature in C. (B) Temperature changes of CC, CC-LNP, and CC-Ag@LNP films under artificial solar irradiation (0.1 W/cm 2 ).(C) Absorption spectra of films calculated by A = 100% --T --R, where T = in-line (direct) transmittance (%) and R = reflectance spectra (%) of cross-linked chitosan (CC), CC-LNP, and CC-Ag@LNP films.The filled (red) spectrum indicates solar radiance at sea level.See also Figure S3.

Figure 7 .
Figure 7. Antibacterial performance of the cross-linked chitosan and nanocomposite films with a constant lignin content of 40 wt% (A) Digital images of bacterial colonies of E. coli after different treatments; scale bar: 3 cm.(B) E. coli treated by CC, CC-LNP, and CC-Ag@LNP films for 15 min with or without simulated solar irradiation (0.1 W/cm 2 ).Error bars represent standard deviation based on the entire population (n = 3).*p < 0.05 and **p < 0.01.NS (p > 0.05), statistically not significant.See also Figure S4, TablesS1 and S2.