Hybrid Tellurium–Lignin Nanoparticles with Enhanced Antibacterial Properties

The surge of antibiotic-resistant bacteria is leading to the loss of effectiveness of antibiotic treatment, resulting in prolonged infections and even death. Against this healthcare threat, antimicrobial nanoparticles that hamper the evolution of resistance mechanisms are promising alternatives to antibiotics. Herein, we used Kraft lignin, a poorly valorized polymer derived from plant biomass, to develop novel hybrid tellurium–lignin nanoparticles (TeLigNPs) as alternative antimicrobial agents. The sonochemically synthesized TeLigNPs are comprised of a lignin matrix with embedded Te clusters, revealing the role of lignin as both a reducing agent and a structural component. The hybrid NPs showed strong bactericidal effects against the Gram-negative Escherichia coli and Pseudomonas aeruginosa, achieving more than 5 log bacteria reduction, while they only slightly inhibited the growth of the Gram-positive Staphylococcus aureus. Exposure of TeLigNPs to human cells did not cause morphological changes or reduction in cell viability. Studies on the antimicrobial mechanism of action demonstrated that the novel TeLigNPs were able to disturb bacterial model membranes and generate reactive oxygen species (ROS) in Gram-negative bacteria.


INTRODUCTION
Antimicrobial resistance (AMR) is a naturally occurring phenomenon of bacteria to ensure their survival after exposure to drugs that would normally eliminate them or inhibit their growth. The overuse of antibiotics, however, is exerting selective pressure on bacteria, favoring the surge of drugresistant strains. As a consequence, the effectiveness of antibiotics for the treatment of bacterial infections has rapidly decreased, resulting in prolonged illness and even death. 1 The US and Europe have already reached 35 and 25 thousand annual deaths, respectively, related to AMR. 2,3 If solutions are not found, the global number of deaths per year by 2050 will extend to a dramatic 10 million cases. 4 Therefore, there is an urgent need to develop alternative and efficient antimicrobials to tackle microbial infections, while preventing the appearance of AMR.
In the search for antibiotic alternatives, metal and metalloid nanoparticles (NPs) are promising antimicrobial agents due to their higher reactivity compared to their bulk counterparts. 5 Unlike antibiotics, these NPs target multiple bacterial components simultaneously due to their unspecific antimicrobial mode of action including oxidative damage, disruption of the bacterial lipidic membrane, and inhibition of metabolic enzymes, thereby hampering the evolution of resistance mechanisms. 5,6 Silver is by far the most explored element among the reported antimicrobial metal NPs, 7,8 while others such as Zn, Cu, and Ti have been less extensively studied. 9,10 However, findings on the bacterial resistance mechanism against AgNPs 11 and other metals and metal oxides 12 define the need to expand the metal NP toolbox against AMR. Despite the known toxicity of tellurite ions (TeO 3 2− ) against Gram-negative bacteria, 13,14 the development of Te-based antimicrobial nanomaterials displaying the advantages of metal NPs has only gained interest in recent years. 15−18 Moreover, toxic reducing agents used in the traditional physicochemical methods for the synthesis of tellurium NPs (TeNPs) are cytotoxic to human cells and environmentally harmful. 19,20 To overcome the main limitations of traditional synthesis, efforts have been made to develop nanobiotechnological approaches for the synthesis of TeNPs including tellurite-reducing microorganisms 21−23 and plant-derived reducing agents. 15,16,24 In these cases, the biomolecules involved in tellurium reduction can act as capping agents, providing enhanced stability of the synthesized TeNPs and increased biocompatibility, thus overcoming the major drawbacks of traditional synthesis methods. 20,25 In this work, industrial lignin was used for the first time as a reducing agent in a green sonochemical approach to obtain hybrid tellurium−lignin NPs (TeLigNPs). Lignin, the second most abundant biopolymer on Earth, is rarely valorized in its macromolecular form and usually burned for energy uses. Within the context of nanobiotechnology research, lignin is being used for the development of safe-by-design approaches yielding green biocompatible nanomaterials such as porous nanotemplates for infusing silver ions, 26,27 carriers for drug delivery, 28,29 and anticancer targeted NPs. 30 The developed ultrasound-assisted synthesis is a one-pot, fast water-based process, performed under mild conditions and without the need for any chemical modifications of the starting materials. TeLigNPs were extensively characterized in terms of morphology and composition to determine the role of lignin in NP formation. The novel NPs were evaluated as antimicrobial agents by investigating their mode of action and antimicrobial efficacies against Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa and Escherichia coli. Finally, the potential toxicity of the novel TeLigNPs was assessed using human cell models.
2.2. Synthesis of TeLigNPs. Protobind 6000 lignin was dispersed in water (1% w/v). To increase lignin solubility, the pH was increased to 9 by addition of NaOH. Thereafter, sodium tellurite powder was diluted in the prepared lignin solution at 100 mM final concentration. The resulting solution was subjected to ultrasound (20 kHz, 50% amplitude, Ti-horn) for 30 min at 60°C (VCX 750 ultrasonic processor, Sonics). The generated NPs were purified by centrifugation at 25 000g for 30 min. The pellet was washed for complete removal of tellurite ions by resuspension in Milli-Q water and were further centrifuged at 25 000g for 30 min. Afterward, the washed pellet was resuspended again in Milli-Q water, and low-intensity ultrasound was used to completely disaggregate the NPs in the suspension. Finally, centrifugation at 500g for 10 min removed larger particles and the remaining insoluble lignin.
2.3. Characterization of TeLigNPs. The morphology and distribution of the TeLigNPs were determined by transmission electron microscopy (TEM) using carbon-coated silicon dioxide grids and a JEOL JEM-2100 LaB6 microscope coupled with energydispersive X-ray spectroscopy (EDX) operating at an accelerating voltage of 200 kV. EDX was used for the elemental analysis of the TeLigNPs, and chemical maps were acquired using high-angle annular dark-field (HAADF) scanning TEM (STEM). Nanoparticle size and distribution were obtained using ImageJ software (version 1.52a). The crystalline structures of the TeLigNPs were obtained from highresolution TEM (HR-TEM) experiments. The ζ-potential of NPs in water was determined using a Zetasizer Nano Z (Malvern Instruments Inc., U.K.).
The content of tellurium in the TeLigNPs was quantified by inductively coupled plasma mass spectrometry (ICP-MS 7800, Agilent Technologies) calibrated by an internal standard with 45 Rh and a standard curve of 125 Te. Prior to the analysis, the samples were digested with 20% (v/v) HNO 3 at 100°C for 1 h, diluted until a final concentration of 2% HNO 3 , and filtered through a 0.2 μm pore size filter.
2.4. Growth Inhibition of S. aureus, E. coli, and P. aeruginosa by TeLigNPs. The antibacterial activity of the TeLigNPs was assessed toward Gram-positive S. aureus and Gramnegative E. coli and P. aeruginosa following the serial dilution method. Overnight bacterial cultures were diluted in NB to an OD 600 = 0.01 (∼10 5 −10 6 CFU·mL −1 ). Then, 50 μL of the TeLigNPs at different concentrations (Te content ranging from 0.04 to 2.39 ppm) were mixed with 50 μL of bacterial suspension in 96-well polystyrene plates. The samples were incubated for 24 h at 37°C with shaking. Bacterial growth in the presence of NPs was assessed by measuring the OD at 600 nm in a microplate reader (Infinite M200, Tecan, Austria). Bacterial inoculum without NPs was used as a growth control (no inhibition). The OD 600 of the samples at time 0 h was used as a blank. The growth inhibition was calculated as follows bacterial growth inhibition (%)

100
OD OD The minimal inhibitory concentration (MIC) was taken as the lowest concentration of TeLigNPs that inhibited the growth of the bacteria after 24 h of incubation at 37°C. In addition, the number of surviving bacteria during NP treatment was determined after plating 10 μL of the suspensions onto specific agar and further incubation for 24 h at 37°C. After counting the colonies, the log reduction and the percentage of reduction were calculated.

Bacteria Time-Killing Kinetics.
Overnight bacterial cultures were diluted in NB to an OD 600 = 0.01 (∼10 5 −10 6 CFU·mL −1 ). For the assay, 300 μL of the bacteria were mixed with 300 μL of TeLigNPs at different concentrations (Te contents 2.39, 1.20, and 0.60 ppm) and incubated at 37°C with 230 rpm shaking. Samples were taken at different time points, and the surviving bacteria were enumerated using the drop plate method. In total, 10 μL of diluted E. coli and P. aeruginosa suspensions were plated on Coliform ChromoSelect agar and Cetrimide agar, respectively. After 24 h incubation at 37°C, the grown colonies were counted. Bacteria in NB were used as a growth control.
2.6. Cytotoxicity Test. The cytotoxicity of the TeLigNPs was tested toward human fibroblasts (cell line BJ-5ta) and human keratinocytes (cell line HaCaT). The cells were grown in 100 μL of DMEM in a 96-well plate (60 000 cells per well) at 37°C in a humidified atmosphere with 5% CO 2 . After 24 h of cell growth, TeLigNPs at different concentrations were incubated with the cells for 24 h. Afterward, the NPs were removed from the wells, and the cells were recovered for 24 h in 100 μL of fresh DMEM. The cell viability assessment was performed using the AlamarBlue assay. AlamarBlue reagent is a redox indicator dye used for the evaluation of the metabolic activity of cells. After removing the culture medium, 100 μL of AlamarBlue reagent diluted in culture medium (10% v/v) was added to each well. After 3 h incubation at 37°C, the fluorescence was measured (λ ex = 550 nm, λ em = 590 nm). The percentage of cell viability was calculated using the fluorescence values of the wells containing only cells and AlamarBlue reagent as the reference (growth control). Wells containing only AlamarBlue reagent were used as the blank group. The percentage of cell viability was calculated as follows The reactive oxygen species (ROS) generated by bacterial cultures exposed to the TeLigNPs were evaluated using the oxidationsensitive probe H 2 DCFDA, which is activated by intracellular oxidants such as hydrogen peroxide and the hydroxyl radical. 13,31 In the assay, carried out in triplicate, S. aureus, E. coli, and P. aeruginosa were grown in NB to an OD 600 of ∼0.8 and were exposed to TeLigNPs (2.39 ppm of Te) for 30 min at 37°C. The samples were centrifuged at 4000g and washed with PBS. The pellet was resuspended in 1 mL of PBS and 1 μL of 20 mM H 2 DCFDA was added. After 30 min incubation in the dark, fluorescence measurements (λ ex = 490 nm, λ em = 520 nm) were performed and normalized to OD 600 . Bacteria incubated without TeLigNPs were used as controls.
ROS generated in human keratinocytes and fibroblasts were assessed after growing the cells in 100 μL of DMEM in a 96-well plate (60 000 cells per well) at 37°C in a humidified atmosphere with 5% CO 2 . After 24 h, the cells were washed with PBS and exposed to TeLigNPs (2.39 ppm of Te) for 30 min at 37°C. After removing the particles, 100 μL of 2 mM H 2 DCFDA was added. After 30 min incubation in the dark, fluorescence measurements (λ ex = 490 nm, λ em = 520 nm) were performed. Cells incubated without TeLigNPs were used as controls.
2.8. Transmission Electron Microscopy of Bacterial Samples. TeLigNPs were incubated under shaking with E. coli and S. aureus (OD 600 = 0.01) for 2 h at 37°C. The samples were centrifuged at 2000g for 5 min at 4°C and resuspended in a fresh fixative solution containing 2.5% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The samples were incubated with the fixative solution for 1 h at 4°C, washed three times with 0.1 M phosphate buffer, pH 7.4, and fixed in 1% (w/v) osmium tetraoxide. Afterward, the samples were stained with 2% (w/v) uranyl acetate, dehydrated in ethanol, and embedded in Spurr resin. Ultrathin sections were obtained with an Ultracut E (Reichert-Jung) ultramicrotome and counterstained with lead citrate. Then, the slices were deposited on bare mesh copper grids and the sections were observed using a JEOL 1100 transmission electron microscope at 80 kV.
2.9. Gram-Negative Bacteria Model Membranes. A mixture of PE and PG was prepared at an 8:2 (v:v) ratio in CHCl 3 (0.5 mg· mL −1 ) to mimic a Gram-negative bacterial membrane. 32 The Langmuir films were formed in a Langmuir trough equipped with two mobile barriers (model KN2002, KSV NIMA, Sweden) mounted on an antivibration table housed in an insulation box at 23 ± 1°C. Prior to subphase addition (Milli-Q water), the Langmuir trough was cleaned with CHCl 3 and water, and the surface was further cleaned by suction. Afterward, 40 μL of the lipid mixture solution was added dropwise into the trough filled with the subphase, and after 10 min evaporation of CHCl 3 , the barriers were compressed at 15 cm 2 ·min −1 until reaching 33 mN·m −1 , the equivalent of the natural membrane's lateral surface pressure. 33 After the stabilization of the membrane for at least 30 min, 500 μL of TeLigNPs in Milli-Q water were inserted beneath the Langmuir film and the changes of the surface pressure derived from their interactions with the already formed bacterial model membrane were recorded. Blank experiments were carried out using the same procedure, but inserting 500 μL of Milli-Q water instead.

RESULTS AND DISCUSSION
3.1. TeLigNP Synthesis and Characterization. In the search for antibiotic alternatives against AMR bacteria, antimicrobial metal or metalloid NPs, whose mode of action circumvents the surge of resistance mechanisms, are of high interest for the development of novel antimicrobials. Among them, TeNPs have emerged as promising antimicrobial agents. However, these NPs have been mostly obtained by traditional chemical synthesis yielding TeNPs with low biocompatibility and an associated environmental burden. Previously explored environmentally friendly synthetic approaches mostly rely on the extraction and purification of plant-derived reducing agents and thus, despite being green, are competing with food and healthcare industries.
Herein, lignin, which is rarely valorized in its macromolecular form, was chosen as a reducing agent for tellurite. This polymer possesses a variety of chemical groups, including phenolic and aliphatic hydroxyl groups, 34 and it was previously used for reducing silver ions into NPs. 32 However, initial attempts to reduce tellurite by incubation with a lignin solution did not result in NP formation. To increase the reduction capacity of lignin and increase the NP synthesis yield, a sonochemically assisted approach was adopted. In a highintensity ultrasound (US) field, reducing species formed from the sonolysis of organic additives and water accelerate the reduction of the metalloid and increase the yield of produced NPs. 35−38 Moreover, it has been reported that the application of US on lignin causes both side-chain depolymerization and oxidative coupling/polymerization of phenoxy radicals, 39−41 which in turn may complex with metals. 42 The darkening of the lignin−tellurite mixture upon application of US suggested the reduction of tellurite to elemental tellurium (Te 0 ). 16 The synthesized NPs were negatively charged, with a ζ-potential of −32.7 mV and an average NP size of 182 ± 85 nm (Figure 1a). The NPs were comprised of an amorphous matrix embedding several electrodense spherical clusters containing defined crystalline domains (red lines in Figure 1b). The elemental map showed that tellurium was mainly located in the electrodense clusters (Figure 1c,d), while the high-intensity oxygen signal observed in the amorphous matrix indicated the presence of lignin containing numerous methoxy and hydroxyl groups (Figure 1e). These images confirmed that in the sonochemical synthesis of TeLigNPs, lignin not only acted as a reducing agent but also as an NP matrix embedding Te clusters.
3.2. Antibacterial Activity of TeLigNPs. TeLigNPs at 2.39 ppm of Te were able to completely inhibit the growth of E. coli and P. aeruginosa (Figure 2a,b), while only 13% growth inhibition could be achieved against the Gram-positive S. aureus (Figure 2c). These results are in agreement with previous studies reporting higher antibacterial activity of TeNPs against Gram-negative than Gram-positive bacteria, 16,22,23,43 a trend also observed for AgNPs. 44 The evaluation of the MIC of TeLigNPs took into account the concentration of Te, the bactericidal agent in the synthesized hybrid NPs, thus allowing comparison with its bulk counterpart, the tellurite ion (TeO 3 2− ). While an MIC corresponding to 0.07 and 2.39 ppm of Te was determined for E. coli and P. aeruginosa, respectively, the same Te concentrations in the form of tellurite did not reduce the growth of these Gram-negative bacteria ( Figure S1). Indeed, the MICs of tellurite for E. coli and P. aeruginosa were 0.31 and 15.60 ppm, respectively (Table S1). These results indicated that the nanoformulation of Te into hybrid TeLigNPs increased the antimicrobial activity of the metalloid.
TeLigNPs showed a strong bactericidal effect against E. coli and P. aeruginosa, achieving 100 and 99.9995% reduction, respectively, corresponding to more than 5 log reduction (Table 1). To the best of our knowledge, this complete bacterial eradication has not been achieved for previously reported TeNPs, which, in contrast to TeLigNPs, lack a structural polymeric component. 16,22,43 Thus, these results suggest that lignin not only acts as a green reducing agent but also as a nanoparticle structural component that synergistically enhances the antibacterial activity of the Te clusters. 45 The killing kinetics of the TeLigNPs were assessed to study the bactericidal effect of the hybrid NPs against Gram-negative bacteria as a function of time. The killing curves revealed that TeLigNPs with Te concentrations of 2.39 and 1.2 ppm were bactericidal against E. coli within 4 h and achieved complete eradication after 24 h (Figure 3a), in agreement with previous results (Table 1). Regarding P. aeruginosa, the bactericidal efficacy of TeLigNPs at a concentration of 2.39 ppm was achieved after 6 h, corresponding to 5.7 log reduction, and was maintained after 24 h (Figure 3b). However, lower concentrations of the NPs exerted only a bacteriostatic effect. Although an initial reduction was observed after 4 h (4 log), P.  aeruginosa was able to recover and increase up to the initial CFU values.
An ultrastructural analysis of bacteria was performed to study the morphological changes of the cells exposed to TeLigNPs. A clear difference was observed between control E. coli cells and those exposed to the hybrid NPs. Untreated E. coli presented a smooth surface and an electrodense cytoplasmic content (Figure 4a), while E. coli cells treated with TeLigNPs showed different signs of cellular damage such as loss of cytoplasmic integrity, disrupted cell envelope, and aggregation of intracellular content (arrows in Figure 4b,c). In contrast, S. aureus cells exposed to TeLigNPs were undamaged ( Figure 4d) and did not present morphological differences compared to the control S. aureus cells (Figure 4e,f).
3.3. Biocompatibility of TeLigNPs. The biocompatibility of metal-and metalloid-based antibacterial agents is a major issue for biomedical applications in humans. The cytotoxic mechanism of NPs toward eukaryotic cells is associated with oxidative stress, damage to cell membrane and DNA, and consequently apoptosis. 46,47 These cytotoxic effects are exacerbated when the NPs are chemically synthesized since toxic solvents and reducing agents may remain in the NPs. 20,48 The TeLigNPs were synthesized following a safe-by-design approach using lignin as a reducing agent. This biopolymer has been innovatively used to reduce silver nitrate for the production of AgNPs, 36,49,50 to decrease the cytotoxicity of metallic NPs, 27 and has been formulated in nanocomposites for improving the biocompatibility of biomedical devices. 51 The potential toxicity of the novel TeLigNPs was evaluated using human keratinocytes and fibroblasts as cell models. The cell viability of these cell lines upon incubation with tested antibacterial concentrations of TeLigNPs (Te concentrations ranging from 0.07 to 2.39 ppm) did not decrease compared to the untreated cells (Figures 5 and 1a). Remarkably, the cytotoxic effect was only observed on increasing the concentration of TeLigNPs 8-fold (19.12 ppm). Further evaluation by fluorescent microscopy of the cell viability and morphology of human keratinocytes and fibroblasts incubated with TeLigNPs did not reveal cell morphology changes ( Figure  5b). The biocompatibility of the TeLigNPs is associated with the presence of a natural, benign organic matrix that embeds the Te clusters. 20  3.4. Mechanism of TeLigNP Antibacterial Action. The bactericidal mechanism of TeNPs has not been completely elucidated, considering the production of ROS as one of the factors involved in their antibacterial capacity. 22 The toxicity of the tellurium oxyanion in bacteria has been related to superoxide-mediated oxidative stress causing cytoplasmic thiol oxidation, inactivation of iron−sulfur center-containing enzymes, and peroxidation of the membrane lipids, which lead to cell death. 13,52,53 Therefore, the generation of ROS induced by the TeLigNPs was evaluated to understand the antibacterial mechanism of these hybrid NPs ( Figure 6). For both E. coli and P. aeruginosa, the incubation with TeLigNPs resulted in an increase of the fluorescence emission after the addition of the H 2 DCFDA probe, indicating the presence of ROS induced by the chemical activity of the tellurium oxyanion (Figure 6a). An increase in fluorescence was not detected after incubation of S. aureus with TeLigNPs, indicating the absence of cellular damage, in agreement with the previously obtained antimicrobial results (Figure 2). On the other hand, low fluorescence levels detected after incubation of human keratinocytes and fibroblasts with TeLigNPs indicated that these NPs did not induce ROS in the studied cell lines (Figure 6b), in line with the biocompatibility results.
Besides the production of ROS, the ability of TeLigNPs to disturb bacterial membranes was assessed using the Langmuir technique. The injection of the TeLigNPs beneath the prepared Gram-negative model membrane induced an increase of the surface pressure, indicating a membrane-disturbing effect due to the surface activity of the NPs (Figure 7). 54 These results are supported by the observed irregularities in the cell envelope of the Gram-negative E. coli treated with TeLigNPs (Figure 4b,c). Different types of lignin have shown surface activity according to their origin or the processes employed for their purification, 55 while metallic particles without a stabilizer have shown negligible surface activity. Thus, the observed surface activity was attributed to the lignin component of TeLigNPs, a role also previously described for lignin-capped silver NPs. 56  Based on the TeLigNP mode of action and the observed different antimicrobial activities against the tested bacteria, we hypothesize that the bactericidal activity against Gram-negative bacteria is due to an insertion of the hybrid NPs into the outer membrane of these bacteria coupled to membrane lipid peroxidation. The surface activity of TeLigNPs would allow binding and insertion into the lipid bilayer of the outer membrane. In this sense, it has been reported that lignin particles adsorb at hydrophilic/hydrophobic interfaces, 57 and lignin-derived compounds can cross membrane bilayers due to hydroxyl−lipid interactions. 58,59 Once inserted, ROS production by TeLigNPs generates lipid peroxides that decompose into highly reactive short-chain aldehydes able to diffuse in the cytoplasm and oxidize thiol and amino groups of proteins, thus affecting several cellular processes and leading to death. 60,61 On the other hand, Gram-positive bacteria are characterized by a thick external peptidoglycan cell wall surrounding the cytoplasmic membrane, which would prevent the access of the TeLigNPs to the cell membrane, thus resulting in the observed low antimicrobial activity against S. aureus.

CONCLUSIONS
In this work, we developed novel hybrid antibacterial TeLigNPs through a sonochemical synthetic approach using industrial lignin. This biopolymer was not only used as a tellurite-reducing agent but also became a nanoparticle structural element that embeds reduced Te clusters and confers surface activity to the nanohybrid. The novel NPs completely inhibited the growth of the Gram-negative E. coli and P. aeruginosa at a lower tellurium concentration than when using the tellurite salt, thus showing the advantages of the applied nanoformulation strategy. Furthermore, the TeLigNPs were found to possess strong bactericidal activity against the aforementioned Gram-negative bacteria, being able to eradicate 99.9995% of these bacteria within 4 h. On the other hand, the hybrid NPs slightly inhibited the growth of Gram-positive S. aureus. Investigation of the antimicrobial mode of action revealed the capacity of TeLigNPs to induce a membrane-disturbing effect due to surface activity and the generation of ROS in the presence of Gram-negative bacteria. On this basis, we hypothesize that TeLigNPs enter the lipid bilayer of the outer membrane of Gram-negative bacteria and possibly induce lipid peroxidation. These events cause a disruption of the cell envelope and oxidative damage to several metabolic pathways, eventually leading to cell death. In contrast, the external peptidoglycan cell wall of Gram-positive bacteria prevents the access of the TeLigNPs to the lipid membrane, resulting in the observed low growth inhibition. TeLigNPs did not induce cytotoxic effects or morphological changes to human cell lines, demonstrating that lignin can be used to develop safe-by-design tellurium-based nanomaterials. Altogether, we provided an environmentally friendly approach for preparing biocompatible NPs as a potential antibacterial agent. The capacity of killing bacteria, together with their biocompatibility, makes these NPs promising antimicrobial agents for the treatment of bacterial infections. Further in vivo investigations should be pursued to validate the potential of these NPs for the treatment of infectious diseases.
Growth inhibition effect of tellurite against E. coli, P. aeruginosa, and S. aureus and MICs of TeLigNPs and tellurite (PDF)