WO2020149755A2

WO2020149755A2 - Nanosphere materials and methods of synthesizing same

Info

Publication number: WO2020149755A2
Application number: PCT/QA2020/050002
Authority: WO
Inventors: Khaled Mahmoud; Prakash PANDEY; Kashif RASOOL; Abdul Rasheed PATHATH; One-Sun LEE
Original assignee: Qatar Foundation For Education, Science And Community Development
Priority date: 2019-01-15
Filing date: 2020-01-14
Publication date: 2020-07-23
Also published as: WO2020149755A3

Abstract

A nanosphere composite is disclosed. The nanosphere composite includes a chitosan and a ligosulfonate. The chitosan and the ligosulfonate are covalently cross-linked. A crosslinker of the nanosphere composite includes formaldehyde. A method of preparing the nanosphere composite is also disclosed. The method includes providing a first solution comprising a chitosan; providing a second solution comprising a ligosulfonate; mixing the first solution and the second solution to form a mixture; and adding a crosslinking solution comprising formaldehyde to the mixture to produce a suspension.

Description

TITLE

NANOSPHERE MATERIALS AND METHODS OF SYNTHESIZING SAME

BACKGROUND

[0001] The present disclosure generally relates to nanosphere composite materials and methods of synthesizing the same.

[0002] Biodegradable polymeric nanoparticles from renewable sources are recognized as promising materials for biomedical and environmental applications due to their low cytotoxicity, increased dispersibility, biocompatibility, biodegradability, and higher thermal and chemical stability. In particular, the amphiphilic spherical particles exhibit high resistance to nonspecific protein adsorption, bacterial adhesion, and prove excellent antifouling properties, which make them ideal for biological applications.

[0003] Chitosan (CS) is a typical example of biodegradable polymers with high adhesion to the surfaces, high hydrophilicity, nontoxicity, antimicrobial properties and low costs. CS nanoparticles have been widely employed in food packaging, cosmetics, pharmaceutical, biomedical, agricultural, and chemical industries. In addition, CS-based nanocomposites can be used as cost-effective biocides for the inhibition of sulfate-reducing bacteria (SRB), which are major cause of microbially influenced corrosion in oil and gas industry. The higher the degree of deacetylation of chitosan nanoparticles, the higher the positive charge density, which can infer stronger antibacterial activity at a pH of about 5.5-6.5.

[0004] Physical crosslinking protocols such as hydrogen bonding, ionic interactions, and entanglement, such as those described in Fredheim et al., Biomacromolecules, 2003, 4, 232- 239 and Rasool et ah, ACS Sustain. Chem. Eng., 2018, 6, 3896-3906 can be facile and versatile approaches for the preparation of CS nanoparticles.

[0005] However, microspheres prepared by these methods suffer from poor mechanical strength that could affect further modification and performance of microspheres thus, limiting their sustainable application. On the contrary, covalent bonding or multivalent functionalization usually lead to the strongest and most stable nanoparticles (Zhao et ah, Chem. Int. Edit., 2018, 57, 7580-7608). [0006] Lignin is the second most abundant polyphenolic water-insoluble biopolymer, which can be extracted from biomass and can be easily modified to water-soluble derivatives. Additionally, chemical modification can add unique properties to lignin such as thermal moldability, amphiphilicity, and miscibility (Figueiredo et al., Prog. Mater. Sci., 2018, 93, 233-269).

[0007] Lignosulfonate (LS) is a sulfonic acid groups-containing lignin derivative soluble both in water and organic solvents, thus having amphiphilic characteristics. LS has been largely used for antioxidant, anticoagulant, antiulcerogenic, and antitumor products in the field of biomedical applications.

[0008] CS/LS polyelectrolyte complexes were previously prepared through ionic interaction between the positively charged amino groups of CS and the negatively charged sulfonate groups of LS via ultrasonication, as described in Fredheim et al., Biomacromolecules, 2003, 4, 232-239; Kim et al., Colloids Surf. B-Biointerfaces , 2013, 103, 1-8; and Al-Rashed et al ., Macromolecular Chemistry and Physics, 2019, 220, 1800338.

[0009] In other efforts, such as in Tartakovsky et al., J. Ind. Microbiol. Biotechnol. , 1998, 20, 45-47, CS/LS composites were prepared by simply mixing aqueous solutions of CS and LS at pH 4-5 via ionic gelation or polyelectrolyte complex formation. These CS/LS composites that have been prepared by mechanical homogenization, present non-uniform sizes, and shapes and are unstable above pH 4.5, which in turn limits their practical utilization in the drug delivery applications.

[0010] Yan et al. ( International Journal of Biological Macromolecules, 2019, 136, 927- 935) prepared CS cross-linked graphene oxide (GO)/lignosulfonate hybrid hydrogels through simple mixing of GO aqueous suspension with LS solution and CS solution.

[0011] Furthermore, many of the existing fabrication processes involve multiple steps, which can potentially alter the properties of the microspheres once spheroidized and in turn hinder their scale-up production (Hossain et al., Progress in biomaterials, 2014, 4, 1-19). More specifically, current chemical cross-linking approaches involve tedious steps including emulsification with an organic solvent followed by particles hardening with a cross-linking agent (Mitra et al., Indian journal of pharmaceutical sciences, 2011, 73, 355-366.)

SUMMARY

[0012] The inventors surprisingly discovered a simple one-step method for the preparation of CS/LS amphiphilic nanospheres (“CS@LS”) by forming a stable covalent bond between the free hydroxyl groups of both CS and LS moieties. The inventors surprisingly found that these CS@LS nanospheres have enhanced efficiency as environmentally friendly biocides and disinfectants in several industrial applications.

[0013] In light of the present disclosure, and without limiting the scope of the disclosure in any way, in an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise,

[0014] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, a nanosphere composite material is provided. The nanosphere composite material can include a chitosan and a ligosulfonate, wherein the chitosan and the ligosulfonate are covalently cross-linked with each other. The ligosulfonate molecules can form the core of the nanosphere composite material, and the chitosan molecules can be assembled on the surface of the core.

[0015] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, a crosslinker of the nanosphere composite material includes formaldehyde.

[0016] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, the nanosphere composite material is amphiphilic.

[0017] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, a weight ratio of chitosan to lignosulfonate can be from about 1 :2 to about 2 : 1 or about 1: 1.

[0018] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, the nanosphere composite material may have a homogeneous binary network and a particle size in a range from 20nm to about 200 nm.

[0019] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, an antimicrobial material is provided. The antimicrobial material can comprise the nanosphere composite material.

[0020] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, a biocide material is provided for water disinfection and/or MIC inhibition. The biocide material can comprise the nanosphere composite material.

[0021] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, a method of synthesizing a nanosphere composite material is provided. The method can includes mixing a first aqueous solution of chitosan with a second aqueous solution of ligosulfonate and adding a crosslinking solution comprising formaldehyde into the mixture of the first and second solutions.

[0022] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, a weight ratio of chitosan to lignosulfonate can be from about 1 :2 to about 2 : 1 or about 1 : 1.

[0023] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, the cross-linking solution can be prepared by dissolving sodium sulfate, the formaldehyde, and sulfuric acid in water.

[0024] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, the mixture can be stirred after the crosslinking solution was added.

[0025] In an aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, the first solution comprising the chitosan may have a pH from about 3. The first and second solutions can each have a concentration of about 0.1 wt%.

[0026] The reader will appreciate the foregoing details, as well as others, upon considering the following detailed description of certain non-limiting embodiments of the present disclosure.

BRIEF DESCRIPTION OF DRAWINGS

[0027] Fig. 1(a) is a photograph of CS and FS (1 : 1) solution without cross-linker. Fig. 1(b) is a photograph of CS and FS (1 : 1) solution with cross-linker. Fig. 1(c) is a photograph of CS solution with cross-linker. Fig. 1(d) is a photograph of FS solution with cross-linker. Each photograph taken after 2h of reaction.

[0028] Fig. 2(a) shows the UV-spectra of different weight ratio (0-100%) of CS and FS mixture without cross-linking agent (kept for stirring for 2h). Fig. 2(b) shows UV-spectra of supernatant of different weight ratio of CS and FS mixture after cross-linking, obtained by centrifugation at 5000 rpm for 10 min. Fig. 2(c) shows the percentage area under curve showing percentage of FS reacted with CS during cross-linking.

[0029] Fig. 3 shows the preparation scheme for cross-linked CS@FS composite.

[0030] Fig. 4 shows the proposed mechanism for the formation of CS@FS nanospheres.

[0031] Fig. 5(a) is SEM image of CS@FS-2: 1. Fig. 5(b) is SEM image of CS@FS-1 : 1. Fig. 5(c) is SEM image of CS@FS-1 :2. Fig. 5(d) is TEM image of CS@FS-2: 1. Fig. 5(e) is TEM image of CS@LS-1: 1 (Inset: STEM image of a single nanosphere). Fig. 5(f) is TEM image of CS@LS-1:2.

[0032] Figs. 6(a)-6(j) are SEM images for different cross-linked CS and LS composites with varying ratios of LS (0-100%).

[0033] Fig. 7(a) is scanning transmission electron microscopy (STEM) image of CS@LS- 1: 1 nanospheres; Fig. 7(b) is mix energy dispersive spectroscopy (EDS) mapping of CS@LS- 1: 1 nanospheres; Figs. 7(c)-7(f) are EDS elemental mapping of CS@LS-1: 1 nanospheres with separate elements; and Fig. 7(g) is EDS spectrum of CS@LS-1: 1 nanospheres.

[0034] Figs 8(a)-8(c) show the particle size distributions of (a) CS@LS-1:2, (b) CS@LS- 1: 1, and (c) CS@LS-2: 1.

[0035] Fig. 9(a) shows FT-IR spectra for (i) CS, (ii) LS, (iii) Non-cross-linked CS/LS composite, and (iv) Cross-linked CS@LS composite. Fig. 9(b) show solid-state 13C NMR spectra for: (i) Non-cross-linked CS/LS composite and (ii) cross-linked CS@LS composite.

[0036] Fig. 10 shows the Zeta potential of CS/LS-IT and CS@LS-1: 1.

[0037] Fig. 11 shows Nitrogen adsorption-desorption isotherms of CS/LS-IT and CS@LS-1: 1 composites.

[0038] Figs. 12(a)-12(b) show the water contact angle for non-cross-linked CS/LS-1: 1 and cross-linked CS@LS-1: 1: Fig. 12(a) are optical images at different time interval: (i) 0 min, (ii) 1 min, (iii) 2 min, (iv) 3 min, and (v) 4 min; and Fig. 12(b) is a representative graph. The error bar indicates the standard deviation from the three independent measurements.

[0039] Fig. 13 shows the X-ray diffraction patterns for CS, LS, non cross-linked CS/LS- 1: 1 and cross-linked CS@LS-1: 1.

[0040] Figs. 14(a)-14(b) are snapshots from the trajectories of CG MD simulations (total simulation time is 8 ps) of the mixture of CS and LS (Fig. 14(a)) and covalently bonded CS and LS (Fig. 14(b)).

[0041] Fig. 15(A) shows the schematic atomic structure of chitosan, where m = 9 and n = 6 for our simulations. The order of protonated glucosamin (g) and N-acetylglucosamine (n) is randomly chosen as ggngngggngngngn. Fig. 15(b) shows the coarse-grained model of chitosan. Fig. 15(c) shows schematic atomic structure of monolignol. k = 3 for our CG model of lignin. Fig. 15(d) shows the chemical structure of three monolignol connected by b-O-4 bond which is the most abundant in nature. Fig. 15(e) shows the coarse-grained model of monolignol. Fig. 15(f) shows the coarse-grained model of three consecutively bonded monolignols. We used this structure as the CG model of lignin. Fig. 15(g) is a schematic representation of the bond between lignin and chitosan. The bond between lignin and chitosan is represented by green line. Fig. 15(h) shows the structure of the covalently bonded lignin and chitosan used in the CG MD simulations. Lignin is shown in yellow and the bond between chitosan and lignin is represented by the green stick model. For chitosan CG, the bead with a positive charge is shown in blue where other beads are in red.

[0042] Figs. 16(a)-16(d) are SEM images of CS@LS-1: 1 nanospheres kept in water at different temperatures for 2h (a) 22 °C, (b) 40 °C, (c) 50 °C, and (d) 60.

[0043] Figs. 17(a)-17(f) are SEM images of CS@LS-1: 1 nanospheres kept in water at different pH for 2h (a) pH3, (b) pH4, (c) pH5, and (d) pH6, (e) pH7, and (f) pH8.

[0044] Fig. 18 shows the TGA curves for CS, LS, non-cross-linked CS/LS-1: 1 and cross- linked CS@LS-1: 1.

[0045] Fig. 19 shows the differential scanning calorimetry (DSC) curve for: (a) CS, (b) LS, (c) non-cross-linked CS/LS-1 : 1, and (d) cross-linked CS@LS-1: 1.

[0046] Fig. 20(a) is a plot of apparent viscosities versus shear rate, and Fig. 20(b) is a plot of apparent viscosities versus temperature.

[0047] Figs. 21(a)-21(l) show the cell viability analysis of E. coli and B. subtilis cells by flow cytometry dot plots: (a & d) control, (b & e) non-cross linked CS/LS-L 1, and (c & f) cross-linked CS@LS-1: 1. Corresponding SEM images: (g & j) control, (h & k) non cross- linked CS/LS-1: 1, and (i & 1) cross-linked CS&LS-L 1.

[0048] Fig. 22 shows the LDH release from E. coli and B. subtilis cells exposed to different concentrations of CS@LS-1 : 1 nanospheres after 12 h of incubation time.

[0049] Fig. 23 shows E. Coli and B. Subtilis cells growth inhibition by CS@LS hybrids.

[0050] Fig. 24 shows the bactericidal activities of CS@LS-1: 1 nanosphere in aqueous suspensions: Photographs of agar plates onto which E. coli (top panel) and B. subtilis (bottom panel) bacterial cells were recultivated after treatment for 12 h with: (a) 0 pg/mL, (b) 20 pg/mL, (c) 50 pg/mL, (d) 100 pg/mL, (e) 250 pg/mL, and (f) 500 pg/mL of CS@LS-1: 1 nanosphere, respectively. Bacterial suspensions in PBS without nanospheres were used as control.

[0051] Fig. 25 is a graph showing the cell viability measurements of E. coli and B. subtilis treated with CS@LS-1: 1 nanosphere in aqueous suspension.

[0052] Figs. 26(a)-26(f) are photographs of agar plates onto which E. coli (top panel) and B. subtilis (bottom panel) bacterial cells: (a & d) control, (b & e) treated with CS/LS-1: 1, and (c & f) treated with CS@LS- 1: 1 (both at 500 pg/mL).

[0053] Figs. 27(a)-27(c) show the SRBs activity measurements of biomass treated with CS, LS, CS@LS-1: 1, CS@LS-2: 1, and CS@LS-1:2 composites in simulated seawater. Fig. 27(a) shows the relative sulfate reduction and organics oxidation inhibition given as % of the control. Fig. 27(b) is SEM image of the control assay showing a viable bacterial cell. Fig. 27(c) is SEM image of SRB treated with CS@LS- 1 : 1 showing damaged bacterial cell.

DETAILED DESCRIPTION

[0054] The present disclosure provides nanosphere composite materials and methods of synthesizing the nanosphere composite materials.

[0055] These core/shell nanoparticles can be used for environmental remediation, food, and biomedical applications due to their low cytotoxicity, increase in dispersibility, biocompatibility, and higher thermal and chemical stability. Amphiphilic core/shell nanoparticles with outer hydrophilic shell are suitable for drug and gene delivery, removal of contaminants, enzyme immobilization, and antibacterial agent. Chitosan (CS) is a biodegradable polymer with high adhesion to the surfaces, high hydrophilicity, low cost, nontoxicity, and antimicrobial properties. CS nanoparticles can be been widely used in food packaging, cosmetics, pharmaceutical, biomedical, agricultural, and chemical industries. Lignin is an abundant polyphenolic biopolymer extracted from biomass. Lignosulfonate (LS) is the water-soluble derivative of lignin containing sulfonic groups having hydrophilic, chemically reactive, and bioactive features.

[0056] Antibacterial agents can be used to disinfect and eliminate potentially harmful bacteria and can be used in water treatment, health-care, disinfection, and food packaging. Nanomaterials are efficient antibacterial agents and can be used in a large number of commercial products. However, traditional antibacterial agents may not be environment friendly and produce disinfection by products which are even more toxic than there pristine compounds.

[0057] The inventors surprisingly found that lignosulfonate can be used as an anionic polymer for the synthesis of chitosan-lignosulfonate nanocomposite towards the water- soluble, highly efficient, environmental friendly chitosan based antimicrobial nanomaterials.

[0058] The inventors surprisingly discovered methods of synthesizing highly uniform biocompatible nanospheres based on chitosan and lignosulfonate (CS@LS) through chemical cross-linking processes. The sizes of the CS@LS nanospheres can be controlled in the range of 45±3 nm diameter by varying the chitosan and lignosulfonate content. The cross-linked structure of CS@LS was confirmed by FT-IR and solid state 13C NMR. The self-assembly of the cross-linked CS@LS hybrids were further validated by coarse-grained molecular dynamics (CGMD) simulations. [0059] As a non-limiting example, the CS@LS-1 : 1 nanosphere shows excellent antibacterial activity toward both Gram-negative E. coli and Gram-positive B. subtilis compared with non cross-linked CS/LS-1 : 1 hybrid. Flow cytometry dot plots and corresponding SEM images of bacterial cells treated with CS@LS-1: 1 nanosphere shows about more than 3 fold and 2 fold of dead cells population of E. coli and B. subtilis, compared to non cross-linked CS/LS-1 : 1.

[0060] According to an aspect of the present invention, a method of preparing a nanosphere composite material can include at least one step from providing a first solution comprising a chitosan; providing a second solution comprising a ligosulfonate; mixing the first solution and the second solution to form a mixture; and/or adding a crosslinking solution comprising formaldehyde to the mixture to form a suspension. The cross-linking solution can be prepared by dissolving sodium sulfate, the formaldehyde, and sulfuric acid in water. The mixture can be stirred for a period of time after the crosslinking solution was added. The obtained suspension can be washed by, for example, water, and followed by centrifugation to obtain the nanosphere composite material.

[0061] According to an aspect of the present invention, the chitosan and the lignosulfonate can be provided in a weight ratio from about 1 :2 to about 2: 1, from about 1 :2 to about 1 : 1, from about 1 : 1 to about 2 : , preferably about 1 : 1.

[0062] According to an aspect of the present invention, the ligosulfonate can comprise lignosulfonic acid sodium salt or any other suitable lignosulfonic acid salt (EXAMPLES OF OTHER SUITABLE SALTS?).

[0063] According to an aspect of the present invention, the first solution comprising the chitosan may have a pH from about 2 to about 5, from about 2 to about 3, from about 3 to about 4, from about 4 to about 5, from about 2 to about 4, or from about 3 to about 5.

[0064] According to an aspect of the present invention, the chitosan may have an average molecular weight from about 50,000Da to about 190,000Da, from example, from 50,000Da to about 150,000Da, from 50,000Da to about 100,000Da, from 50,000Da to about 80,000Da, from 50,000Da to about 70,000Da, from 60,000Da to about 100,000Da, from 60,000Da to about 80,000Da, from 70,000Da to about 80,000Da.

[0065] According to an aspect of the present invention, the ligosulfonate may have an average molecular weight from about 7,000Da to about 52,000Da, for example, from about 7,000Da to about 50,000Da, from about 8,000Da to about 48,000Da, from about 8,000Da to about 45,000Da, from about 9,000Da to about 42,000Da, from about 10,000Da to about 40,000Da, from about 10,000Da to about 30,000Da, from about l l,000Da to about 25,000Da, from about 15,000Da to about 20,000Da, from about 15,000Da to about 18,000Da.

[0066] According to an aspect of the present invention, the first solution comprising the chitosan in a concentration from about 0.01 wt% to about 10 wt%, for example, from about 0.05 wt% to about 10 wt%, from about 0.1 wt% to about 10 wt%, from about 0.05 wt% to about 5 wt%, from about 0.08 wt% to about 2 wt%, from about 0.09 wt% to about 1.5 wt%, from about 0.09 wt% to about 1.1, about 0.1 wt%.

[0067] According to an aspect of the present invention, the second solution comprising the ligosulfonate in a concentration from about 0.01 wt% to about 10 wt%, for example, from about 0.05 wt% to about 10 wt%, from about 0.1 wt% to about 10 wt%, from about 0.05 wt% to about 5 wt%, from about 0.08 wt% to about 2 wt%, from about 0.09 wt% to about 1.5 wt%, from about 0.09 wt% to about 1.1, about 0.1 wt%.

[0068] According to an aspect of the present invention, a weight ratio of the chitosan to the lignosulfonate is from about 0.0001 : 100 to about 100:0.0001, for example, from about 0.001 : 100 to about 100:0.001, from about 0.01: 100 to about 100:0.01, from about 0.05:50 to about 50:0.05, from about 0.1 : 100 to about 100:0.1, from about 0.5:50 to about 50:0.5, from about 1 : 10 to about 10: 1, from about 1 :5 to about 5: 1, from about 1 :2 to about 2: 1, about 1 : 1.

[0069] According to an aspect of the present invention, a nanosphere composite c material an comprise a ligosulfonate; and a chitosan, wherein the chitosan and the ligosulfonate are covalently cross-linked, ligosulfonate molecules forming the core of the nanosphere composite material, and chitosan molecules being assembled on the surface of the core. The nanosphere composite material is amphiphilic.

[0070] According to an aspect of the present invention, the nanosphere composite may have a homogeneous binary network and may have a characteristic domain size/particle in a range from 20nm to about 300nm, for example, from 20nm to about 200nm, from 20nm to about 40nm, from 20nm to about lOOnm, from 30nm to about 50nm, from 30nm to about lOOnm, from 40nm to about 50nm, from 40nm to about 80nm, from 30nm to about 40nm, from 40nm to about 50nm, from 50nm to about 200nm, from 30nm to about 50nm, from 150nm to about 200nm, from lOOnm to about 200nm, from lOOnm to about 150nm.

[0071] According to an aspect of the present invention, the nanosphere composite material can be used as an antimicrobial material, a biocide material for water disinfection, or a biocide material for MIC inhibition, or any other suitable applications.

[0072] A detailed description of non-limiting embodiments of the present disclosure are further provided in the examples below. [0073] EXAMPLES

[0074] Materials

[0075] Low molecular weight chitosan (CS) (average MW from about 50,000Da to about 190,000 Da and 75-85% deacetylated), lignosulfonic acid sodium salt (LS) (average MW about 52,000 and about 7,000), glacial acetic acid, NaiSCri, EhSCri, and formaldehyde were supplied by Sigma-Aldrich. Deionized (DI) water was obtained from a Milli-Q water purification system (Millipore).

[0076] Synthesis of cross-linked chitosan/lignosulfonate composite (CS ri LS). cross- linked CS and non-cross-bnked CS/LS

[0077] Cross-linked CS@LS hybrids were prepared by chemical cross-linking of CS and LS at different stoichiometric ratios (CS:LS) of about 1: 1, about 1 :2, and about 2: 1. CS (about 0.1 wt%) was dissolved in an aqueous acetic acid solution at pH 3 and stirred overnight at room temperature (25°) using a magnetic stirrer. LS (0.1 wt%) was dissolved in DI water at room temperature (25°C) using a magnetic stirrer. After that, CS and LS solutions were filtered through the syringe filter (pore size 0.45 pm, Millipore, USA) to remove insoluble residues.

[0078] The cross-linking solution was prepared by added sodium sulfate (1.50 g), formaldehyde (0.541 g), and sulfuric acid (1.25 g) in DI water (4.70 mL) under constant stirring for 10 min. at room temperature. CS (30 mL) and LS (30 mL) solutions were mixed together under constant stirring at room temperature for another 30 min.

[0079] The cross-linking solution (450 pL) was added proportionally with the help of a syringe and the resulting solution stirred for an additional 30 min. The obtained suspension was washed five times with DI water, followed by centrifugation at 10,000 rpm to obtain CS@LS-1 : 1 hybrid.

[0080] Similarly, the different weight ratios of CS and LS (2: 1 and 1 :2) were used to prepared as CS@LS-2: 1 and CS@LS-1 :2 composites. The same method was used for the preparation of cross-linked LS and CS, separately.

[0081] The non-cross-linked CS/LS composite was also prepared by mixing aqueous solution (0.1 wt%) of CS and LS in a 1 : 1 ratio at room temperature for 30 min. followed by freeze-drying, to get powder.

[0082] Antibacterial assays

[0083] Antibacterial activity of non-cross-linked CS/LS composite and cross-linked CS@LS composites at different weight ratios were studied by exposing the E. coli and B. subtilis suspensions (about 107 CLU/mL) to material concentrations of 100 pg/mL. In another assay, cell suspensions were spiked with different concentrations (20-500 pg/mL) of CS@LS-1 : 1 nanospheres and incubated at 35°C for 12 h in a shaking incubator. Aliquots of the samples were withdrawn, and 50 pL of 10-fold serial dilutions were plated onto nutrient agar plates. Plates were incubated overnight at 35°C and the cell viability was calculated as the percentage of the control using the following equation:

[0084] Relative cells viability = (Am/Ac) x 100

[0085] where Ac is the number of bacterial colonies of the control sample, and Am is the number of bacterial colonies of the CS@LS nanospheres treated sample.

[0086] Flow cytometry analysis (BD CSampler, Accuri,) was performed by staining control and CS@LS composites treated bacterial cells by propidium iodide (PI), and SYTO 9. The SYTO 9 labels all bacterial cells, whereas, propidium iodide (PI) labels only the dead cells. The analysis was carried out on a by illuminating with a 15 mW argon ion laser (488 nm). The fluorescence was detected via 525 ± 10 nm (green) and 620 ± 10 nm (red) band pass fdters. Signals were amplified with the logarithmic mode for side scattering, forward scattering, and fluorescence. Different bacterial populations were gated according to the viability stages in dot plots of fluorescence. Cell membrane activity of CS@LS treated bacterial cells was studied using cytotoxicity detection kit (Roche Applied Science). The standard assay was performed according to the manufacturer’s instructions. Cells were treated with 20, 50, 100, 200 and 400 pg/mL nanospheres in DI and LDH release was studied. The inhibitory activity of CS@LS against enriched mixed culture sulfate-reducing bacteria (SRB) was studied by exposing SRB to 100 pg/mL of CS@LS-1: 1, CS@LS-2: 1 and CS@LS-1 :2 composites at 35 °C and 150 rpm shaking speed. The mixed-culture SRBs were enriched from sludge biomass using Postage’s C medium as described in Rasool et ak, ACS Sustain. Chem. Eng. , 2018, 6, 3896-3906. Co-substrate oxidation and sulfate reduction was assayed by analyzing total organic carbon (TOC) and residual sulfate concentrations. The impact of nanospheres exposure to SRB surface morphology was examined by SEM images.

[0087] Characterization

[0088] The size, morphology, and structure of prepared CS@LS composites were observed via transmission electron microscopy (TEM), scanning electron microscopy (SEM), Scanning transmission electron microscopy (STEM), and dynamic light scattering (DLS). TEM micrographs were recorded using FEI Talos F200X TEM. The powder samples were dispersed in ethanol and mounted on a lacey Formvar carbon-coated Cu grid. SEM images were obtained using FEI Quanta 650 FEG SEM, after gold sputter coatings on samples. The rheological studies of the CS@LS composite solutions were analyzed using a Discovery HR 2 rheometer (TA Instrument, New Castle, DE) with a temperature-controlled Peltier plate having 1° cone geometry and 60 mm diameter.

[0089] The ultraviolet-visible (UV-vis) absorbance spectra were collected using a Multiskan Sky Microplate Spectrophotometer (Thermo scientific). The particle size of prepared CS@LS composites was measured by laser diffractometry using a Nanotrac II (Microtrac) at 25°C with an incident wavelength of 780 nm and 180backscattering angle. The Fourier-transform infrared (FT-IR) spectrum for synthesized CS@FS composites was obtained by the KBr pellet method using Nicolet™ iS50 FTIR Spectrometer in the range of 3800-500 cm-1. The solid-state 13C NMR spectrum was recorded using the instrument VNMRS 400 operating at 100.52 MHz for 13C. Wide-angle X-ray diffractograms (WAXRDs) were recorded using a Bruker D8 Advance (Bruker AXS, Germany) X-ray diffractometer with Cu-Ka radiation (l = 1.54056 A) at a voltage of 40 kV and a current of 15 mA with a step scan and scanning speed of 0.02° per step and 1° min-1, respectively. Zeta potential was measured using a Malvern Zetasizer Ultra. The Brunauer-Emmett-Teller (BET) surface area was calculated by the N2 adsorption/desorption isotherm at 77 K using a BET surface area analyzer (Micromeritics ASAP 2020). The water contact angle was measured using KRUSS Drop Shape Analyzer DSA25 equipped with a video camera and an image analysis software. The powder samples of both CS/LS-E 1 and CS@LS-1: 1 were dispersed in DI water using bath sonication for 2h and deposited on 0.22 pm hydrophilic polyvinylidene difluoride (PVDF) membrane (47 mm diameter, Merck Millipore, Ireland) using a vacuum-assisted filtration. The resulting films on PVDF were used to measure the water contact angle. Thermal stability of prepared CS@LS composites was studied using a thermogravimetric analyzer (TGA) (TA Instruments Discovery TGA) under N2 atmosphere with 20 °C min-1 heating rate from 50°C to 500°C.

[0090] Results

[0091] The inventors surprisingly found that the size and shape of CS@LS composites were affected by the CS and LS ratio as well as the cross-linking agent.

[0092] As detailed above, aqueous solutions of 0.1 wt% CS (in 0.1 % acetic acid) and 0.1 wt% LS (in water) were mixed together, in CS:LS ratios of 1: 1, 2: 1, and 1:2, under stirring at room temperature (25°C) for 30 minutes. A cloudy suspension was immediately formed after adding the cross-linking agent composed of formaldehyde and sulfuric acid (HCHO/H2SO4) in 450 pL aliquots. The remaining HCHO was decomposed by adding NaHSCh solution.

[0093] For comparison, the formation of chemically cross-linked CS, LS, and non-cross- linked CS/LS was also attempted. In the absence of the crosslinking agent, CS/LS mixture remained as a transparent light orange solution with no evidence of precipitation (Fig. 1(a)), while after crosslinking a cloudy suspension was formed (Fig. 1 (b)). In the case of CS and LS controls, the addition of a crosslinking agent formed a cloudy solution of CS while LS solution remains transparent after stirring for 2h (Fig. 1 (c) and Fig. 1(d)).

[0094] Figs. l(a)-l(d) are the photographs of (a) CS and LS (1 : 1) solution without cross linker, (b) CS and LS (1: 1) solution with cross-linker, (c) CS solution with cross-linker, and (d) LS solution with cross-linker. Each photograph taken after 2h of reaction.

[0095] The UV-vis spectra were used to quantify the residual LS at different stoichiometric ratios for both reactions to form CS@LS and CS/LS, respectively.

[0096] Fig. 2(a) shows the UV-spectra of different weight ratio (0-100%) of CS and LS mixture without cross-linking agent (kept for stirring for 2h). Fig. 2(b) shows the UV-spectra of supernatant of different weight ratio of CS and LS mixture after cross-linking, obtained by centrifugation at 5000 rpm for 10 min. In Fig. 2(c), the percentage areas under curve show the percentages of LS reacted with CS during cross-linking.

[0097] As shown in Figs. 2(a) and 2(b), a significant decrease was observed in the residual LS intensity at 280 nm in the case of CS@LS compared to CS/LS up to 50:50 CS:LS, indicating the successful formation of CS@LS composite. By comparing the peak area of residual LS absorption, up to 71% of starting LS was utilized at 50:50 CS:LS. After this, more unreacted LS was observed in the supernatant. (Fig. S2 (c)).

[0098] The inventors believe that as shown in Figs. 3-4, chemical cross-linking could involve a two-step mechanism: by forming first a hemiacetal bond between formaldehyde and -OH groups of CS or LS and then the hemiacetal reacts with another -OH group of CS or CL to form the acetal (-0-C-0-) bridge. In this acidic medium (pH 4), it is unlikely for the protonated amino groups (-NHC) of CS to participate in the crosslinking reaction.

[0099] Particle morphologies of the different CS@LS composites were examined by SEM and TEM (Figs. 5(a)-5(f) and 6(a)-6(j)). The CS formed aggregated pseudo-spherical microspheres after chemical cross-linking at the same experimental conditions (Fig. 6(a)). Increasing the LS ratio in the composite leads to the formation of aggregated nanoparticles (Figs. 5(a) & 6(b)-6(e)). After reaching 50 wt% ratio of LS to composite, the well-defined and well-dispersed nanospheres were formed (Figs. 5(b) & 6(f)). Further increasing the LS ratio leads to the formation of porous fibers like structures with few embedded nanoparticles (Figs. 5(c) & 6 (g)-6(j)). The TEM image of CS@LS-2: 1 showed aggregated nanoparticles with an average diameter in the range of 30-50 nm (Fig. 1(d)). The TEM image of CS@LS- 1 : 1 showed spherical shape with an average diameter in the range of 150-200nm. STEM image of CS@LS-1 : 1 nanosphere showed hydrophilic/ hydrophobic phase separation. The bright dots on the surface are most likely resembling the hydrophilic domains of the functional groups (-NH2, -S03- and -OH) (Fig. 5(e)). Further, the TEM image of CS@LS- 1 :2 exhibited a fdm-like structure with embedded nanoparticles with an average diameter in the range of 20-40 nm (Fig. 5(f)). The cross-linking attempt of only LS could not form any particles.

[00100] Energy dispersive spectroscopy (EDS) elemental mapping confirmed the uniform distribution of carbon, nitrogen, sulfur, and oxygen on the surface of CS@LS-1 : 1 nanospheres (Figs. 7(a)-7(g)). CS@LS-1 : 1 had an average hydrodynamic size of -228 nm as compared to -265 nm and 964 nm for CS@LS-1:2 and CS@LS-2: 1, respectively (Figs. 8(a)- 8(c)). This particle size distribution pattern was in good agreement with SEM and TEM measurements. Therefore, CS@LS-1 : 1 was considered as the optimum ratio to prepare well- defined nanospheres.

[00101] The chemical structure of cross-linked CS@LS-1 : 1 as compared with other ratios were confirmed by FTIR and solid-state 13C NMR spectra. The FTIR spectra of CS, LS, non-cross-linked CS/LS and CS@LS-1 : 1 are given in Figs. 9(a)-9(b). CS@LS-1 : 1 showed a vibrational peak at -1634 cm-1 attributed to the C=C aromatic stretches from LS moiety, overlapped with the N-H stretching bands of primary amine from CS moiety. In addition, absorption bands aroused at 1517 and 1036 cm-1 representing the C=C aromatic skeletal and the sulfonate groups in the CS@LS, respectively. A new absorption peak at 1106 cm-1 in CS@LS- 1 : 1 was aroused, which is due to the formation of the diether (-0-C-0-) bond and confirmed the cross-linked structure of the composite. On the other hand, this peak was absent in non-cross-linked CS/LS (Fredheim et al. Biomacromolecules, 2003, 4, 232-239).

[00102] The solid-state 13C-NMR further supported the proposed cross-linked structure of CS@LS-1 : 1. As shown in Fig. 9(b), peaks at d = -24, 57, 61, 75, and 102 ppm were assigned to the chitosan moiety present in both CS/LS-I T and CS@LS-1 : 1 composites. The resonance peaks between d = 143 to 150 ppm were assigned to aromatic regions from lignin moiety in both cases. More importantly, two new peaks at d = -94, and 97 ppm, which are corresponding to the formation of the diether linkage (-0-CH2-0-) between CS and LS, confirmed the successful covalent cross-linking. The presence of the sulfonate groups in CS@LS-1 : 1 was confirmed by the resonance peak at d = -72 ppm, assigned for the -C-S03- groups. The FT-IR and 13C-NMR spectra of CS@LS-1 : 1 were confirmed that no formation of -NH2 cross-linked bridge (-NH-CH2-0-) between CS and LS motilities. [00103] Zeta potential can explain some detailed information about the surface composition and surface charge density of the prepared nanospheres. Zeta potential value depends upon the net charge, nature of functional groups located on the nanosphere surface, and pH (Liao et al., Colloids Surf. A Physicochem. Eng. Asp., 2009, 348, 270-275).

[00104] Fig. 10 shows the zeta potential of CS/LS-L 1 and CS@LS-1: 1 at pH 2-10. Both samples have shifted from positive to negative zeta potential value in a nearly linear manner as the solution with pH increase. The isoelectric point (IEP) of non-cross-linked CS/LS-L 1 and cross-linked CS@LS-1: 1 was 7.5 and 5.4, respectively. The lower IEP for CS@LS-1: 1 may be due to the formation of covalent bonds between CS and LS as well as the distribution of -S03- and -NH3 charges on the nanospherical surface (Ashraf et al., Langmuir, 2016, 32, 3836-3847).

[00105] The textural properties of CS/LS-L 1 and CS@LS-1: 1 composites were examined by BET analysis. The surface area of the CS@LS-1: 1 composite was 1.3522 m2g-l, which is significantly higher than those of CS/LS-L 1 composite (0.9045 m2g-l). The specific pore volume and pore diameter of the CS@LS-1: 1 composite were 0.002723 cm3 g-1 and 11.41 nm, respectively (Fig. 11 and Table 1).

[00106] Table 1: Textural parameters of CS/LS-L l and CS@LS-1: 1 composites.

^" fSri 0522 ΪMKΪZ723 GGΪΐ

[00107] The hydrophilicity of the prepared composites was assessed by the water contact angle measurement. The lower contact angle is an indication of higher hydrophilicity, which can facilitate the attraction of more water molecules and thus improve protein adsorption, bacterial adhesion, and maintain antifouling properties (Jiang et al., ACS Nano, 2016, 10, 8732-8737; Wei et al., Nano Lett., 2012, 12, 22-25; and Leal et al., Nanoscale, 2017, 9, 8176- 8184).

[00108] As observed in Figs. 12(a)-12(b), CS/LS-L l and CS@LS-1: 1 show water contact angles of -72.3° and 79.7°, due to the hydrophilic nature of chitosan and -S03- groups of LS, respectively. The lower hydrophilicity of CS@LS-1: 1 compared to CS/LS-L l is most likely due to the decrease in the overall density of hydroxyl groups as they engage in the cross- linking. Furthermore, water contact angle has decreased with the aging of water droplet from 72.3° to 51.3° and 79.7° to 59.6° for CS/LS-L l and CS@LS-1: 1 from 0 to 4 min, respectively (Fig. 12(b)). [00109] Interestingly, the XRD pattern of CS/LS-1: 1 showed semi-crystalline nature where characteristic CS and LS peaks were still observed but wider with lower intensity as compared with their precursors. In the case of CS@LS-1: 1, however, the peak at 10.0° has disappeared and the peak intensity at -20.0° decreased and broadened, while overlapped with characteristic LS peak at 22.7°. Such weaker and broader peak can be explained by the formation of a new binary framework that could disrupt the original structure of both CS and LS (Fig. 13).

[00110] To understand the self-assembly of CS and LS at the atomistic level, the inventors built the coarse-grained (CG) model of CS and LS based on MARTINI force field (Marrink et al., J. Phys. Chem. B, 2007, 111, 7812-7824) and performed molecular dynamics (MD) simulation.

[00111] Specifically, the coarse-grained (CG) model of chitosan and lignin is built based on the MARTINI force field (Figs. 15(A)-15(H)) (Marrink et al., J. Phys. Chem. B, 2007, 111, 7812-7824; Monticelli et al, J. Chem. Theory Comput., 2008, 4, 819-834). Bond, angle, and dihedral energy functions are used for the intramolecular bonded interactions, whereas Lennard-Iones (LI) and Coulomb functions are used for the intermolecular non-bonded interactions. The details of CG type for defining LI parameters are shown in Table 2.

[00112] Table 2. Name, type, and charge of CG model used for chitosan and lignin

[00113] The CG model of chitosan is composed of 9 protonated-glucosamine (g) and 6 N- acetylglucosamine (n) where the inventors adapted a random sequence of them (ggⁿgⁿgggⁿgⁿgⁿgⁿ) and their types and properties are based on the CG model of carbohydrates (Benner et al, Macromolecules, 2016, 49, 5281-5290; Lopez et al, J. Chem. Theory Comput., 2009, 5, 3195-3210). For building the model of lignin, the inventors adapted the CG model composed of three monolignol molecules where the bond between the monolignol is the b-O-4 bond that is most abundant in nature. Even though the structure of lignin is a cross-linked polymer with high molecular masses and the degree of polymerization is difficult to measure, the inventors adopted a simple model of lignin composed of three monolignols (Figs. 15(C)- 15(F)) to minimize the simulation time.

[00114] The inventors prepared two systems for comparing the self-assembly of non-cross- linked chitosan and lignin (system 1) and cross-linked chitosan and lignin (system 2) to understand how cross-linking between CS and FS can affect self-assembly of the structures (Figs. 14(a)-14(b)). System 1 is composed of 10 molecules of chitosan, 10 molecules of lignin, 9000 water beads, 1000 ant freezing water beads, and 90 beads of chloride to electrically neutralize the system. System 2 is composed of 10 molecules of cross-linked chitosan and lignin, 9000 water beads, 1000 anti -freezing water beads, 1 and 90 beads of chloride to electrically neutralize the system. These two systems were identical except for the cross-link (covalent bond) between CS and FS in System 2 (Figs. 15(A)-15(H)). The initial positions of CS and FS in both systems were randomly chosen, and CG MD simulation was performed for 8 ps.

[00115] All simulations were performed with GROMACS simulation package (version 5.0.7) with a time step of 25 fs in the NPT ensemble (Van der Spoel et al., J. Comput. Chem. , 2005, 26, 1701-1718). The pressure and temperature were maintained at 1 bar and 300 K, respectively, by means of the Berendsen method (Berendsen et al., J. Chem. Phys., 1984, 81, 3684-3690). The neighbor list was updated every 10 steps using a neighbor list cutoff of rcut = 1.2 nm. When interpreting the simulation results with the MARTINI model, a standard conversion factor of 4 was used to specify the speedup in CG diffusion dynamics compared to real water due to the smoothing of the potential energy landscape (Marrink et al., J. Phys. Chem. B, 2007, 111, 7812-7824; Baron et al., ChemPhysChem, 2007, 8, 452-461). The inventors used an effective time rather than the actual simulation time unless specifically stated. The total simulation time for each system is 8 ps, and no coordinates were constrained during the simulation.

[00116] Figs. 14(a)-14(b) are snapshots from the trajectories of CG MD simulations (total simulation time is 8 ps) of: Fig. 14(a) the mixture of CS and FS and Fig. 14(b) covalently bonded CS and FS. CS is shown in red and blue where FS in yellow. The cross-link between CS and FS is shown in the green balland-stick shape. The molecular structure of each system is magnified in the green circle. The concentration and the system size are the same in both systems. Water and ions are not shown for clarity. Dotted lines are used for representing the periodic boundaries of the simulation box. For clarity, only self-assembled structures are shown without periodic boundaries for the snapshots at t = 4, 6, and 8 ps in Fig. 14(b).

[00117] In system 1, CS and FS moieties formed intermittent amorphous structures after ~2 ps but dissipated quickly (Fig. 14(a)). The inventors predicted that the Van der Waals interaction energies between CS and FS are not high enough to form a stable self-assembled structure in System. In the case of cross-linked CS@FS (Fig. 14(b)), a self-assembled structure started to form at t = ~2 ps and eventually lead to a semi-spherical structure.

[00118] FS moieties were favorably assembled at the inner core of the sphere where CS formed the outer shell. Even though the Van der Waals interaction energy is the same as System 1, the self-assembled structure of System 2 is ordered in a significantly different fashion. A possible explanation could be that the newly formed acetal bond between the CS and FS moieties restricts the diffusion of FS in System 2 and the dissipation of FS is slow enough so that FS molecules form a stable core of the spherical structure. In order to validate this assumption, the inventors measured the diffusion coefficient of FS during the first 4 ps of CG MD simulation using the Einstein relation Equation (1), where < > is ensemble average, d is the dimension of the system, and r(t) is the position of species (i) at time (t).

[00120] The calculated diffusion coefficient of LS was 14.4 ^c 10-7 cm2/s when free (System 1), but it was 1.1 ^c 10 ⁷ cm²/s when cross-linked to CS (System 2). This indicates that LS movement is significantly restricted when it is covalently bonded to CS, and the dissipation of LS is slow enough to allow the self-assembly of LS and let CS moiety wraps around the LS core. On the other hand, Van der Waals interaction energy between molecules is not high enough to form a stable assembled structure for System 1.

[00121] The stability of CS@LS nanospheres in aqueous suspension was investigated by SEM imaging at different temperatures (20-60°C) and pH (3-8). The shape of CS@LS-1: 1 nanospheres were stable with a slight morphological change between 20°C to 50°C. After 60°C, a distortion of the particles was observed most likely due to the swelling nature of the chitosan at higher temperatures (Figs. 16(a)-16(d)). The CS@LS-1: 1 was also stable between pH (3- 8). At a higher pH, the nanospheres suffered from noticeable shrinking and shape deformation (Figs. 17(a)-17(f)). The thermal stability of CS@FS-1: 1 was not significantly different from the CS/FS-L l, while both CS/FS-L l and CS@FS-1: 1 exhibited higher thermal stability as compared with CS at 500°C (Fig. 18). [00122] Fig. 18 shows the TGA curves for CS, LS, non-cross-linked CS/LS- 1 : 1 and cross- linked CS@LS-1: 1. Weight loss up to 150 °C was observed due to the elimination of bulk and bound water from the CS@LS hybrids. Weight loss between 200 and 300 °C, was attributed to the decomposition of oxygen-containing functional groups (CO, CO2, SO3H, etc.). Beyond 300 °C, decomposition of CS and LS polymer backbone have started and finally, and 45 wt% residual mass was observed at -499 °C, for CS, LS, non-cross-linked CS/LS-L l, and cross-linked CS@LS-1 : 1 hybrids, respectively. The non- cross-linked CS/LS- 1 : 1 and cross-linked CS@LS-1 : 1 hybrids show more thermal stability than pristine chitosan might be the presence of LS moieties.

[00123] The DSC curves of CS, LS, CS/LS-L l, and cross-linked CS@LS-1 : 1 are shown in Fig. 19. All samples show a broad endothermic peak, around 50-150 °C, which is probably attributed to the loss of absorbed water. CS showed a broad exothermic peak, around 260-310 °C centered at near 285 °C, it might be the decomposition of CS polymer backbone, which is in good agreement with the TGA analysis as shown in Fig. 18. The cross-linked CS@LS-1 : 1 showed a new endothermic peak at 220 °C, which might be ascribed to reduced hydrogen bonding between CS and LS as well as the formation of covalent bond thus molecular organization due to chemical cross-linking. However, non-cross-linked CS/LS-L l showed dominating exothermic peak, around 250-300 °C centered at near 280 °C related to CS component, which affected to the LS endothermic peak centered at 290 °C.

[00124] Rheological properties were investigated by studying the apparent viscosities as a function of shear rate for 1 mg/mL CS, LS, cross-linked CS@LS-1 : 1 and non-cross-linked CS/LS-L l solution. At shear rate of 100 s ¹, the apparent viscosities were 1.479, 2.225 and 1.647 mPa.s for LS, cross-linked CS@LS-1 : 1 and non-cross-linked CS/LS-L l solutions, respectively (Fig. 20(a)). This is much lower than the CS viscosity of 4.246 mPa.s and it showed Newtonian behavior. However, the cross-linked CS@LS-1 : 1 and non-cross-linked CS/LS-L l solutions showed non-Newtonian behavior with shear-thickening following the behavior of LS.

[00125] In addition, the change of apparent viscosity of CS@LS hybrid solutions with temperature was dominated by LS behavior as shown in Fig. 20(b). When the temperature increases from 25 °C to 60°C, the viscosity decreases from 0.824 to 0.558 mPa.s, 1.335 to 0.772 mPa.s, and 1.176 to 0.658 mPa.s for LS, cross-linked CS@LS-1 : 1 and non-cross-linked CS/LS-L l solutions, respectively. This was marginal variation when compared to the viscosity drop of CS solution at the same temperatures (from 4.149 to 2.059 mPa s). This intense variation is probably due to the destruction of chitosan structures and the by the polymer degradation upon heating. However, LS shows minor variation in the viscosity upon heating due to the cross-linking reaction occurred between the byproducts formed. The cleavage of aryl ether linkages in the lignin backbone upon heating exposed more active sites on the lignin aromatic ring that facilitates the cross-linking reaction.

[00126] The cross-linked CS@LS-1: 1 dispersion in aqueous media also demonstrated lower apparent viscosity than CS and higher apparent viscosity (2.23 mPa.s) than non-cross- linked CS/LS-T 1 (1.65 mPa.s) and LS (Figs. 20(a)-20(b)). This lower apparent viscosity could be attributed to the formation of dense molecules with smaller hydrodynamic volumes compared to pure CS.

[00127] Strong antimicrobial properties are considered a major success indicator for the effective utilization of CS@LS nanospheres in multiple applications. The bactericidal activities of CS/LS-L l and CS@LS-1: 1 were compared using E. coli and B. subtilis as the model Gram (-) and Gram (+) bacteria, respectively. Figs. 21(a)-21(l) depict the flow cytometry dot plots and corresponding SEM images for both bacterial cells treated with CS/LS-L l and CS@LS-1: 1, respectively.

[00128] From flow cytometry plots, more than 94.0% viability was observed for both E. coli and B. subtilis cells for the control assay (green fluorescence region) indicating the healthy cells (Figs. 21(a) & 21(d)).

[00129] For the bacterial culture spiked with the synthesized CS@LS nanospheres at CS@LS-2: 1, growth inhibition of E. coli and B. subtilis cells were 64.7 and 40.5% respectively, and the growth inhibition of E. coli and B. subtilis cells by CS@LS-1:2, was 70.6 and 31% respectively. For the bacterial culture spiked with the synthesized CS@LS-1 : 1, growth inhibition of E. coli and B. subtilis cells increased to 82.4% and 61.9%, respectively, exhibiting much stronger inhibition.

[00130] In presence of CS@LS-1: 1 composite, over 2 fold higher mortality was observed (red fluorescence region) for E. coli and B. subtilis cells, respectively (Figs. 21(b), 21(c), 21(e), & 21(f)). From the corresponding SEM images, significant cell damage was observed when the bacterial cells are treated with CS@LS-1: 1 compared with CS/LS-L l (Figs. 21(g)- 21(1)). Moreover, an obvious deformation was noticed in the shape of both bacteria with irregularly condensed masses, while most bacterial cells were significantly fragmented due to the disruption of the cytoplasmic membrane.

[00131] The weight ratios of CS and LS clearly affect the bactericidal activity of the synthesized nanocomposites, and the most effective antimicrobial material appeared to be at CS and LS weight ratio of 1: 1 and was used for the further studies as CS@LS nanospheres. [00132] Bacterial cell membrane disruption was further investigated by lactate dehydrogenase (LDH) release assay. Fig. 22 shows the LDH release from E. coli and B. subtilis cells exposed to different concentrations of CS@LS-1: 1 nanospheres after 12 h of incubation time. Batch reactor without any CS@LS-1: 1 nanospheres was used as control. Error bars represent the standard deviation of three independent experiments. A concentration of 400 pg/mL of CS@LS-1: 1 hybrid showed an increase in relative LDH release of about 184.0 % and 197.32 % of that of control for E. coli and B. subtilis, respectively (Fig. 22).

[00133] The LDH release results complement the flow cytometry and SEM analysis suggesting that cell membrane disruption could be a major cell inhibitory mechanism (Haile, Molecular Dynamics Simulations: Elementary Methods , John Wiley & Sons, Inc, New York, 1992).

[00134] As observed in Fig. 23, the most effective antimicrobial efficiency was observed for CS@LS-1: 1 with growth inhibition of 82.4% and 61.9% for E. coli and B. subtilis cells, respectively. The optical images of bacterial colonies and cell viabilities percentage supported the dose-dependent inhibition mechanism of CS@LS- 1: 1. The number of colonies grown on the agar plates was considerably decreased with increasing nanospheres concentration and reached to 0 at 500 pg/mL of CS@LS-1: 1, indicating -100% growth inhibition (Figs. 24-25).

[00135] Fig. 24 shows the optical images of bacterial colonies and percentage cell viability after incubating with different concentrations (0-500 pg/mL) of CS@LS-1: 1. Fig. 25 shows the cell viability measurements of E. coli and B. subtilis treated with CS@LS-1: 1 nanosphere in aqueous suspension. Bacterial suspensions (105 CFU/mL) were incubated with different concentrations of CS@LS-1: 1 (0-500 pg/mL) at 35°C for 12 h at 150 rpm shaking speed. Survival rates were obtained by the colony forming count method. Error bars represent the standard deviation.

[00136] The results showed the dose-dependent bactericidal properties of CS@LS-1: 1 as the number of colonies grown on the LB agar plates considerably decreased with increasing concentration of CS@LS-1: 1. The percentage cell viability of both E. coli and B. subtilis was decreased and reached 0 at 500 pg/mL concentration, indicating 100% growth inhibition. Gram (-) E. coli appeared to be more resistant than Gram (+) B. subtilis at each CS@LS-1: 1 concentration. The outcomes are in line with earlier studies, where higher bactericidal properties of nanoparticles were reported against Gram (+) bacteria compared to Gram (-) bacteria. The reason for the dissimilarities in bacterial activity may be due to the difference in the cell wall structure of two bacterial strains.

[00137] However, bactericidal activities of CS/LS-T 1 show only about 60-64% bacterial cell growth inhibition for both E. coli and B. subtilis cells at the same concentration (500 pg/mL) (Figs. 26(a)-26(f)). The antibacterial activity of CS@LS-1: 1 can be attributed to their surface charge and functional groups of CS and LS. The inventors believe that CS@LS-1: 1 could attach to the bacterial cell membrane through -NH2 groups of CS and increase the cell membrane permeability leading to cell destruction as confirmed by the LDH release. In addition, the antibacterial activity of CS@LS-1: 1 was largely enhanced by enforcing the hydrophobic properties due to the presence of lignin backbone that can increase the interaction with proteins on the bacterial cell membrane.

[00138] To explore these uniform nanospheres as cost-effective biocides for water disinfection and MIC inhibition, the inventors studied their inhibitory effect on the anaerobic SRB. The SRB growth inhibition was assayed in terms of co-substrate oxidation and sulfate reduction in the presence of 100 mg/L of CS, LS, CS/LS- 1: 1, CS@LS-1: 1, CS@LS-2: 1 and CS@LS-1:2.

[00139] Figs. 27(a)-27(c) show the SRBs activity measurements of biomass treated with CS, LS, CS@LS-1: 1, CS@LS-2: 1, and CS@LS-1:2 composites in simulated seawater. SRBs Biofilm (200 mg.VSS/L) were incubated with 100 pg/mL of each composites. Fig. 27(a) shows the relative sulfate reduction and organics oxidation inhibition given as % of the control. Batch reactor without CS@LS composite was used as control. Error bars represent the standard deviation of three independent assays. Fig. 27(b) is SEM image of the control assay showing a viable bacterial cell, and Fig. 27(c) is SEM image of SRB treated with CS@LS-1: 1 showing damaged bacterial cell.

[00140] As depicted in Fig. 27(a), the CS@LS 1: 1 showed highest inhibition of sulfate reduction and total organic carbon (TOC) removal with a 48.8% reduction of sulfate to sulfide compared to the control assay (96%), CS (82%) and LS (88%). TOC removal by SRB was the lowest in the presence of CS@LS-1: 1 composite indicating a co-substrate utilization efficiency of 54.26%. On the other hand, sulfate reduction efficiency was 76.81 and 70.49% in presence of CS@LS-2: 1 and CS@LS-1:2, respectively. Moreover, CS@LS 1: 1 demonstrated about 33% and 40% higher sulfate reduction inhibition than chemically cross- linked CS and LS, respectively. Similarly, the TOC removal in presence of CS@LS 1: 1 was reduced by 11.14% and 15.94% compared to CS and LS, respectively. This high performance can be related to the enhanced bactercidal properties of CS@LS-1: 1 nanosphere. SEM demonstrates the SRB morphology before and after exposure to CS@LS-1: 1. SRB cells were intact, smooth and viable in the control assay, (Fig. 27(b)), whereas, in presence of CS@LS- 1: 1 the majority of cells were aggregated and suffered from prevalent surface damage (Fig. 27c)). On the basis of these results, CS@LS-1: 1 can demonstrate as a“green” biocide that can be beneficial for water and oil & gas industries in reducing biofouling and minimizing the risks of piping corrosion, reservoir souring, and improve surface facilities lifetime.

[00141] Minimum inhibitory concentration of synthesized CS@LS hybrids was evaluated by the turbidity method. The minimum inhibitory concentration of CS@LS nanosphere was determined by a turbidimetric method. In this method, a series of test tubes each containing 5 mL of LB broth was prepared. CS@LS nanospheres (1.5 mg/mL) were nicely dispersed in distilled water by ultra-sonication with a pH of about 6.5 and added in a test tube containing 5.0 mL of LB broth. After mixing, half the mixture was transmitted to the second tube, and similar transformations were repeated. Therefore, each test tube has a test sample solution with half of the concentration of the previous one. All the tubes were inoculated with 10 pL of the freshly prepared bacterial suspension of E. coli and B. subtilis. The positive control was incubated with gentamicin, whereas, the blank control tubes only contained LB media. The assays were incubated at 35 °C for 24 h, the test tubes were studied for the visible signs of bacterial growth or turbidity. The lowest concentration of nanospheres that inhibited the growth of bacteria was considered as the minimum inhibitory concentration.

[00142] It was found that 162.5 pg/mL is the first concentration with clarity for the aqueous LB broth for both bacterial strains. So the minimum inhibitory concentration of the CS@LS- 1: 1 nanospheres was found to be 162.5 pg/mL for both E. coli and B. subtilis. Furthermore, bactericidal activities of non-cross-linked CS/LS-L l and cross-linked CS@LS-1: 1 hybrids were compared.

[00143] In summary, the inventors have surprisingly discovered and developed uniform and confined biodegradable nanospheres with amphiphilic characteristics using a simple one-step chemical cross-linking process. Cross-linked (CS@LS) nanospheres are assembled through a stable covalent acetal bond between the OH group of both CS and LS. The most stable and uniform nanospheres were obtained at a ratio of CS:LS of about 1: 1, forming a homogeneous binary network with a characteristic domain size in the range of 150-200 nm.

[00144] MD simulations validated the self-assembled structure of the cross-linked CS@LS. The lignin molecules form the core of the nanospheres while chitosan is mainly assembled on the surface. Additionally, the excellent antibacterial activity of CS@LS-1: 1 toward both Gram (-) E. coli and Gram (+) B. subtilis demonstrates that CS@LS nanocomposites can be used for biomedical and environmental remediation applications.

[00145] Moreover, CS@LS-1: 1 showed substantial inhibitory effects on sulfate reduction and organics oxidation of SRB at 100 pg/mL.

[00146] Lastly, these nanocomposites can provide stable, sustainable, cost-efficient biomimetic frameworks for the biomedical and environmental applications, especially where size and shape confinements are desired.

[00147] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims

Claims

CLAIMS The invention is claimed as follows:

1. A method of preparing a nanosphere composite material, the method comprising: providing a first solution comprising a chitosan;

providing a second solution comprising a ligosulfonate;

mixing the first solution and the second solution to form a mixture; and

adding a crosslinking solution comprising formaldehyde to the mixture to produce a suspension.

2. The method of claim 1, wherein a weight ratio of the chitosan to the lignosulfonate is from about 1 :2 to about 2: 1.

3. The method of claim 1, wherein a weight ratio of the chitosan to the lignosulfonate is about 1 : 1.

4. The method of claim 1 further comprising preparing the cross-linking solution by dissolving sodium sulfate, the formaldehyde, and sulfuric acid in water.

5. The method of claim 1 further comprising stirring the mixture after the crosslinking solution was added.

6. The method of claim 1 further comprising washing the suspension and centrifuging the washed suspension.

7. The method of claim 1, wherein the ligosulfonate comprises lignosulfonic acid sodium salt.

8. The method of claim 1, wherein the first solution comprising the chitosan has a pH from about 2 to 5.

9. The method of claim 1, wherein the first solution comprising the chitosan has a pH from about 3.

10. The method of claim 1, wherein the chitosan has an average molecular weight from about 50,000Da to about 190,000Da.

11. The method of claim 1, wherein the ligosulfonate has an average molecular weight from about 7,000Da to about 52,000Da.

12. The method of claim 1, wherein the first solution comprising the chitosan in a concentration from about 0.01 wt% to about 10 wt%. 0.1

13. The method of claim 1, wherein the second solution comprising the ligosulfonate in a concentration from about 0.01 wt% to about 10 wt%. 0.1

14. The method of claim 1, wherein a weight ratio of the chitosan to the lignosulfonate is from about 0.0001 : 100 to about 100:0.0001.

15. A nanosphere composite material comprising:

a ligosulfonate; and

a chitosan,

wherein the chitosan and the ligosulfonate are covalently cross-linked, ligosulfonate molecules forming the core of the nanosphere composite material, and chitosan molecules being assembled on the surface of the core.

16. The nanosphere composite material of claim 15, wherein the nanosphere composite material is amphiphilic.

17. The nanosphere composite material of claim 15 comprising the chitosan and the lignosulfonate in a weight ratio from about 1 :2 to about 2: 1.

18. The nanosphere composite material of claim 15 comprising the chitosan and the lignosulfonate in a weight ratio of about 1 : 1.

19. The nanosphere composite material of claim 15, wherein the nanosphere composite material has a homogeneous binary network and a particle size in a range from 20nm to about 200 nm.

20. An antimicrobial material comprising the nanosphere composite material of claim 15.

21. A biocide material for water disinfection comprising the nanosphere composite material of claim 15.

22. A biocide material for MIC inhibition comprising the nanosphere composite of claim 15.