New sustainable chemically modified chitosan derivatives for different applications: Synthesis and characterization

Rana A. Elzamly; Hemat M. Mohamed; Mansoura I. Mohamed; Howida T. Zaky; David R.K. Harding; Nadia G. Kandile

doi:10.1016/j.arabjc.2021.103255

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Original article

2021

:14;

202108

doi:

10.1016/j.arabjc.2021.103255

New sustainable chemically modified chitosan derivatives for different applications: Synthesis and characterization

Rana A. Elzamly^{^a}, Hemat M. Mohamed^{^a}, Mansoura I. Mohamed^{^a}, Howida T. Zaky^{^a}, David R.K. Harding^{^b}, Nadia G. Kandile^{^a,⁎}

a Chemistry Department, Faculty of Women for Art, Science and Education, Ain Shams University, Heliopolis Post Cod. No. 11757, Cairo, Egypt

b Chemistry, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand

⁎Corresponding author. nadiaghk@women.asu.edu.eg (Nadia G. Kandile)

Received: 2021-5-2, Accepted: 2021-6-3,

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Abstract

The functionalization of chitosan (CS) by terephthaloyl chloride (TPC), glutaraldehyde (GA), and 4(4-(ethyl carbamoyl) benzoyl chloride) benzene sulfonamide was performed under different reaction conditions to yield the new hydrogels (I, II, III) respectively. However, hydrogel (I-NPs) was prepared from reaction of chitosan with (TPC) via ionic gelation technique using sodium tripolyphosphate (TPP) as a cross-linker. Moreover, hydrogel (I) was loaded (Au, Ag and ZnO) nanoparticles to give the nanoformulations (I-Au NPs, I-Ag NPs and I-ZnO NPs) respectively. Structural and morphological analysis of the new chitosan derivatives hydrogels and NPs formulations were characterized by FTIR, elemental analysis, TGA, DSC, XRD, SEM and TEM. From swelling study, chitosan derivatives hydrogels revealed higher swelling degree compared to (CS) with increasing time, temperature and pH values which reached maximum at pH 7 then decreased at pH 10. In addition, the maximum sorption capacities of Congo Red (CR) in aqueous solution were in the range 81–88%. Moreover, adsorption equilibrium isotherm results displayed favorable Langmuir model than Freundlich model. Furthermore, chitosan derivatives hydrogels showed broad spectrum antimicrobial activities against Gram-negative bacteria, Gram-positive bacteria and fungi with the inhibition zone diameter ranged from 13 to 25 mm compared to (CS) hydrogel which revealed inhibition zone diameter ranged from 11 to 16 mm, especially the nano formulation hydrogel (I-Ag NPs) showed the highest antimicrobial activity. The results were promising suggesting that the new modified chitosan derivatives could be potential for dye removal and as antimicrobial agents.

Keywords

Chitosan

Terephthaloyl chloride

Nanoparticles

Congo Red

Antimicrobial activity

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1 Introduction

Chitosan (CS) is a useful cationic biomaterial and an exceptional candidate to be used to obtain multifunctional biopolymers, due to its unique biological and chemical properties, such as its polycationic nature, biocompatibility, biodegradability, non-toxicity, hydrophilicity, and mucoadhesive properties (Mohammed et al., 2020). Chitosan has great potential for use as active films with widely potential applications in pharmaceutics, composite materials, drug delivery, adsorption of contamination from water, food industry, tissue engineering, packaging material, cosmetics, wound healing and antibacterial agents due to its biological properties such as antimicrobial (Qiu et al., 2019; Omidi and Kakanejadifard, 2019).

Due to the presence of reactive amino group on its backbone (Lucas et al., 2021), chitosan can be modified to provide a powerful means to promote new biological activities and enhance its physicochemical properties (Panda et al., 2019). It has been established that the primary amino group of chitosan can be used also to crosslink the polymer by using a variety of crosslinkers such as glutaraldehyde (Kandile et al., 2009), multifunctional carboxylic acids (Govindaraj et al., 2019), and tripolyphosphate (TPP) (Kandile and Mohamed, 2019; Ahmed et al., 2020). Cross-linking can also affect the crystalline nature of chitosan and enhances the adsorption characteristics (Abdul et al., 2017). However, crosslinking can be used as an example of chemical modification (Bakshi et al., 2020) which is a promising method to develop chitosan and improve its properties, such as chemical stability, and mechanical resistance (Mohammed et al., 2020).

Moreover, certain applications such as chelation of metal ions, and water purification require chitosan in the form of a super absorbing hydrogel (Govindaraj et al., 2019; Kandile et al., 2015). The crosslinked chitosan is more hydrophilic, has high swelling properties especially their high stability in acidic media and high thermal properties (Kandile et al., 2009; Kavianinia et al., 2014).

The unique properties of chitosan nanoparticles for their small size could be made chitosan nanoparticles exhibit higher antibacterial activity than chitosan (Ahmed et al., 2020; El-Alfy et al., 2020). Hydrogel nanoparticles (NPs) have received considerable attention in terms of therapeutics and diagnosis due to their unique physicochemical properties such as hydrophilicity, flexibility, mobility, high water absorptivity, and biocompatibility that revolutionize medical treatment with more potent, less toxic, environment, agriculture, and smart outcomes (Kandile and Mohamed, 2019).

The environmental pollution from dyes as poisonous/hazardous chemicals in wastewater is one of the most important problems (Jawad et al., 2020) such as water can harm human health and the environment (Hou et al., 2020). Different industries widespread are utilized the organic dyes including textiles, food, paper, pulp, cosmetics, paints, leather and pharmaceuticals (Huong et al., 2020). The methods used for water treatment to control and minimize water pollution, such as physical, chemical, biological methods, etc., have some drawbacks like high cost of operation, complicated procedure with formation of toxic by product. The adsorption method is considered a better approach for water purification due to its low-cost, ease of operation, simplicity of design and environment friendly properties (Soni et al., 2020; Tanhaei et al., 2020).

It is well-known that chitosan exhibits high antimicrobial activity against a wide variety of microorganisms and stability of chitosan can be enhanced by the incorporation of conducting polymers, metal nanoparticles and oxide agents such as gold, silver and ZnO nanoparticles (Bernabé et al., 2020).

The aim of this work is modification of chitosan to develop new hydrogels to enhance its physicochemical, swelling, dye removal from wastewater and antimicrobial properties. The modified hydrogels (I, II, III and I-NPs) were prepared from the reaction of (CS) with terephthaloyl chloride (TPC), glutaraldehyde (GA), 4(4-(ethyl carbamoyl) benzoyl chloride) benzene sulfonamide and sodium tripolyphosphate (TPP) respectively at different reaction conditions. The modified hydrogel (I) was loaded Ag, Au and ZnO nanoparticles to give the nanoformulations (I-Au NPs, I-Ag NPs and I-ZnO NPs). All chitosan derivatives were characterized by FTIR, elemental analysis, TGA, DSC, XRD, SEM and TEM for NPs hydrogels. Moreover, the swelling behavior of the hydrogels under different parameters such as time, pH and temperature were evaluated. In addition, the efficiency of the hydrogels to remove Congo Red (CR) from aqueous solution under different conditions such as time, pH values, and initial concentration of dye were studied. Finally, the inhibition zone (IZ), minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were used to evaluate the antimicrobial activity of the hydrogels and loaded (NPs) towards three Gram-positive bacteria and three Gram-negative bacteria in addition to two plant pathogenic fungi.

2 Materials and methods

2.1

2.1 Materials

Medium molecular weight (CS) (MW 100–300 kDa), (TPC), (GA) (50%) and 4-(2 Aminoethyl) benzenesulfonamide (AEBSA) were purchased from Aldrich. Tetrachloroauric acid (HAuCl₄) and ZnO nanoparticles were purchased from Alfa Aesar. Glacial acetic acid, dimethyl formamide (DMF), dichloromethane, ethanol (95%), trisodium citrate sodium tripolyphosphate (TPP), and silver nitrate were obtained from Adwic, Egypt. The Congo red was purchased from Chemie Loba. All aqueous solutions were prepared using distilled and deionized water.

2.2

2.2 Methods

2.2.1

2.2.1 Synthesis of 4(4-(ethyl carbamoyl) benzoyl chloride) benzene sulfonamide (3)

To solution of TPC (1) (0.25 g, 0.001 mol) dissolved in DMF (1 mL), (AEBSA) (0.25 g, 0.001 mol) dissolved in CH₂Cl₂ (10 mL) was added dropwise followed by stirring for 24 h. The obtained solid was filtered off and crystallized using ethanol to give white crystals m.p > 300 °C, yield 73%. Anal. Calcd. for C₁₆H₁₅N₂ClO₄S: C, 52.38; H, 4.09; S, 8.73%; Found: C, 52.53; H, 4.22; S, 7.90%. Mass spectrum (m/z) calcd. 366.5, found 366.7 M⁺ (22%).

2.2.2

2.2.2 Synthesis of chitosan hydrogel (CS)

Chitosan (0.5 g, 1% wt /v) was dissolved in acetic acid solution (50 mL, 1% v/v) under stirring until complete dissolution, and the pH of this solution was 3. Then sonicated for 15 min. The solution was evaporated under vacuum and left to dry in oven at 110 °C. The dried film was washed with NaOH (1%) to neutralize the excess of acetic acid solution and finally with distilled water and dried to give the (CS) hydrogel.

2.2.3

2.2.3 Modification of chitosan with TPC to give hydrogel (I)

A clear solution of acetic acid solution (50 mL, 1% v/v) contained chitosan (CS) (0.5 g,1% wt/v) was prepared. TPC (1) (0.25 g) was dissolved in DMF (1 mL) and added to (CS) solution followed by stirring at room temperature for 24 h. The reaction mixture was evaporated under vacuum and the film left to dry in oven at 110 °C to afford chitosan derivative (I) as white hydrogel. The hydrogel (I) was washed with NaOH (1%) and distilled water then dried in oven at 110 °C.

2.2.4

2.2.4 Modification of chitosan with TPC in presence of TPP to give (I-NPs)

Briefly, (0.5 g,1% wt/v) of (CS) was dissolved in (50 mL, 1% v/v) aqueous acetic acid under stirring at room temperature until complete solubility. TPC (0.25 g) was dissolved in DMF (1 mL) and added to (CS) solution. Then, aqueous solution of TPP (20 mL, 1% wt/v) was added dropwise to the mixture and stirred at room temperature for 24 h. The reaction mixture was evaporated under vacuum and the film left to dry in oven at 110 °C to give chitosan nano derivative (I-NPs) as white hydrogel. The hydrogel was washed with NaOH (1%) and distilled water then dried in oven at 110 °C.

2.2.5

2.2.5 Modification of chitosan with TPC and GA to give hydrogel (II)

(CS) solution was prepared by dissolving (0.5 g, 1% wt/v in 50 mL acetic acid 1% v/v) under stirring at room temperature. TPC solution (0.25 gm dissolved in 1 mL DMF) was added to (CS) solution followed by addition of GA (5 mL, 50%). The reaction mixture was carried out under stirring at room temperature for 24 h and left to dry in oven at 110 °C. The dried film was washed with NaOH (1%) and distilled water then dried in oven at 110 °C.

2.2.6

2.2.6 Modification of chitosan with (3) to give hydrogel (III)

A mixture of (CS) solution (0.5 g,1% wt/v was dissolved in 50 mL, 1% v/v acetic acid) and 4(4-(ethyl carbamoyl) benzoyl chloride) benzene sulfonamide (3) (0.25 gm, 0.001 mol was dissolved in 1 mL DMF) and stirred at room temperature for 24 h then left to dry in oven at 110 °C to give chitosan derivative (III) as white film .The dried film was washed with NaOH (1%) and distilled water then dried in oven at 110 °C.

2.2.7

2.2.7 Synthesis of gold nanoparticles (Au-NPs)

The reduction of HAuCl₄ with trisodium citrate was used to prepare gold nanoparticles (Au NPs). The HAuCl₄ solution (2 mL, 1% wt/v) was diluted to (200 mL) and heated to boiling temperature then the trisodium citrate solution (2.5 mL, 1% wt/v) was added dropwise under stirring until the color changed to purple. Deionized water was used for preparation of all solutions (Azzam et al., 2016, Kandile et al., 2020).

2.2.8

2.2.8 Synthesis of silver nanoparticles (Ag-NPs)

The colloidal silver nanoparticles (AgNO₃) solution was prepared by reducing silver nitrate with trisodium citrate. The AgNO₃ solution (50 mL, 1X10^-3 M) was brought to boil and then trisodium citrate (5 mL, 1 %wt/v) was added drop by drop. The reaction mixture was vigorously stirred and heated until it turned light yellow in color (Azzam et al., 2016; Kandile et al., 2010).

2.2.9

2.2.9 Loading of hydrogel (I) on (Au, Ag and ZnO-NPs)

Hydrogel (I) (0.1 g) was soaked in acetic acid (5 mL, 1 %v/v) and then, Au or Ag NPs solution (10 mL) or ethanolic solution of ZnO nanoparticles (25 mL, 0.1% wt/v) was added drop by drop and stirred for 24 h. The nanoparticles formulations were collected using centrifuge at (8,000 rpm for 30 min) and then left to dry in oven at 110 °C (Azzam et al., 2016; Al-Naamani et al., 2016).

2.3

2.3 Instrumentation

2.3.1

2.3.1 Ultraviolet visible spectroscopy

The UV measurements for the solution of Au NPs, Ag NPs, and ZnO NPs were carried out by UV–vis photometer UV-2800BMS Scientific Technical Corporation (PVT) Ltd.

2.3.2

2.3.2 FTIR spectroscopy studies

The Perkin Elmer 200 (FTIR) spectrophotometer instrument was utilized in recording the FT-IR spectrum. The samples were grinded with KBr to form a disk and measured in the wavelength range from 4000 to 450 cm⁻¹ during 64 scans, with 2 cm⁻¹ resolutions.

2.3.3

2.3.3 X-Ray diffraction studies

The X-ray diffraction (XRD) patterns of hydrogels was measured using X-ray powder diffractometer with Ni filter Cu Kα radiation source (λ = 0.154 nm), set at scan rate = 10°/ min, using a voltage of 40 kv and a current of 30 mA.

2.3.4

2.3.4 Thermal stability studies

2.3.4.1

2.3.4.1 Thermogravimetric analysis

Thermogravimetric analysis (TGA) was performed at a temperature starting from 25 °C to 800 °C under inert nitrogen atmosphere with heating rate of 10 °C min⁻¹ using the instrument: SDT Q600 V20.9 Build 20, USA.

2.3.4.2

2.3.4.2 Differential scanning calorimetric

Differential scanning calorimetry (DSC) 131 evo (SETARAM Inc., France) was calibrated using the standards (Mercury, Indium, Tin, Lead, Zinc and Aluminum). Nitrogen and Helium were used as the purging gases. The test was programed including the heating zone from 25 °C to 500 °C with a heating rate 10 °C / min. The samples were weighted in Aluminum crucible 120 ul and introduced to the DSC. The thermos-gram results were processed using (CALISTO Data processing software v.149).

2.3.5

2.3.5 Morphological studies

2.3.5.1

2.3.5.1 Scanning electron microscope (SEM)

The surface morphology of hydrogels was investigated using scanning electron microscope (SEM) images. The samples were mounted on a metal stub with double stick adhesive tape and coated under vacuum with gold. The gold film thickness was 150 Å. The samples were then viewed in a Quanta 250 FEG (Field Emission Gun) attached with EDX Unit (energy dispersive X-ray analysis). All samples were examined using an accelerating voltage of 20 KV magnification 14 × up to 1,000,000 and resolution from the Gun FEI Company, Netherlands.

2.3.5.2

2.3.5.2 Transmission electron microscopic (TEM)

Shape and size of the nanoparticles were practically obtained using (TEM); JEOL-JEM-1200. The preparation of specimens for (TEM) measurements occurred by sonication for 15 min. then placed onto a carbon-coated copper grid. The coated grids were viewed under a JEM - 1200 EX 2, Electron Microscope Jaban Specimens for (TEM) measurements.

2.4

2.4 Determination the swelling degree (SD)

The swelling degree of all prepared hydrogels was affected by changing the time from 10 to 100 min, pH ranged from 3 to 10 and the temperature changed from 40 to 100 °C. The swollen hydrogels after reached equilibrium, were taken out from the aqueous medium and immediately weighed after the excess fluid lying on the surface was removed with a filter paper. The swelling degree was calculated using the following [Eq. (1)] (Ruvalcaba et al., 2009):

(1)

S D = (W_{1} - W_{o}) / W_{o}

where W₁ and W_o: weights of wet and dry samples, respectively.

2.5

2.5 Sorption study of Congo Red dye

0.1 g of each hydrogels was immersed in distilled water and left to swell then filtered and added to (25 mL, 100 mg/L) of Congo Red dye solution which affected by different conditions include, (i) effect of time intervals was ranging from 5 to 120 min (ii) effect of pH by changing from pH 3 to 10 which measured by pH meter (Adwa) after controlled by adding few drops of 0.1 N HCl or 0.1 N NaOH (iii) effect of initial dye concentration was ranging from 20 to 100 mg/L. The concentration of Congo Red dye in the solution was measured using UV–visible spectrophotometer at wavelength 497 nm. The efficiency of dye removal was calculated using the following [Eq. (2)]:

(2)

F % = [1 - (C_{e} / C_{o})] \times 100

where F was the efficiency (%), C_e was the final concentration of dye at equilibrium after sorption and C_o was the initial concentration of dye in the solution.

Sorption capacity (q_e), the amount of the dye adsorbed by the adsorbent, was calculated using the following [Eq. (3)]:

(3)

q_{e} = [(C_{o} - C_{e}) V] / W

where q_e was the amount of dye at equilibrium (mg/g), V was the volume of the dye solution (L), C_o was the initial concentration of dye (mg/L), C_e was the final concentration of dye at equilibrium after sorption and W was the dry weight of the adsorbent (g).

2.6

2.6 Antimicrobial activity studies

2.6.1

2.6.1 Antibacterial and antifungal evaluation

The disc diffusion method (Khalil and Farag, 1994) was used for assessing the antibacterial and antifungal activity of hydrogels against Staphylococcus aureus (S. aureus, ATCC 6538), Bacillus subtilis (B. subtilis, ATCC 6633), and Staphylococcus epidermidis (S. epidermidis, ATCC 12228) as Gram positive bacteria and versus Escherichia coli (E. coli, ATCC 11229), Proteus (ATCC 33420), and Klebsiella pneumonia (K. pneumoniae, ATCC 10145) as Gram negative bacteria using nutrient agar method. Antifungal activity was carried out against (Aspergillus Niger, ATCC 13497) and (Candida, ATCC 10231). Ciprofloxacin was used as standard drug. Briefly, disc of 10 mm diameter was cut from the hydrogel. Nutrient agar plates were incubated with microbial culture. The cut discs of hydrogels were placed onto the surface of inoculated plates. The plates were incubated at 37 °C for 48 h and 72 h against bacteria and fungi, respectively. The inhabitation zone (distance from disc circumference in mm) was determined for each disc.

2.6.2

2.6.2 Minimum inhibition and bactericidal concentration

To assess the antibacterial activity of (CS) and modified (CS) hydrogels minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were evaluated according to broth tube dilution method with slide (Abou-Zeid et al., 2011) as follow, a series of culture tubes were prepared all containing the same volume of medium inoculated with test microorganism. Decreasing concentration of (CS) or modified (CS) hydrogels was added to the tubes usually a stepwise dilution (two-fold serial dilution) was used starting from highest to lowest concentrations. One tube was left without (CS) or modified (CS) hydrogels to serve as positive. The cultures were incubated at 37 °C to 24 h. The tubes were inspected visually to determine the growth of organisms by the presence of turbidity & the tubes in which (CS) or modified (CS) hydrogels is present in minimum concentration sufficient to inhibit the microbial growth which remains clear was noted as MIC of the (CS) or modified (CS) hydrogels. The MBC was also calculated, for this, a volume of 100 µL was taken from the tubes where no growth had been detected, spread on nutrient agar plates & incubated at 37 °C to 48 h. MBC was definite as the concentration at which there was ≥ 99.9% (3 log) decrease in viable cells.

3 Results and discussion

In the present work, functionalization of (CS) was performed by reaction with TPC, TPP, GA and (3) under different reaction conditions to give chitosan derivatives hydrogels (I, I-NPs, II and III) in order to enhance the utilization of (CS) in different applications.

3.1

3.1 Synthesis of 4(4-(ethyl carbamoyl) benzoyl chloride) benzene sulfonamide (3)

4(4-(ethyl carbamoyl) benzoyl chloride) benzene sulfonamide (3) was prepared from the reaction of (TPC) (1) with (AEBSA) (2) Scheme 1 and its structure was confirmed by elemental analysis and mass spectrum (experimental part). FTIR (KBr) for compound (3) showed characteristic absorption bands at 3360 and 3102 cm⁻¹ assigned to the —NH amide and —NH (NH₂) bands. Two bands at 3064 cm⁻¹ and 2985 cm⁻¹ referred to C—H aromatic and aliphatic stretching respectively. Also, sharp band assigned to C⚌O at 1683 cm⁻¹ and two bands at 1285, 1136 cm⁻¹ referred to SO₂.

3.2

3.2 Synthesis of hydrogels (I, I-NPs, II, III)

Zhao et al. (2012) reported that chitosan was coated with PVDF nanofibers and non-woven PET substrate to give the three-tier composite membrane then modified with TPC and cross-linked with GA. However, in the present study, (CS) was reacted with TPC in absence or presence of GA via stirring the reaction mixture in dilute acetic acid for 24 h to yield chitosan derivatives (I and II) respectively.

Hydrogel (I) was synthesized by the elimination of HCl molecules through interaction of two chlorine atoms of TPC and NH₂ groups of (CS) to form two amide units in the structure of hydrogel (I) Scheme 2. Also, (CS) was reacted with TPC in the presence of GA as crosslinker to produce chitosan derivative hydrogel (II) Scheme 2. Moreover, chitosan derivative hydrogel (III) was prepared via the reaction between (CS) and compound (3) by elimination reaction of HCl molecule to yield a new amide bond in the structure of the hydrogel (III) Scheme 2. The nanohydrogel (I-NPs) was prepared from reaction of chitosan with TPC in presence of TPP as crosslinker via the ionotropic gelation technique in which the free polycation groups of chitosan (CS) interact with a polyanionic TPP (Kandile and Mohamed, 2019) Scheme 3. The new hydrogels (I, I-NPs, II and III) were characterized by FTIR, ¹HNMR, ¹³CNMR, elemental analysis, TGA, DSC, XRD, SEM and TEM.

3.3

3.3 Characterization of hydrogels (I, I-NPs, II and III)

3.3.1

3.3.1 FTIR spectra

FTIR spectra of hydrogels (CS, I, I-NPs, II and III) were shown in (Fig. 1). The FTIR spectrum of (CS) showed a broad band at 3419 cm⁻¹ assigned to —OH and —NH stretching of (—NH₂) bands. The band at 2921 cm⁻¹ referred to the aliphatic C—H stretching. Two adsorption bands at 1651 cm⁻¹ and 1589 cm⁻¹ were shown for the C⚌O of acetyl group and N—H bending vibrations of the (—NH₂) groups and the band at 1071 cm⁻¹ referred to C—O stretching (Kandile and Mohamed, 2019; Zhao et al., 2012).

FTIR for CS, I, I-NPs, II and III hydrogels.

The hydrogels (I, I-NPs, II and III) showed similar bands of the FTIR spectrum to (CS). The hydrogel (I) revealed two new bands at 3064 and 1686 cm⁻¹ for C—H aromatic and high intensity C⚌O amide group (Kandile and Nasr, 2011a,b). The FTIR spectrum of (I-NPs) displays peaks at 3413 cm⁻¹ (—OH), 3373 cm⁻¹ (—NH₂ stretching), 2998 cm⁻¹ (C—H stretching), 1683 cm⁻¹ (C⚌O amide stretching), 1574 cm⁻¹ (—NH₂ of amide), 1285 cm⁻¹ (C—N stretching), 1086 cm⁻¹ (C—O—C stretching) and finally 1019 cm⁻¹ (C—O stretching). The increase in the intensity of N—H amide indicated an electrostatic association between PO₄^3- group of TPP and NH₃⁺ group of (I) (Hadidi et al., 2020). While the hydrogel (II) showed the band of C—H aliphatic stretching shifted to 2949 cm ^-1, sharp band assigned to C⚌O at 1687 cm⁻¹ and a new band referred to C⚌N at 1574 cm⁻¹, also, hydrogel (III) showed two bands pointed to SO₂ group at 1287 and 1136 cm⁻¹.

3.3.2

3.3.2 ¹H NMR &13C NMr

The hydrogel (I) was confirmed by ¹H NMR and ¹³C NMR. The ¹H NMR spectrum (DMSO, d₆) showed a singlet at δ 8.7 (1H, NH), a doublet at δ 8 (4H, Ar-H), a broad signal at δ 3.8 (1H, OH), a singlet at δ 2.9 (1H, CH Hemiacetal), a singlet at δ 2.7 (2H, CH2) and a singlet at δ 2.5 (1H, CH aliphatic). The ¹³C NMR (DMSO, d₆) data showed peaks at δ167.1, 134.9, 129.9, 40.4, 40.2, 40, 39.8, 39.6, 39.4, 39.2, 34.5 ppm.

3.3.3

3.3.3 Degree of deacetylation of chitosan (DD)

The degree of deacetylation (DD) influences the physical, chemical and biological properties of (CS) therefore it is considered as one of the main parameters characterizing (CS) (Hussain et al., 2013). The percentage of free amino groups on (CS) was calculated using elemental analysis of C, H, N percent for (CS) found in Table 1 using the following [Eq. (4)] (Braz et al., 2018):

(4)

DD = (1 - \frac{(C / N) - 5.145}{6.861 - 5.145}) \times 100

where 5.145 is related to the completely N- deacetylated (CS) (C₆H₁₁O₄N repeat unit) and 6.861 to the fully N-acetylated chitin (C₈H₁₃O₅N repeat unit). Thus, the degree of deacetylation (DD) of chitosan used in this study was calculated to be 68.8%.

Table 1 The degree of deacetylation of (CS) and the degree of substitution of hydrogels (I, I-NPs, II and III).

Hydrogel	C%	H%	N%	S%	C/N	DD%	DS
CS	34.59	6.11	6.09	–	5.68	68.8	–
I	40.19	5.01	4.44	–	9.05	–	0.42
I-NPs	34.24	5.59	4.04	–	8.47	–	0.35
II	48.55	6.23	2.28	–	21.29	–	1.2
III	42.05	5.75	6.59	2.95	6.38	–	0.044

3.3.4

3.3.4 Degree of substitution of the prepared hydrogels (DS)

Table 1 showed the degree of substitution (DS) which calculated for (I, I-NPs, II and III) using the following [Eq. (5)] (Baran and Mentes, 2015; Braz et al., 2018):

(5)

DS = \frac{{(C / N)}_{m} - {(C / N)}_{i}}{n}

where (C/N) _m and (C/N) _i were the C/N ratios for the modified (CS) and non-modified (CS) respectively and (n) was the number of carbon atoms introduced after the modification.

The degree of substitution of hydrogels (I, I-NPs, II and III) were 0.42, 0.35, 1.2 and 0.044 respectively. From Table 1 it was observed that the hydrogel (II) showed the highest degree of substitution value (1.2) due to the presence of active dialdehyde cross-linker GA. The hydrogels (I and I-NPs) revealed moderately degree of substitution values (0.42 and 0.35) due to the existence of TPP as cross-linker which decreased the percentage of carbon therefore the molar ratio and the degree of substitution was decreased. The lowest degree of substitution value (0.044) was detected for the hydrogel (III) due to the presence of the bulky sulfonamide compound (Braz et al., 2018).

3.3.5

3.3.5 Thermal stability studies

3.3.5.1

3.3.5.1 Thermogravimetric analysis (TGA)

TGA is used to determine thermal stability and measured over time as the temperature changes. The TGA thermograms of hydrogels (CS, I, I-NPs, II and III) were in the range from 25 °C to 800 °C (Fig. 2) and Table 2. The TGA curve of (CS) showed two stages of weight loss. The first stage occurred at 92.88 °C due to loss of water molecules with a weight loss of about 11.43%. The actual degradation of (CS) found at 299.44 °C with a weight loss of about 61.04% (Kumar and Koh, 2012). However, three stages appeared in TGA curve of hydrogel (I). The first stage showed weight loss 2.23% at 57.14 °C due to the moisture. The second stage is the beginning decomposition of the hydrogel which revealed weight loss 11.82% at 178.74 °C. The third stage displayed weight loss 5.21% at 744.26 °C. Moreover, the thermal stability of hydrogel (I) increased compared to (CS) hydrogel. TGA curve of (I-NPs) hydrogel displayed two weight losses at 63.69 and 326.30 °C with 15.26 and 64.61% respectively. The first weight loss was attributed to the water evaporation and the second weight loss pointed to the thermal degradation of the backbone of the hydrogel. Furthermore, the hydrogel (II) revealed four stages of weight loss. The first stage of weight loss 4.42% was at 72.83 °C corresponded to the moisture. The second decomposition stage occurred at 157.83 °C with a weight loss 22.75%. The third stage accomplished at 320.76 °C with a weight loss 29.07% and the last degradation stage shown at 454.26 °C with a weight loss 20.94%.

TGA for hydrogels CS, I, I-NPs, II and III.

Table 2 Thermal properties for hydrogels CS, I, II, III and I-NPs.

Sample code	T_g (°C)	T_c (°C)	T_d (°C)	Temp.	Weight loss %	Temp.	Weight loss %	Temp.	Weight loss %	Temp.	Weight loss %
CS	–	–	300.97	92.88	11.43	–	–	299.44	61.04	–	–
I	196.66	–	–	57.40	2.223	178.47	11.82	–	–	744.26	5.21
I-NPs	128.66	–	306.61 365.85 442.66	63.69	15.26	–	–	326.30	64.61	–	–
II	158.44	174.25	312.94	72.83	4.42	157.83	22.75	320.76	29.07	454.26	20.94
III	186.77	–	242.57 459.37	–	–	214.52	11.28	349.41	57.53	680	25.31

On the other hand, TGA of hydrogel (III) showed three stages of weight loss. The first one found at 214.52 °C with a weight loss 11.28%, the second stage occurred at 349.41 °C with a weight loss of about 57.53% and the third one of weight loss 25.31% was at 680 °C. The TGA results demonstrated that the thermal stability of (CS) increased after modification with TPC, GA and compound (3) (Kandile et al., 2009) and the hydrogel (I) showed the highest thermal stability at 744.26 °C with weight loss 5.21%.

3.3.5.2

3.3.5.2 Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) is widely used for examining polymeric materials to determine their thermal transitions included the glass transition temperature (T_g), crystallization temperature (T_c), and decomposition temperature (T_d). The results of (DSC) analysis of hydrogels (CS, I, I-NPs II and III) were in the range from 25 °C to 500 °C and given in (Fig. 3) and Table 2.

DSC for hydrogels CS, I, I-NPs, II and III.

(CS) revealed an endothermic peak at 92.11 °C due to the loss of water molecules, an exothermic peak at 300.97 °C due to its decomposition (Hassan et al., 2018) and crystallinity percent which was about 64%. The endothermic glass-transition temperature (T_g) of hydrogel (I) was observed at 196.66 °C may be related to the crosslinking, which would decrease the flexibility and the ability of the chains to undergo segmental motion (Chen et al., 2020; Mokhtari et al., 2020). The (DSC) curve of hydrogel (I-NPs) showed an endothermic peak (T_g) at 128.66 °C and another three endothermic peaks (T_d) at 306.61, 365.85 and 442.66 °C may be attributed to the ionic crosslinking with (TPP) promoted a structural change in the nanoparticles.

On the other hand, the (T_g) of hydrogel (II) was found at 158.44 °C, an exothermic peak referred to a crystal temperature (T_c) at 174.25 °C and an endothermic peak (T_d) at 312.94 °C this may be due to the presence of amide groups and the cross-linker GA. The (DSC) data of hydrogel (III) showed an endothermic peak at 69.96 °C due to remove of water molecules, an endothermic peak (T_g) at 186.77 °C and two endothermic peaks displayed the decomposition of its structure at 242.57 and 459.37 °C. The (DSC) results proved that hydrogel (I) showed the highest (T_g) at 196.6 °C and the (T_g) displayed increasing order from (I > III > II > I-NPs).

3.3.6

3.3.6 X-Ray diffraction

X-ray diffraction (XRD) was used to study the amorphous and crystallinity properties of the hydrogels (CS, I, I-NPs, II and III). (Fig. 4) showed the interfering peaks at 2θ were in the range of 4–90. X-Ray diffraction data for hydrogel (CS) gave two sharp peaks at 2θ = 10 and 20 (Kumar and Koh, 2012) which appeared in hydrogel (I) in addition to new peaks at 2θ = 30, 35, 40 and 45. These major changes suggested a more crystalline for hydrogel (I) than (CS) due to the presence of amide groups leading to some degree of crystallinity (Kandile and Nasr, 2011a,b).

XRD for hydrogels CS, I, I-NPs, II and III.

The curve of hydrogel (I-NPs) showed at 2θ = 10, 20, 32, 45 and 57 indicating an increase in the crystallinity nature which achieved after crosslinking with (TPP) (Kandile, and Mohamed, 2019).

Moreover, the (XRD) pattern of hydrogels (II and III) indicated greater crystallinity than (CS) which showed three sharp peaks at 2θ = 45, 65 and 80 for (II) and two new peaks around 2θ = 30 and 45 for hydrogel (III). This may be due to the increasing degree of crosslinking in the hydrogels (Li et al., 2019).

3.3.7

3.3.7 Morphological characterization

3.3.7.1

3.3.7.1 Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) is one of the high-resolution electron microscopy techniques which used to study and characterize the surface morphology of the hydrogels. The (SEM) images of the hydrogels (CS, I, II and III) were shown in (Fig. 5 A-D). (Fig. 5 A) showed smooth, dense and flat morphology of (CS) (Li et al., 2019), while (Fig. 5 B) revealed that hydrogel (I) had a rough surface. Moreover, the surface morphology of hydrogels (II and III) had irregular structure (Fig. 5 C and D). However, the (SEM) images of the hydrogels (I, II and III) showed a different morphology compared to (CS) and these different results may be attributed to the increase of crosslinking degree (Li et al., 2013).

SEM images of hydrogels (A) CS, (B) I, (C) II and (D) III.

3.3.7.2

3.3.7.2 Transition electron microscopy (TEM) of nanohydrogel (I-NPs)

The shape of hydrogel (I-NPs) was characterized by using transition electron microscopy (TEM).

(Fig. 6) showed that the hydrogel (I-NPs) revealed spherical shapes in similar to shape of (CS)-NPs (Kandile and Mohamed, 2019) confirmed that the particles shape was not affected by the crosslinking of (CS) with (TPC).

3.3.8

3.3.8 The swelling study (SD)

Swelling is one of the basic characteristics of three-dimensional, hydrophilic, and polymeric networks hydrogels which are capable of imbibing large amounts of water or biological fluids and exhibit a thermodynamic compatibility with water which allows them to swell in aqueous media (Rahman et al., 2019).

3.3.8.1

3.3.8.1 Effect of time

The swelling degree of the hydrogels at different time (10–100 min) in the aqueous medium was represented in (Fig. 7A). At initial time of immersion (t = 10 min), the swelling appeared in lower values due to the weak interaction between the polymer functional groups and the water molecules.

Effect of immersion time on the swelling degree of hydrogels (CS, I, I-NPs, II and III).

Moreover, longer exposure time facilitated more interaction between the polymer functional groups and the water molecules which increased the adsorbed amounts of the water on the polymer and consequently increased the swelling degree. (Azmy et al., 2019). Therefore, the swelling degree reached its maximum values of 510, 1.8, 1 and 495 for hydrogels (I, I-NPs, II and III) at 100 min respectively. (Fig. 7A) showed that swelling degree of the hydrogels increased gradually by increasing the immersion time. Generally, the synthetized (CS) hydrogels showed excellent swelling properties reached to 510 and 495 in case of hydrogels (I and III). Obviously, chitosan hydrogels (I & III) revealed swelling degree higher than the prepared hydrogels (II and I-NPs). This is due to the polarity of carbonyl groups and sulphur atom and their proclivity for forming hydrogen bonds with water molecules.

3.3.8.2

3.3.8.2 Effect of pH

The effect of pH value of the medium pH (3–10) on the swelling degree of the prepared hydrogels (I, I-NPs, II and III) indicated that the gradual increase in the acidity or the alkalinity of the medium decreased the swelling degree of the modified hydrogels (Azmy et al., 2019). The highest swelling degree of the prepared hydrogels was obtained at pH (7) and the lowest swelling degree was achieved at pH (10). Also, the swelling degree of the modified hydrogels (I, I-NPs, II and III) were increased by decreasing the acidity of the medium to reach their maximum values of 410, 1.6, 2.7 and 249 respectively at pH (7) while increasing the alkalinity of the medium showed decreasing in the swelling degree values of 78, 1, 1.9 and 119 respectively at pH (10) (Fig. 7B).

Effect of pH value on the swelling degree of hydrogels (CS, I, I-NPs, II and III).

3.3.8.3

3.3.8.3 Effect of temperature

The effect of increasing temperature from 40 to 100 °C on the swelling degree of the hydrogels (I, I-NPs, II and III) was shown in (Fig. 7C) which clarified that the regular increase in the temperature of the aqueous medium is gradually increased the swelling degree. This may be attributed to the effect of the temperature on the creation of the hydrogen bonds formation between the water molecules and the various functional groups (C⚌O, —OH, —S—, and NH₂) in the modified hydrogels (Azmy et al., 2019). Wherever the rising of temperature from 40 to 60 °C, the swelling degree of the prepared hydrogels ranged from 0.06 to 181.12 and hydrogel (III) revealed the highest swelling degree. Moreover, the increasing of temperature from 70 to 100 °C, swelling degree ranged from 0.84 to 328.26 and the hydrogel (I) showed the highest swelling degree.

Effect of temperature on the swelling degree of hydrogels (CS, I, I-NPs, II and III).

3.4

3.4 Loading of hydrogel (I) on (Au, Ag and ZnO NPs)

3.4.1

3.4.1 UV–VIS spectroscopy of (Au, Ag and ZnO NPs)

The formation of gold, silver and ZnO nanoparticles were confirmed by UV– visible absorption. UV–vis absorption spectra of Au NPs, Ag NPs and ZnO NPs were shown in (Fig. 8). The UV–vis absorption of the colloidal suspension showed a strong broad band at 528 nm in the case of Au NPs which related to the surface plasmon resonance (SPR) of gold nanoparticles (Kandile et al., 2020). On the other hand, in case of Ag NPs the characteristic peak at 418 nm is related to SPR of silver nanoparticles (Azzam et al., 2016; Abd-Elaal et al., 2015). However, ZnO NPs revealed band around 378 nm (Kandile et al., 2020).

UV–vis spectrum of a) Au NPs, b) Ag NPs and c) ZnO NPs.

3.4.2

3.4.2 FTIR spectra of (I-Au NPs, I-Ag NPs and I-ZnO NPs) formulations

The modified (CS) nanoformulations (I-Au NPs, I-Ag, and I-ZnO NPs) showed absorption bands in the range of 3443–3404 cm⁻¹ related to the stretching vibrations of —OH and —NH bonds. The peaks ranged from 1685 to 1634 cm⁻¹ refered to the stretching vibration of C⚌O amide groups. The absorption peaks ranged from 1639 to 1574 cm⁻¹ ascribed to bending vibration of –NH₂ group. The bands in the range of 1075–1059 cm⁻¹ assigned to C–O stretching group (Fig. 9).

FTIR for I-NPs, I-ZnO NPs, I-Ag NPs and I-Au NPs.

6. exists an overlapping band from 3300 to 3500 cm

It is worthy noting that there are significant changes in FTIR spectra of hydrogel (I) with the addition of Au NPs and Ag NPs such as the decrease of intensity (—OH and —NH stretching vibrations) and the increase in the intensity of the C–O stretching. These features established the (I-Au and I-Ag) nanocomposites formation (Chen et al., 2020; Mokhtari et al., 2020). The FTIR spectrum of (I-ZnO NPs) revealed new absorption peaks at 692 cm⁻¹ and 449 cm⁻¹, in addition to the shifted broader and stronger band at 3404 cm⁻¹ indicated the strong attachment of ZnO to the amide groups of hydrogel (I) (Abd El hady, 2012).

3.4.3

3.4.3 Transition electron microscopy (TEM) of (Au, Ag and ZnO) nanoparticles and nano formulations

Transition electron microscopy (TEM) was used to characterize the shapes and particle size of (Au, Ag and ZnO) nanoparticles and modified (CS) nanoformulations (I-Au NPs, I-Ag NPs and I-ZnO NPs) (Fig. 10 A-F). TEM images were measured at 100 nm and indicated that the Au NPs, Ag NPs and ZnO NPs were predominantly spherical in shape and poly dispersed (Azzam et al., 2016; Kandile et al., 2020) (Fig. 10 A-C).

TEM images of (A) Au NPs, (B) Ag NPs, (C) ZnO NPs, (D) I-Au NPs, (E) I-Ag NP and (F) I-ZnO NPs.

TEM images showed spherical shapes for (I-Au NPs) with particle size in the range of 22–42 nm (Murawska et al., 2012; Kandile et al., 2020) and (I-Ag NPs) with particle size in the range of 17–25 nm (Fig. 10 D-E) (Jiang et al., 2011). Furthermore, TEM images indicated that particle size distribution for (I-ZnO NPs) was in the range of 50–60 nm (Al-Naamani et al., 2016) (Fig. 10 F).

3.5

3.5 Dye removal studies

Dyes are priority pollutants and its presence in wastewater can cause severe problems to aquatic life and human beings. Therefore, the removal of dyes from wastewater is important in order to minimize their hazardous effects on the environment. Removal of dye from wastewater can be achieved by using adsorption methodology which is highly effective and economical (Jawad et al., 2020; Hou et al., 2020).

The efficiency of the prepared hydrogels to adsorb the Congo Red (CR) dye from aqueous solution was evaluated under different parameters such as contact time, pH and initial dye concentration.

3.5.1

3.5.1 Effect of time

The effect of contact time on the sorption capacity (qe mg/g) and the efficiency (f %) of Congo Red on the surface of prepared hydrogels were shown in (Fig. 11A) with keeping the parameters such as adsorbent dose (100 mg), solution pH (7), and temperature (25 °C) constant during this study (Abdul et al., 2017). The results indicated that the hydrogel (I-Au NPs) showed a fast sorption in 5 min with sorption capacity (476 mg/g), (47%). With increasing in time reaching to 30 min, hydrogel (III) revealed the highest sorption capacity (663 mg/g), (66%). After 120 min, nanoformulation (I-Au NPs) displayed the highest sorption capacity (886 mg/g), (88%) followed by hydrogels (I, III and I-NPs) ,871(87%), 834(83%) and 818 (81%) respectively. Obviously, the modified (CS) derivatives (I, I-NPs, III, & I-Au NPs) revealed the highest efficiency (f %) and sorption capacity (qe) with increasing in time compared to (CS) due to the large amount of surface area available in nanoparticles for dye adsorption and the capacity of the adsorbent gradually became exhausted with time as the few remaining vacant surface sites became difficult to be occupied (Wanyonyi and Onyari, 2014).

Effect of time on the CR dye sorption capacity.

3.5.2

3.5.2 Effect of pH

The effect of pH on the sorption of Congo Red dye showed that the hydrogel (I) had the highest efficiency and sorption capacity 941 (94%) at pH (2) decreasing to 710 (71%) at pH (10) while the nanoformulation (I-Au NPs) revealed the sorption capacity from 924 (89%) to 39 (4%) as the pH value increased from (2) to (10). Generally, the sorption capacity was high at low pH which could be attributed to the adsorption mechanism of Congo Red on the modified (CS) derivatives via dipole – dipole interaction which based on the electrostatic forces between negatively charged sulfonic groups of Congo Red dye molecule and the protonated amino groups in (CS) backbone (Abdul et al., 2017) (Fig. 11B).

Effect of pH on the CR dye sorption capacity.

3.5.3

3.5.3 Effect of initial dye concentrations

The effect of the initial Congo Red concentrations (C_o) was investigated by the Congo Red concentrations ranging from 20 to 100 mg/L at pH (7), temperature (25 °C), adsorbent dose (100 mg) and contact time (120 min). The results showed that modified (CS) derivatives (I-ZnO NPs and I-NPs) had the highest sorption capacity 149 and 143 respectively at C_o = 20 mg/L. With increasing the initial concentration to C_o = 40 mg/L, hydrogels (I and I-NPs) displayed the highest sorption capacity 368 and 238 respectively. The hydrogel (I) revealed the maximum sorption capacity (8 5 4) at C_o = 100 mg/L. The results from (Fig. 11C) revealed that the sorption capacity increased with increasing initial Congo Red dye concentrations. This sorption phenomenon occurs because a high initial Congo Red concentration provides a sufficient adsorption environment, allowing a powerful adsorption force to overcome the mass transfer resistance between the adsorbate and adsorbent (Kim et al.,2019).

Effect of initial CR dye concentration on sorption capacity.

3.5.4

3.5.4 Adsorption isotherms

The adsorption isotherm models are the tools used to explain the distribution of the dye molecules over the adsorbent surface and the interactive behavior between adsorbent and dye. The isotherm models have been evaluated to calculate the adsorption capacity of an adsorbent using two parameter equations namely Langmuir and Freundlich models.

The Langmuir isotherm model assumed dye adsorption in a monolayer on a homogeneous adsorbent surface with finite and unique adsorption active sites (Abdul et al., 2017). The linear form of Langmuir [Eq. (6)] employed as following:

(6)

C_{e} / Q_{e} = 1 / K_{L} Q_{m} + C_{e} / Q_{m}

where Ce (mg/L) was the equilibrium concentration of Congo Red dye in the solution, q_e (mg/L) was the equilibrium adsorption capacity, Q (mg/g) was the maximum adsorption capacity of the adsorbent, K_L was the Langmuir constant. The values of Q_m and K_L were calculated from the curve of C_e/q_e versus C_e and represented in Table 3.

Table 3 Adsorption isotherm model parameters for the sorption of CR dye on hydrogels (I, I-NPs, III and I-Au NPs).

Hydrogel	Langmuir isotherm					Freundlich isotherm
	Equation	R²	K_L	Q_m	R_L	Equation	R²	N	K_f	1/n
I	Ce/qe = 0.016Ce + 0.0034	0.988	4.7	62.5	0.004–0.00135	Logqe = 0.105LogCe + 2.905	0.8326	9.524	798	0.105
I-NPs	Ce/qe = 0.0329Ce + 0.2137	0.9927	0.154	30.4	0.0128–0.075	Logqe = 0.1379LogCe + 2.2883	0.747	7.251	194.23	0.1379
III	Ce/qe = 0.0091Ce + 0.0245	0.9886	0.371	109.9	0.0053–0.026	Logqe = 0.1077LogCe + 2.8947	0.7972	9.285	784.7	0.1077
I-Au NPs	Ce/qe = 0.0264Ce + 0.0059	0.9972	4.475	37.87	0.005–0.0004	Logqe = 0.0381LogCe + 2.5064	0.9841	26.24	320.93	0.038

The dimensionless constant called separation factor R_L was used to calculate the shape of the Langmuir isotherm and defined by [Eq. (7)]:

(7)

R_{L} = 1 / (1 + K_L C_{o})

where C_o is the initial dye concentration, and K_L is the Langmuir constant. The value of R_L indicates the type of isotherm. If the value of R_L = 1, it indicates linear isotherm, if R_L = 0, it indicates isotherm to be irreversible, if the value lies (0 < R_L < 1) it indicates favorable isotherm and if R_L > 1it is unfavorable.

A multilayer adsorption of an adsorbate onto a heterogeneous adsorbent surface was predicted by the Freundlich isotherm model (Abdul et al., 2017). The linear [Eq. (8)] for Freundlich isotherm can be expressed as:

(8)

l o g q_{e} = l o g K_{f} + 1 / n l o g C_{e}

where K_f was the Freundlich isotherm constant, and n was related to the adsorption intensity. The value of 1/n indicates the type of isotherm. If the value of 1/n lies in between (0 < 1/n < 1) it showed that isotherm is favorable, if 1/n = 0, it indicates isotherm is irreversible and if 1/n > 1, it is unfavorable. The values of the Langmuir and Freundlich isotherm constants and correlation coefficients were recorded in Table 3 which showed that hydrogels (I, III, I-Au NPs and I-NPs) had a much higher coefficient constants R² of 0.988, 0.9886, 0.9972 and 0.9927 by Langmuir model than 0.8326, 0.7972, 0.9841 and 0.7476 respectively by Freundlich model indicating a monolayer coverage adsorption.

3.6

3.6 Antimicrobial evaluation

Antimicrobial activity can be defined as a collective term for all active principles (agents) that inhibit the growth of bacteria, prevent the formation of microbial colonies, and may destroy microorganisms. Antibacterial activity is the most important characteristic of the hydrogels to provide appropriate protection against microorganisms (Elmogahzy, 2020). Despite that many different mechanisms for microbial inhibition by (CS) have been proposed. The most accepted one is the interaction of the positively charged (CS) with the negatively charged residues at the cell surface of bacteria such as E. coli and S. aureus, causing extensive cell surface alterations and altering cell permeability. This interaction would probably inhibit the normal metabolism of microorganisms and finally causing the death of these cells (El-Dahma et al., 2017).

3.6.1

3.6.1 Antibacterial evaluation

3.6.1.1

3.6.1.1 Inhibition zone diameter (IZ)

The antibacterial activity of the (CS) derivatives hydrogels and modified (CS) nanoformulations against S. aureus, B. subtilis and S. epidermidis as Gram positive bacteria, E. coli, Proteus and K. pneumonia as Gram negative bacteria were evaluated using (IZ) which displayed in (Fig. 12A). The results showed that the inhibition (IZ) values which ranged from 13 to 18 mm for hydrogels (I, II and III) were higher than (IZ) values of hydrogel (CS) which ranged from 11 to 16 mm.

Inhibition zones of the hydrogels and modified (CS) nanoformulations against Gram +ve and Gram −ve bacteria.

Moreover, after loading process (IZ) was increased with values (19–25 mm) and modified (CS) nanoformulation (I-Ag NPs) showed the highest antibacterial activity and similar effect of the antibiotic Ciprofloxacin with values (23–26 mm), may be attributed to the small sizes of nanoparticles which make them have a larger contact surface with bacteria enhancing their penetration and bacterial effects (Bakshi et al., 2020, Kandile and Mohamed, 2021). Also, modified (CS) nanoformulation (I-Ag NPs) is able to attach to the membrane of bacteria by electrostatic interaction and disrupt the integrity of the bacterial membrane (Hajipour et al., 2012).

3.6.1.2

3.6.1.2 MIC & MBc

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were evaluated to assess the antibacterial activity of the (CS) derivatives hydrogels and modified (CS) nanoformulations against S. aureus and B. subtilis as Gram positive bacteria, E. coli and Proteus as Gram negative bacteria as shown in (Figs. 12B and 12C).

MIC for the hydrogels and modified (CS) nanoformulations.

MBC for the hydrogels and modified (CS) nanoformulations.

The MIC and MBC results in (Figs. 12B and 12C) showed that hydrogels (I and III) had MIC and MBC values better than (CS) except MIC value for Proteus revealed by hydrogel (I) and hydrogel (III) displayed superior effect ,while hydrogel (II) showed only better MIC values for E. coli and Proteus.

Accordingly, the loaded hydrogel (I-ZnO NPs) showed similar effect as (CS) for Gram positive while for Gram negative showed better values than (CS). However, the modified (CS) nanoformulations (I-Au NPs and I-Ag NPs) revealed greater MIC and MBC values than (CS). Furthermore, the modified (CS) nanformulation (I-Ag NP) displayed the same effect as Ciprofloxacin against B. subtilis as Gram positive owing to the presence of silver nanoparticles, which accumulated on the bacterial membrane and formed pits, allowing biologically important lipopolysaccharide molecules and bacterial membrane proteins to leak out, eventually, this results in the demise of bacterial cells (Li and Zhuang, 2020).

3.6.2

3.6.2 Antifungal evaluation

The results of antifungal evaluation of the hydrogels (CS, I, I-NPs, II, III) and modified chitosan nanoformulations (I-Au NPs, I-Ag NPs, I-ZnO NPs) against Aspergillus Niger and Candida were shown in (Fig. 12D). The results revealed that hydrogels (CS, I and II) had no activity against Aspergillus Niger and Candida, while the hydrogel (III) displayed good (IZ) toward Aspergillus Niger and Candida, but hydrogel (I-NPs) revealed only high IZ against Aspergillus Niger.

Inhibition zones of the hydrogels and modified (CS) nanoformulations. against Aspergillus Niger and Candida.

Moreover, modified (CS) nanoformulations (I-Au NPs, I-Ag NPs, I-ZnO NPs) showed high values ranged from 9 to 30 mm which displayed the same effect as Ciprofloxacin with values (25, 30 mm). However, the modified (CS) nanoformulations (I-Ag NPs) showed the highest activity values compared to Ciprofloxacin against Aspergillus Niger. The findings imply that nanoformulations have antifungal activity due to the interaction of nanoparticles with the fungal cell wall and membrane. It leads to membrane breakdown and loss of integrity and creation of holes, which causes cell death (Kim et al., 2009).

4 Conclusion

In the present work we reported the synthesis and characterization of chemically modified chitosan hydrogels (I, I-NPs, II and III) via the reactions between CS, TPC, TPP, GA and 4(4-(ethyl carbamoyl) benzoyl chloride) benzene sulfonamide (3) under different reaction conditions. Hydrogel (I) was loaded Au, Ag, and ZnO nanoparticles to perform the modified chitosan nanoformulations (I-Au NPs, I-Ag NPs and I-ZnO NPs). The synthesized hydrogels (I, II, III and I-NPs) were improved thermal stability of (CS). The crystallinity of the new modified hydrogels (I, II, III and I-NPs) showed a higher degree of crystallinity than (CS) and showed higher degree of crystallinity than (CS). Also, the surface morphology of the hydrogels and modified (CS) nanoformulations revealed different shapes compared to (CS).

Moreover, the chitosan hydrogels (I, I-NPs, II and III) showed excellent swelling properties under different factors such as time (10–100 min), pH (3–10) and temperature (40–100 °C).

Evaluation of the efficiency of sorption of Congo Red dye by the hydrogels and its modified (CS) nanoformulations in aqueous solution was studied under different parameters such as contact time, pH and initial dye concentration. It was observed that the hydrogels (I, I-NPs, and III) and modified (CS) nanoformulation (I-Au NPs) revealed the highest sorption capacity and efficiency with increasing in time compared to (CS). While the hydrogels (I) and modified (CS) nanoformulation (I-Au NPs) showed the highest adsorption capacity and efficiency under different pH values. However, the hydrogels (I, I-NPs) gave the highest adsorption capacity values with increasing the initial concentration from 20 to 100 mg/L.

Equilibrium isotherms studied for Langmuir and Freundlich isotherm and the results indicated that the Langmuir isotherm model gave a better fit to the experimental data than the Freundlich isotherm model and the RL value showed that the adsorption was favorable. Finally, results of evaluation of the antimicrobial activity of chitosan derivatives hydrogels and modified chitosan nanoformulations showed high inhibitory effects in comparison with (CS) hydrogel. Modified chitosan nanoformulation (I-Ag NPs) was the most active and gave higher values for antibacterial and antifungal than the Reference antibiotic Ciprofloxacin. From the results of this study, it can be suggested that the new modified chitosan derivatives can be used for the following potential applications: modified (CS) derivatives (I, INPs, and I-Au NPs) as potential adsorbent agent for Congo Red dye and modified chitosan nanoformulation (I-Ag NPs) as antimicrobial agent.

Acknowledgments

We thank Central Lab,Ain Shams University for samples analysis for this research.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Hou Y., Yan S., Huang G., Yang Q., Huang S., Cai J., . Fabrication of N-doped carbons from waste bamboo shoot shell with high removal efficiency of organic dyes from water. Bioresour. Technol.. 2020;303:122939
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Mohammed I.A., Jawad A.H., Abdulhameed A.S., Mastuli M.S., . Physicochemical modification of chitosan with fly ash and tripolyphosphate for removal of reactive red 120 dye: statistical optimization and mechanism study. Int. J. Biol. Macromol.. 2020;161:503-513.
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Panda P.K., Yang J.M., Chang Y.H., Su W.W., . Modification of different molecular weights of chitosan by p-coumaric acid: preparation, characterization and effect of molecular weight on its water solubility and antioxidant property. Int. J. Biol. Macromol.. 2019;136:661-667.
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Show Sections

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[20] Hassan, M.A., Omer, A.M., Abbas, E.A., Baset, W.M., Tamer, T.M., 2018. Preparation, physicochemical characterization and antimicrobial activities of novel two phenolic chitosan Schiff base derivatives. Sci. Rep. 8, 11416. http://www.nature.com/scientficreports.

[21] Hou Y., Yan S., Huang G., Yang Q., Huang S., Cai J., . Fabrication of N-doped carbons from waste bamboo shoot shell with high removal efficiency of organic dyes from water. Bioresour. Technol.. 2020;303:122939
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[22] Huong D.T.M., Chai W.S., Show P.L., Lin Y.L., Chiu C.Y., Tsai S.L., Chang Y.K., . Removal of cationic dye waste by nanofiber membrane immobilized with waste proteins. Int. J. Biol. Macromol.. 2020;164:3873-3884.
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[37] Kim U.J., Kimura S., Wada M., . Highly enhanced adsorption of congo red onto dialdehyde cellulose crosslinked cellulose-chitosan foam. Carbohydr. Polym.. 2019;214:294-302.
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New sustainable chemically modified chitosan derivatives for different applications: Synthesis and characterization

Abstract

Keywords

Chitosan

Terephthaloyl chloride

Nanoparticles

Congo Red

Antimicrobial activity

1 Introduction

2 Materials and methods

2.1 Materials

2.2 Methods

2.2.1 Synthesis of 4(4-(ethyl carbamoyl) benzoyl chloride) benzene sulfonamide (3)

2.2.2 Synthesis of chitosan hydrogel (CS)

2.2.3 Modification of chitosan with TPC to give hydrogel (I)

2.2.4 Modification of chitosan with TPC in presence of TPP to give (I-NPs)

2.2.5 Modification of chitosan with TPC and GA to give hydrogel (II)

2.2.6 Modification of chitosan with (3) to give hydrogel (III)

2.2.7 Synthesis of gold nanoparticles (Au-NPs)

2.2.8 Synthesis of silver nanoparticles (Ag-NPs)

2.2.9 Loading of hydrogel (I) on (Au, Ag and ZnO-NPs)

2.3 Instrumentation

2.3.1 Ultraviolet visible spectroscopy

2.3.2 FTIR spectroscopy studies

2.3.3 X-Ray diffraction studies

2.3.4 Thermal stability studies

2.3.4.1 Thermogravimetric analysis

2.3.4.2 Differential scanning calorimetric

2.3.5 Morphological studies

2.3.5.1 Scanning electron microscope (SEM)

2.3.5.2 Transmission electron microscopic (TEM)

2.4 Determination the swelling degree (SD)

2.5 Sorption study of Congo Red dye

2.6 Antimicrobial activity studies

2.6.1 Antibacterial and antifungal evaluation

2.6.2 Minimum inhibition and bactericidal concentration

3 Results and discussion

3.1 Synthesis of 4(4-(ethyl carbamoyl) benzoyl chloride) benzene sulfonamide (3)

3.2 Synthesis of hydrogels (I, I-NPs, II, III)

3.3 Characterization of hydrogels (I, I-NPs, II and III)

3.3.1 FTIR spectra

3.3.2 1H NMR &13C NMr

3.3.3 Degree of deacetylation of chitosan (DD)

3.3.4 Degree of substitution of the prepared hydrogels (DS)

3.3.5 Thermal stability studies

3.3.5.1 Thermogravimetric analysis (TGA)

3.3.5.2 Differential scanning calorimetry (DSC)

3.3.6 X-Ray diffraction

3.3.7 Morphological characterization

3.3.7.1 Scanning electron microscopy (SEM)

3.3.7.2 Transition electron microscopy (TEM) of nanohydrogel (I-NPs)

3.3.8 The swelling study (SD)

3.3.8.1 Effect of time

3.3.8.2 Effect of pH

3.3.8.3 Effect of temperature

3.4 Loading of hydrogel (I) on (Au, Ag and ZnO NPs)

3.4.1 UV–VIS spectroscopy of (Au, Ag and ZnO NPs)

3.4.2 FTIR spectra of (I-Au NPs, I-Ag NPs and I-ZnO NPs) formulations

3.4.3 Transition electron microscopy (TEM) of (Au, Ag and ZnO) nanoparticles and nano formulations

3.5 Dye removal studies

3.5.1 Effect of time

3.5.2 Effect of pH

3.5.3 Effect of initial dye concentrations

3.5.4 Adsorption isotherms

3.6 Antimicrobial evaluation

3.6.1 Antibacterial evaluation

3.6.1.1 Inhibition zone diameter (IZ)

3.6.1.2 MIC & MBc

3.6.2 Antifungal evaluation

4 Conclusion

Acknowledgments

Declaration of Competing Interest

References

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3.3.2 ¹H NMR &13C NMr