Novel nanocomposite hydrogel for wound dressing and other medical applications

Abstract

Graft copolymerization of diallyldimethylammonium chloride (DADMAC) vinyl monomer together with N,N′-methylene-bis-acrylamide (MBA) crosslinking agent onto water soluble carboxymethyl cellulose (CMC) was carried out using ammonium persulfate (APS) initiator. The copolymerization resulted in the formation of hydrogels. The characteristics and properties of these hydrogels were dependent on the conditions affecting the copolymerization reaction and these, in turn, controlled the pore size and porous structure of the hydrogels. Thus, increasing the monomer concentration caused a major enhancement in the swelling ratio of the hydrogel provided that the monomer was used at a concentration of 40% or more. The opposite was true for initiator concentration: the swelling ratio of the hydrogel decreased significantly by increasing APS concentration from 0.05 to 0.25 mol L⁻¹. With respect to MBA crosslinker, a maximum swelling ratio of 30 could be achieved with hydrogel prepared using MBA at a concentration of 0.1 mol L⁻¹; hydrogel prepared in the presence of MBA at 0.05 mol L⁻¹ exhibited zero swelling ratio while hydrogel prepared using MBA at 0.3 mol L⁻¹ displayed a swelling ratio of 10%. The maximum swelling ratio for hydrogel was achieved at pH 7 and a significant decrease in the swelling ratio of hydrogel was observed within the pH range 2–6 as well as at pH 8. The hydrogel could also be successfully attached to modified cotton fabric, namely partially carboxymethylated cotton (PCMC) through ionic crosslinking. The in situ formation of CuO nanoparticles inside the matrix of CMC–DADMAC nanocomposite hydrogel attached to cotton fabric was also investigated and was confirmed using X-ray diffraction and scanning electron microscopy studies. Furthermore, the functional performance of the novel CuO nanocomposite hydrogel as wound dressing was tested for antibacterial activities; the nanocomposite hydrogels demonstrated excellent antibacterial effect. The work was further extended to include the synthesis and characterization of Ag/CMC–DADMAC nanocomposite hydrogel. The latter displayed high antibacterial activity.

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1. Introduction

Hydrogels, also known as super absorbents, are preferably synthesized by grafting vinyl monomers onto natural polysaccharides and then compounding with inorganic nanoscale metals. This approach forms the basis of the method of choice because it affords unique environmental and commercial advantages. Up to now, most dual temperature and pH-sensitive hydrogels that can swell in an acidic pH environment and deswell at alkaline pH have found applications in cases such as drug release and dye adsorption.^1–4 For example, the drug (chloramphenicol) must be released more rapidly from hydrogel in a pH 1.4 buffered solution (close to the pH of the stomach) than in a pH 7.4 environment (close to the pH of the intestine),¹ in which the drug release is controlled by the swelling/deswelling behavior of the hydrogel. To achieve such functions, cationic hydrogels are needed. Diallyldimethylammonium chloride (DADMAC) is a water-soluble quaternary ammonium compound that can be cyclopolymerized to its corresponding polymer and is widely used in water treatment, paper manufacturing, mining, and biology.⁵ It was found in previous studies that polyDADMAC hydrogel could absorb water several hundred times its dry weight.^6,7 Furthermore, the quaternary ammonium compound is antibacterial which is advantageous in medical applications.⁸

Among all cellulose ethers, only carboxymethyl cellulose (CMC), available as the sodium salt (NaCMC), is a polyelectrolyte, and thus a smart cellulose derivative which shows sensitivity to pH and ionic-strength variations, plus good swelling capability.^7,9

As textiles become more functional, stimuli-responsive polymers have also found their application in the creation of intelligent or smart textiles. These environmentally responsive fabrics can be tailored by chemical modification of the textile's surface using polymeric chains. Smart textiles may provide us with considerable convenience, support, and even pleasure, in our daily activities.¹⁰

Hydrogel-based hybrid materials incorporating an inorganic phase in the form of nanoparticles (NPs) are receiving an increasing amount of attention, thanks to the synergic properties of the hydrogels and their inorganic components.¹¹ Different types of inorganic nanoparticles have been incorporated to prepare hydrogel-based hybrid systems with tailored mechanical or functional properties.^12–14 Hydrogels can afford free-space between the networks in the swollen stage that serve for nucleation and growth of nanoparticles and act as nanoreactors or nanopots. This approach was established by Wang et al.¹⁵ and Murali Mohan et al.¹⁶ to obtain 3–5 nm sized gold and silver nanoparticles within the poly(N-isopropylacrylamide) (PNIPAM) based hydrogel networks. Much research and development effort has been devoted to the production of hydrogels containing metal nanoparticles which are highly suitable for biomedical applications.¹⁷

Very recently, the development of different stimuli-responsive hydrogels was the subject of our research activities. For example, thermal responsive hydrogels based on a semi-interpenetrating network of poly(N-isopropylacrylamide) (poly(NIPAM)) and cellulose nanowhiskers were discussed.¹⁸ Investigations into the synthesis and characterization of novel CMC hydrogels and CMC hydrogel–ZnO-nanocomposites were performed.¹⁹ We have also reported on the development of CMC hydrogels loaded with silver nanoparticles for medical applications.¹⁷

The current research has been undertaken with a view to developing smart textiles with tunable water absorbance, changing with the environment. The development of such a textile hydrogel is based on radical solution polymerization of DADMAC monomer on to carboxymethylcellulose (CMC) using ammonium persulfate (APS) as an initiator and N,N′-methylene-bis-acrylamide (MBA) as a crosslinking agent. To the best of our knowledge, this copolymer has not been used before in textile applications or as a carrier for nanoparticles. Copolymerization to achieve hydrogel formation was carried out under a variety of conditions as was the application of the hydrogels to partially carboxymethylated cotton (PCMC) fabric. State of the art facilities were used for analysis and characterization of the products obtained. We prepared a novel wound dressing containing CuO/CMC–DADMAC nanocomposite hydrogel as well as Ag/CMC–DADMAC nanocomposite hydrogel.

2. Experimental

2.1 Materials

Mill-scoured and bleached cotton fabric was kindly supplied by Misr Co. for spinning and weaving, Mehala El kubra, Egypt.

Carboxymethyl cellulose (CMC) (M_w = 10 000 Da), diallyldimethylammonium chloride (DADMAC; 97%) (Merck, Germany), N,N′-methylene-bis-acrylamide (MBA; 99%), ammonium persulfate (APS; 98%), and all other chemicals were of laboratory grade.

2.2 Method

2.2.1 Synthesis of CMC–DADMAC copolymer hydrogel through graft polymerization. Graft copolymerization of DADMAC onto CMC was effected using APS as a free radical initiator. In a 100 mL flask, a predefined weight of CMC was dissolved in 10 mL of degassed distilled water. The flask was placed in a water bath at a temperature of 65 °C. A given amount of monomer, DADMAC (20–70%), was added to the flask and the mixture was stirred for 10 min. Then the initiator solution APS (0.05–0.2 mol L⁻¹) and crosslinking agent MBA (0.05–0.3 mol L⁻¹) were added simultaneously to the mixture, and the mixture was left for 30 min in a shaking water bath for gelation to occur. After gelation was complete, the gels were cut into disks 10 mm in diameter and 2 mm in thickness, and then immersed in an excess of deionized water for 4 days to remove the residual unreacted monomer. The swollen hydrogels were dried at room temperature for 2 days.

2.2.2 Preparation of partially carboxymethylated cotton fabric (PCMC). The aim of textile material activation is to impart ionic character to cotton fabric by the incorporation of a cationic hydrogel system. Among several possibilities, the carboxymethylation was carried out to activate the surface of the cotton fabric. The fabric was partially carboxymethylated by a method similar to those previously reported.²⁰ Accordingly, bleached cotton fabric samples were impregnated with 5 M aqueous NaOH for 10 min at room temperature followed by squeezing to a wet pick up of 100%. The samples were dried at 60 °C for 5 min. Thus alkali-treated samples were steeped in an aqueous solution of sodium salt of monochloroacetic acid (0.3 M) for 5 min at room temperature. These samples were then squeezed to 100% wet pick up, sealed in plastic bags and heated at 80 °C for 1 h. Samples were then washed and dried at room temperature.

2.2.3 Surface functionalisation of cotton fabric with hydrogels. As mentioned in Section 2.2.2, the aim of cotton fabric functionalisation was to impart ionic character to cotton cellulose by chemical methods. Anionic cotton has been produced by reaction with monochloroacetic acid (CAA) to give partially carboxymethylated cellulose. The preparation and polymerization reaction of the hydrogel were carried out in the presence of the PCMC sample until a thin film of hydrogel polymer was formed on the fabric by ionic crosslinking between the fabric and the formed hydrogel. Then the hydrogel-coated samples were dried at 30 °C.

2.2.4 Preparation of cotton fabric loaded with CuO/CMC nanocomposite hydrogels. Preparation of cotton fabric loaded with CMC nanocomposite hydrogels containing CuO was as follows. Typically, 1 g of fabric containing CMC hydrogel was immersed in copper sulphate solution for 24 h. The cotton fabric loaded with copper ion CMC hydrogels was washed with distilled water to remove the copper ions attached to the hydrogel surface. Following cleaning, the fabric containing CMC hydrogel loaded with Cu ions was placed in 100 mL of 0.2 M NaOH solution with heating at 100 °C for 10 min to oxidize Cu⁺ to CuO. After oxidation of the bound Cu ions, the fabric will contain hydrogels loaded with CuO nanoparticles with greenish brown color. Then the fabric was washed with distilled water and finally dried at ambient temperature.

2.2.5 Preparation of silver nanoparticles within hydrogels. Another purpose of this research was to prepare a novel composite hydrogel with antibacterial activity. Fifty milligrams of dry hydrogel discs were equilibrated in distilled water for 2 days and the swollen discs were transferred to a beaker containing 50 mL of AgNO₃ (0.01 mol L⁻¹) aqueous solution and then allowed to equilibrate for 1 day. During this equilibration stage, silver ions were exchanged from solution to the gel network through the free-space between the cross-linked networks or anchored to the –COO⁻, –NH₂, –OH groups of the polymeric chains of the hydrogel. Then the silver salt-loaded hydrogels were wiped off using tissue paper and transferred to a beaker containing 50 mL of cold aqueous NaBH₄ solution (0.1 mol L⁻¹). The beaker was left in a refrigerator (4 °C) for 2 h in order to reduce the silver ions to silver nanoparticles and the hydrogel–silver nanocomposites were separated from the NaBH₄ solution. The silver nanoparticles produced in the hydrogels are often termed hydrogel–silver nanocomposites. During this process, there was no change in the shape or size of the hydrogels in any of the samples. All of the hydrogels and hydrogels loaded with silver salt, and hydrogel–silver nanocomposites were stored in a refrigerator until their use.²¹

2.3 Characterization and analysis

2.3.1 Swelling behavior. The swelling behavior of the prepared hydrogel was calculated using the ratio (Q) for the gels as per the equation:¹⁹

Q = W_e/W_d

where W_e is the weight of the swollen hydrogel and W_d is the dry weight of the pure hydrogel.

2.3.2 FTIR spectroscopy. FTIR analyses were recorded on a PerkinElmer FTIR spectrophotometer, using the potassium bromide disk technique, in the range 4000–400 cm⁻¹. The disk was prepared from ground samples (2 mg) and KBr (45 mg) using 400 kg cm⁻² pressure for 10 min.

2.3.3 Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis. The surface morphology of the prepared hydrogel was examined with a JEOL JXA-840 scanning electron microscope (SEM). The prepared hydrogel samples were coated with a thin layer of palladium gold alloy after mounting on double-sided carbon tape. Elemental analysis of the particles was performed with a SEM equipped with an energy dispersive X-ray (EDX) spectrometer, which can provide a rapid qualitative and quantitative analysis of the elemental composition.

2.3.4 X-ray diffraction (XRD). The X-ray diffraction method was used to identify CuO and Ag nanoparticles loaded in the polymer matrix. XRD patterns were examined with a Philips PW 3050/10 goniometer and recorded with a Philips X'Pert MMP diffractometer. The diffractometer was controlled and operated by a PC with ProFit line profile analysis software and used a Mo Kα source (wavelength 0.70930 Å), operating with Mo-tube radiation at 50 kV and 40 mA.

2.3.5 Antibacterial activity. The antimicrobial activity of the prepared hydrogel was evaluated using the agar diffusion test according to AATCC Standard Test Method 147-1988.

3. Results and discussion

3.1 Mechanism of hydrogel formation

Initially, the ammonium persulfate (APS) initiator is decomposed under heating to generate sulfate anion radicals. The radicals extract hydrogen from the hydroxyl group of the sodium carboxymethylcellulose to form alkoxy radicals on the substrate. The monomer molecules, which are in close proximity to the reaction sites, become acceptors of carboxymethylcellulose radicals resulting in chain initiation and thereafter themselves become free radical donors to neighboring molecules. In this way, the grafted chain grows.^22,23 Since a crosslinking agent, i.e. MBA, is present in the system, the end vinyl groups of the MBA crosslinker may react synchronously with polymer chains during chain propagation. The copolymer consists of a crosslinked structure. DADMAC forms mostly via five-member ring formation. Scheme 1 shows the two structures, i.e. structure 1 and structure 2, suggested for DADMAC monomer. Also shown is the mechanism of formation for CMC–poly(DADMAC) crosslinked copolymer hydrogel along with the reactions involved therein.

Scheme 1 Mechanism for CMC–poly(DADMAC) hydrogel synthesis where structures 1 and 2 represent the proposed structures for DADMAC monomer.

3.2 Hydrogel characterization

3.2.1 FTIR spectral analysis of hydrogels grafted to cotton. FTIR analyses of CMC and DADMAC monomer, as well as the hydrogels prepared, are shown in Fig. 1–3. Fig. 1 depicts the characteristic bands of DADMAC which appear at 3413, 3025, 2980, 1639, 1479, 1159, 609 cm⁻¹ as numbered in Fig. 1 (1, 2, 3, 8, 10, 17, 19). A comparison of Fig. 1 with Fig. 2 for CMC and Fig. 3 for CMC grafted to DADMAC signifies that the hydroxyl group band appearing in both figures at 3427 cm⁻¹ shifts in intensity from 59 to 21.15. This lowering in intensity suggests that breakage of hydrogen bonds takes place due to the presence of a lower amount of hydroxyl groups by virtue of their involvement in the graft polymerization reaction. We can also notice the presence of a new band at 960 cm⁻¹ which is the characteristic band for CH₂ bonded to the quaternary ammonium group of DADMAC.

Fig. 1 FTIR spectrum for diallyldimethylammonium chloride monomer.

Fig. 2 FTIR spectrum for carboxymethylcellulose (CMC).

Fig. 3 FTIR spectrum for CMC–DADMAC copolymer hydrogel.

3.2.2 Morphology of CMC–DADMAC copolymer hydrogel using SEM. Fig. 4 shows SEM images of the surface and cross-sectional morphologies of CMC–DADMAC copolymer hydrogels. The plain hydrogel is characterized by a clear and flat surface (Fig. 4a); however, the freeze dried hydrogel has a porous structure (Fig. 4b). Three-dimensional network structures are also formed. These pores are thought to constitute the regions where water permeation takes place. It is well established that a porous surface is essential for the transport of oxygen from outside to inside, for example, wound dressing; meanwhile, a three-dimensional structure is equally crucial for absorbing and retaining large amounts of water in the hydrogel materials.

Fig. 4 SEM images for CMC/DADMAC hydrogels. (a) SEM micrograph of the outer surface of the CMC/DADMAC hydrogel. (b) SEM image of freeze-dried sample of hydrogel (cross-section) prepared at pH 7.

3.3 Synthesis of CMC/DADMAC hydrogels: effect of process parameters

3.3.1 Monomer concentration. Fig. 5 shows the effect of concentration of DADMAC monomer on the swelling ratio of the hydrogel prepared as per the procedure previously described for copolymerization of this monomer on CMC in the presence of MBA crosslinking agent. As is evident (Fig. 5), the crosslinking and copolymerization were carried out in highly concentrated aqueous solutions of the monomer mixture. This is due to an interesting aspect of DADMAC polymerization in that this monomer can only be polymerized in highly concentrated solutions, because of the strong coulombic repulsion between the quaternary ammonium groups, as evident from Butler's pioneering work on the cyclopolymerization of DADMAC monomer.²³ For this reason, the terpolymerization reactions were performed at high monomer concentrations (40% w/w) at 65 °C. Particularly notable is that increasing the monomer concentration above 40% causes outstanding enhancement in the swelling ratio. This means that the concentration of DADMAC monomer plays a key role in determining the value of the swelling ratio of the hydrogel. The pore size and porous structures of the hydrogels under investigation are a direct result of DADMAC concentration.

Fig. 5 Effect of concentration of DADMAC monomer on the swelling ratio of CMC–poly(DADMAC) crosslinked copolymer. Reaction conditions: 0.5 g CMC; 0.16 mol L⁻¹ MBA; 0.04 mol L⁻¹ APS; 65 °C; 2 h.

3.3.2 Initiator concentration. Fig. 6 shows the effect of APS initiator concentration on the swelling ratio of CMC–poly(DADMAC) crosslinked copolymer. Obviously, an increase in APS initiator concentration is accompanied by a decrease in swelling efficiency. This reflects the serious impact of initiator concentration on the morphology, in particular, the porous structure, of the hydrogel. As stated earlier, initiator present above a certain concentration would lead to decreased grafting due to fast termination rate and, in turn, lower molecular weight of the graft. With this in mind, it is a logical conclusion in the current study that the molecular weight of grafted chains of poly(DADMAC) branches and the sequence of these branches on the CMC backbone would depend greatly on the initiator (APS) concentration. Besides decreasing the molecular weight of the grafted chains, a higher concentration of initiator causes oxidation of the CMC backbone thereby leading to CMC with lower molecular weight as a result of glucosidic bond scission; meanwhile, extra carboxylic and/or aldehydic groups are created by oxidation of CMC hydrogels. In short, at higher initiator concentration, the grafted chains and CMC backbone undergo changes in their molecular structure during synthesis of the hydrogel, and these changes affect the pore size and porous structure of the hydrogel such that the swelling ratio of the hydrogel decreases. Once this is the case, the pore size and porous structure of the hydrogels will differ accordingly. The onset of such changes in the physical and chemical structure of the hydrogel would certainly be reflected in the swelling ratio of the hydrogel in question.

Fig. 6 Effect of APS initiator concentration on the swelling ratio of CMC–poly(DADMAC) crosslinked copolymer. Reaction conditions: 0.5 g CMC; 60% monomer concentration; 0.16 mol L⁻¹ MBA; 65 °C; 1 h.

3.3.3 MBA concentration. Of the factors affecting synthesis of the hydrogel, crosslinker concentration is the most influential factor on water absorption of the hydrogel. As can be seen from Fig. 7, the swelling ratio increases to reach a maximum at 0.09 mol L⁻¹, then the water absorption rapidly decreases when increasing the concentration of MBA crosslinker from 0.09 to 0.3 mol L⁻¹. This is rather in conformation with Flory's theory²⁴ – the increase in MBA concentration results in an increase in crosslinking density which in turn diminishes the network voids of holding water thereby decreasing the pore sizes of the hydrogel. As a consequence, the swelling tendency of the hydrogel decreases.

Fig. 7 Effect of concentration of MBA crosslinking agent on the swelling ratio of CMC–poly(DADMAC) (copolymer) hydrogel. Reaction conditions: 0.5 g CMC; 60% monomer concentration; 0.04 mol L⁻¹ APS; 65 °C; 1 h.

3.3.4 Effect of medium pH. Fig. 8 shows variations of water absorption expressed as swelling ratio of CMC–DADMAC graft copolymer with different pH solutions. As is evident, there are no apparent variations in the swelling ratio of the hydrogel when changing the pH of the swelling medium from pH 2 to pH 6. On the other hand, a sharp increase in swelling ratio is observed at pH 7 followed by an abrupt decrease at pH 8.

Fig. 8 Swelling ratio of CMC–DADMAC copolymer hydrogel versus pH of the aqueous swelling medium.

In neutral water as the swelling medium, CMC is a negatively charged polyelectrolyte in the swelling system, and the strong electrostatic repulsions among CMC carboxylate anions (COO⁻) could result in a more expanded network in the hydrogel. The latter assumes the highest swelling ratio at pH 7, a point which could be associated with an increasing number of ionic groups in the hydrogels which causes an increment in their swelling capacity due to additional osmotic pressure provided by counterions inside the gel. However, the swollen gel rapidly shrinks because of protonation of –COO⁻ groups under acidic pH values (pH < 5), where most of the carboxylate anions are protonated. On the one hand, the hydrogen-bonding interaction among carboxylate groups is strengthened and additional physical crosslinking is generated. As a result, the network tends to shrink and consequently swelling values are decreased. The decreased absorbency at higher basic pH values (pH > 8) is related to the ‘screening effect’ of excess cations in the swelling media.

3.4 Functionalization of cotton textile by CMC–DADMAC copolymer hydrogels

The main challenge in developing smart textile materials is confined to techniques for successful attachment of the hydrogel layer to the textile substrate. Recent research has disclosed that hydrogel particles can be covalently bonded to cotton using appropriate crosslinking agents.²⁵ In the current work, the hydrogel was attached to the surface of partially carboxymethylated cotton (PCMC) fabric through ionic crosslinking. Anionic PCMC fabric was synthesized by reacting it with monochloroacetic acid in alkaline medium. This method not only keeps the elastic form of the hydrogel but also confirms its attachment to the cotton fabric. The presence of hydrogel on the surface of PCMC fabric is indeed visually confirmed by SEM.

The surface morphology of PCMC fabric coated with the hydrogel is shown in Fig. 9. As can be seen from Fig. 9a–c, the fibers are covered with irregular fragments, rendering the surface of the fabric rough and homogeneous. This means a thin hydrogel layer is coating the fabric. Fig. 9d illustrates the surface morphology (cross-section) of the fabric coated with a thin layer of hydrogel.

Fig. 9 The surface morphology of fabric coated with a thin layer of hydrogel.

By studying the swelling characteristics of cotton–hydrogel samples, we have concluded that the incorporation of hydrogel onto the surface of the cotton fabric causes significant changes in its swelling behavior. Most probably, the ionic crosslinking between PCMC and the cationic hydrogel decreases the affinity of the hydrogel towards water and the swelling ratio decreases to a value of 10 at pH 7, while in both acidic (pH 5) and alkaline (pH 9) environments, the swelling ratio decreases to a value of 8. This decrease at particular pH values can be attributed to the action of the concentrated solution of caustic soda during functionalisation of the cotton surface. However, the hydrogel-coated cotton still acquires good swelling characteristics.

3.5 Preparation of carboxymethylcellulose/CuO bio-nanocomposite hydrogels

CMC interacts with many metal cations including Al³⁺, Cu²⁺, Co²⁺, Mo⁶⁺, and Zn²⁺,²⁶ due to the porous structure of hydrogels and the existence of carboxylate groups (–CO₂⁻), thus the CMC hydrogels can easily bind to the Cu²⁺ cations in aqueous solutions of copper sulphate via electrostatic interactions. With a suitable basic agent such as NaOH, copper ions are oxidized to CuO nanoparticles. The reaction process can be expressed as follows:

Cu²⁺ + 4HN₃·H₂O → [Cu(NH₃)₄]²⁺ + 4H₂O (1)

[Cu(NH₃)₄]²⁺ + 2NaOH → Cu(OH)₂↓ + 4HN₃ + 2Na²⁺ (2)

Cu(OH)₂ → CuO + H₂O (3)

3.5.1 Characterization of cotton loaded with hydrogel containing CuO nanoparticles. The main challenge here was to entrap CuO nanoparticles within the matrix of the hydrogel and to provide a proof for its synthesis. To achieve this goal, samples of cotton fabric loaded with hydrogel containing CuO nanoparticles were submitted to XRD analysis, SEM examination and EDS spectral analysis. Results obtained are given next.
3.5.1.1 X-ray diffraction (XRD) analysis. The X-ray diffractogram of CuO/CMC nanocomposite hydrogel on fabric in the 2θ range of 2–70° is shown in Fig. 10. The diffractogram of CMC/CuO nanocomposite hydrogel is characterized by diffractions at 2θ values at about 35°, 38°, 49°, 53°, 58°, and 62° which are assigned to the (110), (002), (112), (020), (202) and (−311) diffractions of CuO crystals, respectively. All of the peaks match well with those of monoclinic phase CuO crystals and confirm the formation of CuO particles in the CMC hydrogel matrix.

Fig. 10 XRD pattern of CuO nanoparticles inside the matrix CMC hydrogel.

3.5.1.2 Scanning electron microscopy (SEM). In comparison with Fig. 9d which showed the surface morphology of cotton coated with a hydrogel layer, Fig. 11a,b shows SEM images of cotton fabric coated with CMC hydrogel containing CuO nanoparticles. It was observed that a large amount of needle-like aggregates of CuO nanoparticles are trapped within the hydrogel matrix forming a homogenous layer on the surface of the cotton fabric and at depth inside it.

Fig. 11 (a and b) Scanning electron microscope (SEM) images. (c) EDS spectrum of CuO nanoparticles.

3.5.1.3 EDS analysis. The EDS spectrum of CuO nanoparticles (shown in Fig. 11c) clearly demonstrates the presence of Cu and O peaks with 4.45 wt% and 92.9 wt%, respectively, confirming the presence of CuO nanoparticles, which is consistent with the XRD results.

3.6 Synthesis and characterization of silver nanocomposite hydrogel (Ag/CMC–DADMAC)

When a fully swollen CMC–DADMAC hydrogel in the form of a disk is put in aqueous AgNO₃ solution, Ag⁺ ions replace the H⁺ or Na⁺ ions in the CMC hydrogel. Therefore, Ag⁺ ions are still accessible for reduction into nanosilver by sodium borohydride solution forming silver nanoparticles within the swollen network as illustrated in Fig. 12.

Fig. 12 Schematic representation of steps involved in preparation of Ag/CMC–DADMAC nanocomposite hydrogel.

3.6.1 Characterization of Ag/CMC–DADMAC nanocomposite hydrogel. Scanning electron micrographs of Ag/CMC–DADMAC nanocomposite hydrogel are shown in Fig. 13. It is seen that silver nanoparticles are clearly visible not only on the surface of the Ag/CMC–DADMAC nanocomposite hydrogel (Fig. 13a and b) but also inside the network, as is visible in the cross-sectional view in Fig. 13b. The SEM images show that no significant aggregation of nanoparticles is formed. This can be interpreted in terms of a stable network structure in the hydrogels in addition to strong interaction between the silver particles and the copolymer hydrogel.

Fig. 13 SEM image of a freeze-dried sample of the prepared hydrogel at pH 7.0. (a and b) Images of silver nanoparticles grown inside the hydrogel network. (c) EDS spectrum of the freeze-dried sample of the prepared hydrogel containing silver nanoparticles. (d) Energy Dispersive X-ray Spectroscopy (EDS) maps of the CMC–DADMAC hydrogel loaded with Ag nanoparticles.

Fig. 13b and c also supports the view that the silver nanoparticles are formed throughout the network and along with the polymeric network in addition to existing in free spaces in the networks. This means that the hydrogel acts as a reactor for silver nanoparticles that grow as bright dots distributed between the gel networks with the help of the polymeric chains. This can be seen where the silver nanoparticles are overlying on CMC–DADMAC copolymer chains in the hydrogel network. The Energy Dispersive X-ray Spectroscopy (EDX) of the freeze-dried sample of the prepared hydrogel containing silver nanoparticles (Fig. 13c and d) shows that the Ag nanoparticles are loaded inside the matrix of the hydrogel with high content (71 wt% Ag) with a uniform spatial distribution of Ag nanoparticles on CMC–DADMAC nanocomposite hydrogel.

3.7 Antibacterial activity

The antibacterial activities of CuO/CMC–DADMAC dressing, Ag/CMC–DADMAC nanocomposite hydrogel, and CMC–DADMAC (control sample) were studied against Gram-positive and Gram-negative bacteria. The antibacterial activity was determined in terms of the size of the inhibition zones on agar medium. It was observed that the control sample did not display any antibacterial activity despite the presence of the quaternary ammonium groups in the copolymer hydrogel.^27–29 The effectiveness of the quaternary ammonium groups as an antibacterial seems to be abolished through their intimate association and interaction with CMC in the copolymer hydrogel. That is why the CMC–DADMAC copolymer hydrogel failed to induce antibacterial activity. On the other hand, CuO/CMC–DADMAC nanocomposite hydrogel and Ag/CMC–DADMAC nanocomposite hydrogel can release copper and silver nanoparticles into the pathogenic environment,³⁰ thereby producing highly efficient antibacterial activity (Table 1).

Table 1 Antibacterial activity of CMC–DADMAC copolymer hydrogel and CuO/CMC–DADMAC and Ag/CMC–DADMAC nanocomposite hydrogels^a

Sample	Inhibition zone diameter (mm cm^−1) sample
	E. coli (G –ve)	P. aeruginosa (G –ve)	S. aureus (G +ve)	B. subtilis (G +ve)
a All experimental test data were collected in triplicate, and the average value taken.
CMC–DADMAC dressing hydrogel	Zero	Zero	Zero	Zero
CuO/CMC–DADMAC dressing hydrogel	19	20	20	18
Ag/CMC–DADMAC hydrogel	17	19	18	17

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The results of Table 1 show decisively that both CuO/CMC–DADMAC and Ag/CMC–DADMAC nanocomposite hydrogels acquire high antibacterial activity. The inhibition zones exhibit values of 19 and 20 mm cm⁻¹ for CuO nanocomposite hydrogel dressing and 17 and 19 mm cm⁻¹ for Ag nanocomposite hydrogels upon using E. coli (G –ve) and P. aeruginosa (G –ve) bacteria, respectively. When using S. aureus (G +ve) and B. subtilis (G +ve) bacteria, the values were, respectively, 20 and 18 mm cm⁻¹ for CuO nanocomposite hydrogel dressing and 18 and 17 mm cm⁻¹ in the case of Ag nanocomposite hydrogel.

Within this range of studies, it is logical to assume that the antibacterial activity of the materials under investigation relies, in essence, on the nature of the nanoparticles under investigation as well as on the bacteria which, in turn, determine the speed and mechanism of release of copper oxide or silver ions from the nanoparticles of the nanocomposite hydrogel and the interaction of the released ions with the cell wall of the bacteria. On the other hand, in the case of CuO nanoparticles, the activity could be explained in terms of attachment of CuO nanoparticles to the cell wall of bacteria which damages the cell wall and causes leakage of proteins and other intracellular constituents and ultimately leads to cell death.^30–32

In the case of the presence of Ag nanoparticles in the hydrogel matrix, it was reported that Ag nanoparticles penetrate the cell wall of Gram –ve bacteria.^33,34 As a result, a structural change in the cell membrane occurs. This could lead to an increase in cell permeability which, in turn, leads to uncontrolled transport through the cytoplasm membrane and ultimately to the death of the cell. Another mechanism is based on free radical formation followed by free radical-induced damage to the cell membrane. It is also likely that silver ions move into the cell and, as a result, production of reactive oxygen species takes place which can damage the cell wall. It is further reported that there is a greater tendency for silver ions to interact with thiol groups of vital enzymes as well as phosphorous containing bases³⁵ and, with the presence of silver nanoparticles inside the cells,³⁶ it is logical that certain damage could be realized through interactions with compounds such as DNA. This interaction may stop cell division and DNA replication and end with death of the cell.

4. Conclusion

Hydrogels with unique properties were synthesized through copolymerization of CMC with DADMAC in the presence of APS initiator and MBA crosslinker. The pore size and porous structure of the hydrogels thus obtained could be controlled by making use of variables affecting the formation of the hydrogels. Briefly, CMC-DADMAC copolymer hydrogels had a higher swelling ratio when higher DADMAC monomer concentrations were used. The opposite held true for either APS or MBA where the hydrogels displayed lower swelling ratios with increasing concentrations of APS or MBA. Particularly notable is the plot of the results of swelling ratio versus pH. The swelling ratio of the hydrogel exhibited a striking decrease within the pH range 2–6 as well as at pH 8. On the other hand, a hydrogel with maximum swelling ratio could be achieved at pH 7. The hydrogels under investigation also form the basis for production of wound dressings. The hydrogel was attached to PCMC fabric via ionic crosslinking. Furthermore, the antimicrobial activity of the novel hydrogel was examined on Gram-negative and Gram-positive bacteria according to the agar diffusion test. The CuO/CMC–DADMAC nanocomposite hydrogels showed higher antibacterial activity than Ag/CMC–DADMAC nanocomposite hydrogels against Gram-positive and Gram-negative bacteria. Based on these findings, the prepared nanocomposite hydrogels can be used in different medical fields, i.e. drug delivery, wound dressing as well as wound healing.

Acknowledgements

This project was supported financially by the Science and Technology Development Fund (STDF), Egypt, grant number 4384.

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