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Noncytotoxic Silver and Gold Nanocomposite Hydrogels with Enhanced Antibacterial and Wound Healing Applications.


The hydrogel is a polymeric network with the capability of gripping large quantities of water, due to chemical or physical crosslink between polymeric chains [1, 2]. Recently, most of the researchers assembled the nanoparticles into hydrogel networks, which has involved in biomedical applications [3, 4]. The nanoparticles and nanostructural materials have shown and improvise the following properties like catalytic, optical, electronic, antibacterial, and antioxidant applications. There are several methods has been adopted to prepare the nanocomposite containing metal nanoparticles. The mechanical mixing methods used for the preparation of a polymer by using in situ polymerization in the presence of metal nanoparticles [5-8].

In the last few years, the polymer nanohydrogels has paid significant attention, where the hydrogels are extremely used in the biomedical domain such as drug delivery, tissue engineering and particularly in antibacterial and antifungal applications [9]. Literature reports revealed that IAA combined hydrogel found to have cytotoxic behavior to human cancer cells [10, 11]. Further, IAA based hydrogel with silver nanoparticles found to cytotoxic behavior with respect to fibroblast 3T3 cell line for wound healing. The progressive series of molecular, cellular, and biochemical proceedings of the hydrogels can able to form a complex with dynamic wound healing applications [12].

Gold nanoparticles (AuNPs) used in various fileds that have shown exclusive properties in multidisciplinary research fields [13, 14]. The particles attracts and deeply scatter visible near-infrared light upon excitation of their surface plasmon oscillation (SPR). One specific field of research involves the use of GNPs detection and treatment of cancer cells [15]. Current techniques of cancer analysis and treatment are most expensive and can be very destructive to the body. GNPs, however, offer a low-cost route to targeting only cancerous cells, leaving healthy cells unharmed [16]. These properties include improved or stalled particle aggregation depending on the type of surface alteration, improved photoemission, high electrical, heat conductivity, and enhanced surface catalytic action [17].

Silver nanoparticles found to have antimicrobial properties and pharmacology applications. At present, silver nanocomposite was major missiles against wound infection with the advent of antibiotics [18, 19]. These materials may have low, medium or high potential risk to human safety, depending on the type and extent of usage. Nontoxic nature of NCS only recommended for the appraisal of medical devices is in vitro assessment as biomaterials. Recently, the nanocomposite hydrogel reported based on citric acid (CA), diethyleneglycol (DEG), and indole-3-acetic acid (IAA)/Ag and showed biological activities such as antibacterial, antifungal, antioxidant characteristics [20].

The scope of the present investigation involves in the synthesize silver nanocomposite hydrogel by condensation polymerization without a cross linker. The swelling equilibrium of nanocomposite hydrogel have been calculated at various pH (3-10) and found to have ascending order from acidic media to basic media. The nanoparticles have been identified by FT-IR, transmission electron spectroscopy and scanning electron microscope with electron dispersive X-ray analysis. The effect of cytotoxicity has also been observed for nanocomposite using fibroblast 3T3 cell line can be analyzed by MTT assay.



Anhydrous CA, diethylene glycol, indole-3-acetic acid, silver nitrate, sodium borohydride, HAu[Cl.sub.4], and trisodium citrate were purchased from Sigma Aldrich (Bangalore, India).

Preparation of Ag and Au Nanocomposite Hydrogel

Citric acid (0.025M) was dissolved in 5 mL ethanol using a round bottom flask closed with guard tube and stirred with a magnetic stirrer at room temperature followed by adding DEG slowly and continuously stirred for half an hour by which the glassy white sticky gel obtained named as a prepolymer. In addition, IAA added to the prepolymer and stirred to continue the post-polymerization at 160[degrees]C for 1 h, by which glassy reddish brown sticky gel obtained. The resultant gel was immersed in distilled water for one day, in order to remove the unreacted monomer then filtered and dried in oven at lukewarm condition for 48 h [21]. The amount of unreacted monomers were determined by titration of extract against NaOH (0.05 mol/L) using phenolphthalein indicator which confirms the absence of residual monomer.

Synthesis of Silver and Gold Nanocomposite Hydrogels. In this process, 0.1 g of hydrogel was immersed in water for 24 h and the hydrogel was transferred into a beaker containing 25 mL of AgN[O.sub.3] aqueous solution which is allowed to equilibrate for 2 days, where the [Ag.sup.+] ions are incorporated into hydrogel networks. Later, the silver hydrogel was transferred to a beaker containing 25 mL of concentration NaBH4 for 4 h to reduce the [Ag.sup.+] into [Ag.sup.0] nanoparticles, filtered, and dried at ambient temperature resulted in obtaining brown glassy silver nanocomposite hydrogel (ICD-Ag). Similarly, the same method was used for gold nanocomposites, 0.1 g of hydrogel dipped in the 25 mL HAu[C1.sub.4] aqueous solution. Here, the gold-containing hydrogel ([Au.sup.3+]) changed to [Au.sup.0] nanoparticles by using trisodium citrate as a reducing agent to get a uniformly distributed spherical shape of nanoparticle present in hydrogel network.

However, using NaBH4 as a reducing agent formed an irregular shape of nanoparticle and hence trisodium citrate used as reducing agent for gold nanocomposite [22] by which a glassy browny gold nanocomposite hydrogel (ICD-Au) was obtained and represented in the Scheme 1.


FT-IR studies of the NCHS were recorded on a FTIR-8400S, Shimadzu spectra photometer and thermogravimetric analysis carried out using SDT Q 600 DTA-TGA was used for thermal properties of nanocomposite hydrogels. TGA was measured in the temperature range of 800[degrees]C at a heating rate of 10[degrees]C min under [N.sub.2] atmosphere. Energy-dispersive X-ray (EDX) spectroscopy analysis was done for confirming the presence of silver and gold nanocomposite hydrogels. Transmission electron microscopy (TEM) is being carried out using JEM 2000 and a source of this instrument is LaB6 with resolution point 0.23 nm and voltage is 200 kV. Swelling equilibrium experiments of nanocomposite hydrogel was conducted at various pH ranges 3-10 [21].

Antibacterial Activity

The antibacterial activity of nanocomposite hydrogels was measured as per the reported method available in the literature [21]. The test organisms were subcultured by lining them on nutrient agar, followed by incubation for 24 h at 37[degrees]C. Using sterilized dropping pipettes, samples of different concentrations (500-2,000 [micro]g/well) were carefully added to the wells and allowed to diffuse at room temperature for 2 h, then the plates were incubated at 37[degrees]C for 18-24 h. Gentamicin (10 [micro]g) was used as a positive control and DMSO as a negative control with Staphylococcus aureus, Bacillus cereus, and Escherichia coli.

MTT Assay: Cytotoxicity

The relative cytotoxicity of hydrogel was determined by MTT viability assay developed for high throughput screening (HTS). The MTT substrate is prepared in a physiologically balanced solution, added to cells in culture, usually at a final concentration of 0.2-0.5 mg/mL and incubated for 1-4 h. The quantity of formazan (presumably directly proportional to the number of viable cells) was measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer. Viable cells with an active metabolism, which convert MTT into a purple colored formazan product and when cells die, they lose the ability to convert MTT into formazan and DMSO was added to dissolve formazan crystals.Untreated cells were taken as the control with 100% viability [21].

Scratch Wound Healing Assay

Grow cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (fasting blood sugar). Seed cells into 24-well tissue culture plate at a density that after 24 h of growth, they should reach ~70%-80% confluence as a monolayer. Without changing the medium, scratch the monolayer gently and slowly with a new 1 mL pipette tip across the center of the well. While scratching the surface of the well, the long-axial of the tip should always be perpendicular to the bottom of the well. The resulting gap distance therefore equal to the outer diameter of the end of the tip and the gap distance can be adjusted using different types of tips. Scratch a straight line in one direction and another straight line perpendicular to the first line to create a cross in each well. After scratching, gently wash the well twice with medium to remove the detached cells and replenish the well with fresh medium (Note. Medium may contain ingredients of interest that you want to test, e.g., chemicals that inhibit/promote cell motility and/or proliferation).

Grow cells for additional 48 h (or the time required if different cells are used), wash the cells twice with IX PBS, then fix the cells with 3.7% paraformaldehyde for 30 min. Stain the fixed cells with 1% crystal violet in 2% ethanol for 30 min. Take photos for the stained monolayer on a microscope, set the same configurations of the microscope when taking pictures for different views of the stained monolayer. The gap distance can be quantitatively evaluated using software such as Photoshop or Image. To reduce variability in results, it is suggested that multiple views of each well should be documented and each experimental group should be repeated multiple times.


Fourier Transform Spectroscopy

The FTIR spectra of neat hydrogel and their NCHs have shown in Fig. 1A. The peak of -C=O stretching frequency in the ester group is 1,734.76 cnT1 and a weak shoulder peaks at 2,362.72 and 2,343.85 [cm.sup.-1] related to the aliphatic -CH stretching frequency. The absorption band appeared at 1,523.00 [cm.sup.-1] attributed to -CO[O.sup.-] stretching in the ICD hydrogel [23]. The peak at 747 [cm.sup.-1] was attributed -CH- out of plane bending of an aromatic ring. These results confirmed the incorporation of aromatic moieties presents in ICD. The sharp peak at 3,159 [cm.sup.-1] can be attributed to either the hydrogen bonded -OH in diol and -NH bond or both (overlapped) [24]. The FT-IR spectrum of ICD-Ag and ICD-Au are shown at different peaks, where the spectrum of ICD-Ag (Fig. IB) was attributed to C=0 stretching frequency at 1,732 [cm.sup.-1] in the polyester network and the peak at 2,950 [cm.sup.-1] related to the aliphatic -CH stretching frequency of polymeric backbone.

The absorption band appeared at 1,457 and 1,400 [cm.sup.-1] observed stretching frequency of -CO[O.sup.-] in the ICD-Ag nanocomposite hydrogel [22, 23] and the stretching vibration of C-O observed at 1,201 [cm.sup.-1] [21, 22]. The spectra showed strong band at 1,123 [cm.sup.-1] credited to C-O-C stretching frequency. The main peak of 1,340 [cm.sup.-1] contains silver nanoparticle present in the polymeric network [25, 26]. The peaks at 746 [cm.sup.-1] was attributed -CH-out of plane bending of aromatic ring which has confirmed the incorporation of aromatic moieties presents in ICE-Ag. The sharp peak at 3,194 [cm.sup.-1] can be attributed to either the hydrogen bonded -OH in diol and -NH bond or both (overlapped) [24].

In ICD-Au (Fig. IB) spectrum was observed at 1,731 cnT1 related to C=0 stretching frequency in the polyester network. The peak at 1,457 [cm.sup.-1] indicated the stretching frequency of -COO in the ICD-Au nanocomposite hydrogel [22, 23]. The main peak at 516 [cm.sup.-1] contains gold nanoparticles present in the polymeric network [25, 26]. The peaks at 744 [cm.sup.-1] was attributed -CH-out of plane bending of aromatic ring which has confirmed the incorporation of aromatic moieties presents in ICE-Au. The broad and weak shoulder peak at 3,389 [cm.sup.-1] can be attributed to either the hydrogen bonded -OH in diol and -NH bond or both (overlapped) [24]. The results strongly supported the C=0 stretching frequency of NCHS decreases than ICD pure hydrogel because of the complex between ICD pure hydrogel and metal nanoparticles and strain between pure hydrogel and nanoparticles in hydrogel network [27].

Thermal Analysis

Previously, the thermal stability of pure hydrogel (ICD) has been reported, where the first-stage degradation was observed at 180[degrees]C to 280[degrees]C with 40% weight loss due to moisture cleavage and breakage of aromatic moieties and second stage was observed in the temperature range from 280[degrees]C to 380[degrees]C with 30% weight loss due to depolymerization of hydrogel network [21]. But, incorporating the nanoparticle to hydrogel networks decreases the thermal stability which has shown in Fig. 2A and B. The first step indicated the mass loss nearly 130[degrees]C to 200[degrees]C with 8% weight loss for silver nanocomposite hydrogel and 130[degrees]C to 180[degrees]C with 5% weight loss for gold nanocomposite hydrogel based on loss of water and side chain group. The Second step observed for silver and gold nanocomposite hydrogels were 200[degrees]C to 280[degrees]C with 40% weight loss and 180[degrees]C to 250[degrees]C with 56% weight loss leads to the decomposition temperature of nanoparticles. The Third step of decomposition temperature for ICD-Ag and ICD-Au were noted as 280[degrees]C to 410[degrees]C with 40% weight loss and 250[degrees]C to 390[degrees]C with 20% weight loss indicated decomposition temperature of the polymer matrix.

Comparatively, the thermal stability of pure hydrogel was more than nanocomposite hydrogels due to the interaction between nanoparticles and organic matrix. On the other hand complexation of ICD-Au hydrogel is more than ICD-Ag, so affects the thermal stability of gold nanocomposite hydrogel [28] which results in good agreement with swelling equilibrium.

Field Emission Scanning Electron Microscopy-Energy Dispersive X-Ray Analysis (EDX)

SEM with EDX confirmed the presence of silver nanoparticles in the nanocomposite hydrogel. The silver nanoparticles peak obtained at 2.6 Kev with 0.57% silver, 52.49% carbon, 30.85% nitrogen, and 16.09% oxygen mentioned in EDX analysis (Fig. 3A). The gold nanoparticles peak obtained at 2 Kev with 0.25% gold, 59.81%carbon, 38.72% nitrogen, and 1.22% oxygen (Fig. 3B). No impurity peaks have detected which has proved purity of silver and gold nanoparticles are high and their presence in hydrogels [29].

Transmission Electron Spectroscopy

In Fig. 3, TEM image determined the size of silver and gold nanoparticles in nanocomposite hydrogels. ICD-Ag (Fig. 4A) image has shown the size of nanoparticles distributed with average size of 4-12 nm and gold nanoparticle dispersed with average size of 8-30 nm in ICD-Au (Fig. 4B) which has proved the presence of silver and gold nanoparticles in concern hydrogel network. Comparatively, the size of the nanoparticle of ICD-Ag was smaller than ICD-Au due to less complexity of silver nanocomposite hydrogel. Hence, the smaller size of nanoparticles (ICD-Ag) enhance the biological properties of nano composite hydrogels [30, 31].

Swelling Equilibrium Studies

Figure 5 indicates the swelling equilibrium of silver and gold nanocomposite hydrogels of 48 h at various pH buffer solutions (pH range 3-10). Swelling equilibrium of silver nanocomposite hydrogel (ICD-Ag) at pH 3, 4, 6, 7, 9, and 10 were indicated as 1,480, 1,400, 1,020, 962, 830, and 720, respectively. The gold nanocomposite hydrogel (ICD-Au) at different pH medium 3, 4, 6, 7, 9, and 10 were stated as 1,380, 1,300, 910, 830, 700, and 580, respectively. So, the swelling equilibrium reacts better in the acidic medium compared with basic medium due to the protonated amino group present in the polymeric network [32], decreasing osmotic pressure and electrostatic repulsion. In ICE-Ag, the swelling equilibrium was enhanced due to the smaller size of the nanoparticles, whereas the swelling equilibrium is reduced in ICE-Au due to larger size of nanoparticles and complexation of hydrogel network. So, the resultant of swelling equilibrium leads to ICE-Ag > ICE-Au. The swelling capacity of nanocomposite hydrogels plays an important role in the wound healing and biomedical application due to high absorption capacity of water or solvent [33].

Antibacterial Activity of Nanocomposite Hydrogels

In the present study, silver and gold nanoparticles exhibited good antibacterial activity against pathogens compared with gentamicin (Fig. 6), where ICD-Ag has shown higher antibacterial activity than ICD-Au. Similarly, our results also observed for silver hydrogel, which might be related to more antibacterial than AuNPs, largely due to the inert chemical nature of gold [34]. The zone of inhibition of ICE-Ag hydrogel was Escherichia coli (23 mm), Staphylococcus aureus (27 mm), Bacillus aureus (25 mm) at 2,000 pg/well. In ICE-Au hydrogel contains Staphylococcus aureus (16 mm) and B.c (15 mm), than E. coli (14 mm) at 2,000 ng/well, which has represented in Fig. 9A and B. This results confirmed the good inhibition on grampositive bacteria (Staphylococcus aureus and Bacillus aureus) than a gram-negative bacteria Escherichia coli . This might be due to the bacterial cell wall of gram-positive bacteria is fully composed of nanocomposites with plenty of pores, which allow foreign molecules to come into the cell without difficulty and allow more rapid absorption of ions into the cell [35].

The size of the nanoparticles indicated that the smallest-sized spherical AgNPs (ICD-Ag) were more efficient to kill and destroy both types of bacteria as compared to larger spherical AuNPs (ICD-Au). When paper disks were impregnated with colloidal silver and gold particles of different size and shape, the rate of dissolution of silver and gold cation particles were different. Due to the high surface to volume ratio, the smaller-sized nanoparticles released more silver cations which have more effective to kill the bacteria as compared to larger-sized gold particles. These results are in accordance with already reported outcomes [36]. So, the study proves the antibacterial activity of nanocomposite hydrogels as ICD-Ag > ICD-Au.

Cytotoxicity Evaluation: Qualitative Studying of the Cell Viability

Figures 7-10 shows, the in vitro cytotoxicity of pure hydrogel, silver, and gold nanocomposite hydrogels evaluated using MTT assay in fibroblast cell 3T3 cell line treated with various concentrations of composite hydrogels. The cytotoxicity effect of various concentrations of pure hydrogel (ICD) nanocomposite hydrogels (ICD-Ag, ICD-Au) quantified as percentages of cell viability, including the absorbance values obtained in each concentration. The cell viability of pure hydrogel (Fig. 7) found to be concentrations 100%, 97%, 92%, and 88%, Similiarly, ICD-Ag (Fig. 8) and ICD-Au (Fig. 9) at various concentrations (25, 50, 100, 250 pg/mL) were found at 92%, 90%, 85%, and 76% and 98%, 97%, 90%, and 86% which has been proved that the nontoxic nature of hydrogel. Fig. 10 indicated MTT assay on cell viability above 80% which has been considered as nontoxicity as per guidelines of ISO 10993-5:2009. Generally, the low antibacterial activity of hydrogel leads to high cytotoxicity due to the small size and the relatively large surface area of NPs resulted in increased cytotoxicity and decreased antibacterial activity [37].

Wound Healing Activity of ICD-Ag Hydrogel

The mean percentage of wound closure of ICD-Ag hydrogel was found to be 2, 30, 55 and 75% for 0, 24, 48, and 72 h which have been indicated in Figs. 11 and 12. After 6 days, the curing cum complete closure of the wound have been noticed. In particular, fibroblast treatment induced a statistically increased rate of scratch wound closure due to enhanced cell migration. Wound healing properties associated with cell viability to fight free radicals that have the potential to damage biological tissues by disrupting cell membranes. The ability of the hydrogels to decrease the bacterial load and to inhibit the growth of microbe indicated the wound healing potential and nontoxic nature exhibited high wound closure [38].


The present study focused a highly facile and simple methodology for preparation of silver and gold nanocomposite hydrogels prepared using condensation polymerization without initiator and crosslinker. Further, AuNps and AgNps introduced in hydrogel network resulted in nanocomposite hydrogels. The presence of gold and silver nanoparticles in polymeric hydrogels (ICD-Ag and ICD-Au) was confirmed by FT-IR and ED AX, whereas the size and spherical shape of silver and gold nanoparticles were confirmed by TEM analysis. ICD-Ag has shown higher swelling equilibrium in the acidic medium than ICD-Au, due to complexation of ICD-Au in hydrogel network.

Since the swelling equilibrium have good agreement with TGA results, ICD-Ag has higher thermal stability than ICD-Au. Based on the size of nanoparticles, the smaller size of ICD-Ag has higher antibacterial activity than ICD-Au. Nanocomposite hydrogels (ICD-Ag and ICD-Au) have low cytotoxicity due to more than 80% cell viability comparatively, ICD-Ag has good nontoxic nature. Finally, this work concluded cytotoxicity and antibacterial activity inversely related to each other. Nontoxic nature of this hydrogel applied for wound healing process using 3t3 fibroblast cell line resulted in 75% wound closure.


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G. Chitra, (1, 2) D.S. Franklin, (3) S. Sudarsan, (3) M. Sakthivel, (4) S. Guhanathan (iD)(5)

(1) Department of Chemistry, Periyar University, Salem, Tamilnadu 636011, India

(2) Department of Chemistry, Bangalore College of Engineering and Technology, Chandapura, Bangalore 560081, India

(3) Department of Chemistry, C. Abdul Hakeem College of Engineering and Technology, Melvisharam, Tamilnadu 632509, India

(4) Research and Development Centre, Bharathiar University, Coimbatore 641046, India

(5) PG and Research Department of Chemistry, Muthurangam Government Arts College, Vellore, Tamilnadu, 632002, India

Correspondence to: S. Guhanathan; e-mail(s):,

DOI 10.1002/pen.24824

Published online in Wiley Online Library (

Caption: SCHEME 1. Schematic representation of Nanocomposite hydrogels. [Color figure can be viewed at]

Caption: FIG. 1. FT-IR spectrum of (A) ICD, ICD-Ag and (B) ICD-Au. [Color figure can be viewed at]

Caption: FIG. 2. Thermogravimetric analysis of (A) ICD-Ag and (B) ICD-Au. [Color figure can be viewed at]

Caption: FIG. 3. SEM with EDAX images of (A) ICD-Ag and (B) ICD-Au. [Color figure can be viewed at]

Caption: FIG. 4. TEM images of (A) ICD-Ag and (B) ICD-Au. [Color figure can be viewed at]

Caption: FIG. 5. Swelling equilibrium studies of nanocomposite hydrogels. [Color figure can be viewed at]

Caption: FIG. 6. Antibacterial effect of ICD-Ag and ICD-Au. [Color figure can be viewed at]

Caption: FIG. 7. Cytotoxicity images of ICD. [Color figure can be viewed at]

Caption: FIG. 8. Cytotoxicity images of ICD-Ag. [Color figure can be viewed at]

Caption: FIG. 9. Cytotoxicity images of ICD-Au. [Color figure can be viewed at]

Caption: FIG. 10. MTT assay of ICD, ICD-Ag, and ICD-Au. [Color figure can be viewed at]

Caption: FIG. 11. Wound healing of ICD-Ag. [Color figure can be viewed at]

Caption: FIG. 12. Comparative wound healing of ICD, ICD-Ag, and ICD-Au.
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Author:Chitra, G.; Franklin, D.S.; Sudarsan, S.; Sakthivel, M.; Guhanathan, S.
Publication:Polymer Engineering and Science
Article Type:Report
Date:Dec 1, 2018
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