Lignosulfonic Acid Based Hydrogel Nanocomposites for Antimicrobial Application

Article history: Received on: 25/03/2016 Revised on: 04/04/2016 Accepted on: 17/06/2016 Available online: 29/10/2016 Hydrogels in which poly(acryl amide)/lignosulfonic acid (AAm/LSA) crosslinked with N,N’-methylene bis acrylamide, were prepared by using a redox polymerization technique. These hydrogel networks can act as excellent nano reactors for producing and stabilizing the metal nanoparticles. The current methodology allows us to entrap metal nanoparticles throughout hydrogel networks via AAm/ LSA chains. The synthesized silver nano composite hydrogels (Ag-NCHs) were fully characterized by using UV-visible spectroscopy; Fourier transform infra-red spectroscopy (FTIR), X-ray diffraction (XRD) studies, scanning electron microscopy (SEM), dynamic light scattering (DLS) analysis and Transmission electron microscopy (TEM). The chemical structure and crosslinking in AAm/LSA hydrogels and Ag-NCHs matrix was analyzed by FTIR. SEM and TEM measurements suggested the spherical structure of the silver particles with smooth surface and size of these particles confirmed by DLS. Crystalline nature of the nanoparticles was confirmed by XRD and electron diffraction measurements. Swelling studies were performed in water with the help of an empirical equation to investigate the diffusion mechanism. The developed Ag-NCHs were evaluated for preliminary antibacterial applications against E.coli and Bacillus.


INTRODUCTION
Hydrogels based on both natural and synthetic polymers are currently receiving a great deal of interest, notably due to potential applications in the controlled release of bioactive molecules and tissue engineering.Hydrogels which are polymeric materials that have hydrophilic three-dimensional networks composed of a polymer backbone, water and crosslinking agents, capable of absorbing large amounts of water or biological fluids (Victor et al., 2013;Galed et al., 2003).One of the advantages of the hydrogels is fabrication in their capability of undergoing first-order phase transition under the change of some external parameters such pH (Melendez-Ortiz et al., 2015), ionic strength (Paula et al., 2002), and temperature (Lorenzo et al., 2002).There are several possible mechanisms leading to in situ gel formation: solvent exchange, UV-irradiation, ionic cross linkage, pH change, and temperature modulation (Chaudhary et al., 2014).Generally, polymer nanocomposites are the result of the combination of polymers and inorganic/ organic fillers at the nanometer scale.The interaction between filler components of nanocomposites at nano dimension enables them to act as molecular bridges in the polymer matrix.This is the bases for enhanced mechanical (stiffness and toughness), thermal (flammability resistance), biological, magnetic, optical, electronic, and optoelectronic properties as compared with the corresponding inorganic or polymer component only (Chia-Liang Tsai et al., 2015;Hule et al., 2007).
Lignin is a macromolecular compound more chemically active than the cellulose, hemi cellulose and other natural polymers, due to its functional groups.Globally, lignin is regarded as an unrefined material with a high recovery potential, easy to get from the renewable sources, with low costs and an insignificant pollution degree (Totolin et al., 2010;Sekhar et al., 2014).
Lignin or lignosulfonates are waste discharged from the paper and pulp industries in bulky scale and poses enormous disposal problem (Zhang et al., 2001).Lignosulfonates are precipitated as calcium salts from spent process liquid, black liquid, from sulphite plants by means of lime, 90-95 % of the lignosulfonates is gained.
In the present study the acryl amide and lignosulfonic acid were crosslinked with MBA, it is found that the inclusion of a "ineffective" but abundant and renewable source such as lignin to acrylamide hydrogel networks through graft copolymerization can improve acrylamide hydrogels salt-resistance and impart hydrogels with high metal ion adsorption capacities.
Along with various synthetic hydrophilic polymers used in the preparation of hydrogels, acrylic monomers such as acrylamide (AAm), acrylic acid (AAc), and their derivatives, have been extensively used in the synthesis of hydrogels (Ramli et al., 2013;Maitra et al., 2014).Poly(acryl amide) based copolymers have found to be significant materials in agriculture, horticulture, adsorbents, biomedical and pharmaceutical fields.
It is widely accepted that the improved absorption capacity or absorption rate can be achieved for acrylamide polymers by incorporation of various hydrophilic units.The swelling property of these materials directly measures its function in environmental applications as an adsorbent and as biomaterial in medical, pharmaceutical, and biological applications.The optimized AAm-LSA hydrogels show good water absorbency and ability to high metal ion (Ag + ) adsorption.At the same time, a new way to consume lignin has been developed (Tiina et al., 2014).The antimicrobial property of silver is related to the amount of silver and the rate of its release.Silver in its metallic state is inert but it reacts with the moisture present in the skin and the fluid of the wound and gets ionized.The ionized silver is highly reactive, as it binds to tissue proteins and brings structural changes in the bacterial cell wall and nuclear membrane leading to cell distortion and death (Alexander et al., 2015).Silver also binds to bacterial DNA and RNA and by denaturing inhibits bacterial replication (Sudana Rao et al., 2013;Chopra 2007).

Preparation of AAm/LSA hydrogels:
Hydrogels were prepared by free radical redox polymerization.Briefly 1 gm of acrylamide was dissolved in 10 mL of double distilled water to this various amounts of LSA (0.1, 0.075, 0.6, 0 gm), N.N'-Methylenebisacrylamide (0.1, 0.125 gms), 0.01 gms of KPS followed by adding 1mL of 1% TEMED solution as represented in the scheme 1 and all formulation codes are fitted in Table 1.And then this solution mixture was kept in water both at 65 o C for 3 hours.These hydrogels were cooled at room temperature and washed with distilled water for removal of unreacted particles, dried at 40 o C in hot oven for 24 hours.

Preparation of Ag-NCHs
The dried hydrogels were equilibrated in double distilled water for 2 days.After the hydrogels were immersed into 50 mL of 2 gm/500 mL AgNO 3 solution and allowed to equilibrate for 1 day.The most of the Ag ions were occupied in the free spaces of hydrogel network through -CONH 2 , -OH, -COOH of hydrogel chains.The Ag ion loaded hydrogels were transferred in beaker containing 20 mL of (0.02 mole/100 mL) NaBH 4 solution and allowed for 12 h to reduce silver ions to silver nanoparticles as shown in the graphical abstract (scheme 2).The Ag-NCHs were dried for further studies.
Scheme 1. Schematic representation of formation of hydrogels.

CHARACTERIZATION UV-visible spectroscopic analysis
The synthesized silver nanoparticles by reducing silver metal ion solution with hydrogels and Ag-NCHs matrix were initially characterized by UV visible absorption spectroscopy.The samples were taken in a 1cm quartz cuvette and measured in a LAB INDIA, UV-3092 spectrophotometer containing double beam in identical compartments each for reference and test solution from 300 nm to 600 nm.

Fourier transform infrared spectroscopy
(Perkin Elmer Spectrum Two, UK) analysis was performed to identify the chemical structure and crosslinking in AAm/LSA hydrogels and Ag-NCHs matrix.Hydrogels were finely ground with spectroscopic grade KBr to prepare pellets using a hydraulic pressure of 600 kg/cm 2 to scan the spectra between 4000-400 cm -1 .

X-ray Diffraction studies
X-ray diffraction measurements were recorded using a Rigaku diffractometer (Cu radiation, λ = 0.1546 nm) running at 40 kV and 40 mA and were recorded in an angle of 5-60 °C at a speed rate of 5°/min to estimate the crystallinity of the sample.
X-RD study helps to find the crystallinity of drug in the Ag-NCHs.SEM (MERA\\TESCAN) images of silver nanocomposites were recorded using a JSM 6400 SEM (JEOL Ltd., Akishima, Tokyo, Japan) at X5 and X100 magnifications.
Working distance of 8.5-9.5 mm was maintained and the acceleration voltage used was 10 kV, with the secondary electron image (SEI) as a detector.Mean diameter and size circulation of the nanoparticles were determined by dynamic light scattering (DLS) method using a Brookhaven BI-9000 AT instrument (Brookhaven Instruments Corporation, USA).

Transmission electron microscopy
Samples of synthesized Ag-NCHs were prepared by placing a drop of the colloidal solution on carbon-coated copper grids, allowing the films on the TEM (HR-TEM, JEOL JEM-2010, Accelerating voltage of 200kV) grids to stand for two minutes, removing the excess solution with blotting paper, and letting the grid dry prior to measurement.

Swelling studies
Completely dried AAm/LSA hydrogel and AAm/LSA nanocomposite (Ag-NCHs) (~ 50 mg) were equilibrated in distilled water at 30 o C for 3 days.The equilibrium swelling capacity of AAm/LSA hydrogel and AAm/LSA nanocomposite was calculated following the equation: Where W 1 is weight of swollen hydrogel and W 2 is weight of weight of the dried hydrogel.

Antibacterial activity studies
Synthesized Ag-NCHs are tested for inhibition against the human pathogenic bacteria and antimicrobial activity assay are carried out by Kelman et al. disc diffusion method (Kelman et al., 2001).Mueller Hinton Agar (M173) plates were prepared for rapid growth of Bacillus subtilis (Gram positive) and Escherichia coli (Gram negative) bacteria for this study.The medium was sterilized at 121 o C and 15 lbs for 15 min in an autoclave and plates were equipped with a depth of about 4 mm.The cultures were transferred to the centre of an agar plate autonomously and spread evenly over the surface with a sterile bent-glass rod (purveyor).10μL of Ag-NCHs solution and was impregnated onto filter paper discs of 6 mm diameter, which were prepared by using Whatman grade No.1 Filter paper under aseptic conditions with different concentrations of Ag-NCHs solution (100, 150, 200, 250, 300 mg/mL), then positioned onto a cultured Mueller Hinton agar plates using a mechanical dispenser or sterile forceps.The plates were incubated for 18 to 20 hours at 37 o C in the incubation chamber.Antibacterial activity can be resolute by comparing the zone diameter obtained with the acknowledged zone diameter size for susceptibility (Tetracycline).

FTIR Spectroscopic analysis
Fig ( 1.A.):The spectra of FTIR demonstrate the changes signifying the successful crosslinking reaction within the hydrogel formation (Scheme 1).The spectrum of the starting lignosulfonates contains peaks at 3408 cm -1 due to -OH stretching in phenols; aromatic skeletal vibrations peak at 1600 cm -1 ; at 1451 cm -1 is due to -CH stretching of methyl or methylene groups.The peak at 1040 cm -1 is the result of -OH stretching of primary alcohols.Peaks at 1190 and 550 cm -1 are due to the -S=O stretching in sulfonic acids and -SO 2 scissoring, respectively.A sharp peak at 1666 cm -1 corresponds to conjugated C=O peak of amide group of acrylamide present in hydrogel network.The peak at 2930 cm -1 corresponds to the alkane C-H group in the LSA.The peak at 1117 cm -1 is the result of C-O stretching of primary alcohols.The peaks of alkyne groups are too weak to visible at 2139 cm -1 .Fig ( 1.B.):The spectrum of the AAm/LSA hydrogels was characterized by the presence of the absorption bands typical of the pure components and the silver loaded hydrogels.Due to the interaction of silver nanoparticles with the hydrogel networks, the vibrations are lesser in Ag-NCHs spectrum when compared to pristine AAm-LSA hydrogels.It showed that shifted frequencies of the of groups/bonds due to free O-H stretching (at 3467 cm -1 ), C=O stretching vibrations (around 1670 cm -1 ), 1118 cm -1 for C-O, 2935 cm -1 for alkane C-H, and 1452 cm -1 is due to -CH stretching of methyl or methylene groups.And S=O stretching vibration is shifted to 548 cm -1 .

Identification of silver ion in the hydrogel network
The surface plasmon resonance (SPR) absorption peak plays a major role in the determination of optical absorption spectra of metal nanoparticles that shifts to a longer wavelength with increase in particle size.The size of the nanoparticle implies that it has a large surface area to come in to contact with the bacterial cells; therefore, it will have higher percentage of interaction when compared to bigger particles (Agnihotri et al., 2014;Bondarenko et al., 2013).The formation of Ag-NCHs in the copolymeric hydrogel networks can be expected in our current strategy because the silver salts loaded hydrogels are readily reduced by AAm-LSA hydrogels, which immediately turn into dark brown colour.This change in colour shows that the Ag-NCHs were entrapped inside the networks through strong localization and stabilization established by the polymer chains.The existence of Ag-NCHs in the gel networks were estimated by UV-visible spectroscopy analysis.Fig 2 illustrates the absorption peaks of Ag-NCHs in 300-600 nm range that are assigned to silver nanoparticles which arise from the SPR absorption peak.A significant improvement in the absorption peak at λ max = 425 nm was observed for Ag loaded hydrogels due to the SPR.No additional absorption peaks around 300-600 nm indicates that AAm-LSA hydrogel network is protecting the nanoparticles from the aggregation or cluster formation.Among those formulations Ag-AAm-LSA2 is gave a high intense peak at 425 nm and remaining formulations shows around 425 nm.This analysis confirms the formation of highly dispersed Ag-NCHs in the hydrogels.

X-ray Diffraction studies (XRD):
To study the crystallographic nature of Ag-NCHs, XRD analysis was performed and XRD is shown in

SEM
Scanning electron microscopic photographs significantly showed that the smooth surface of the hydrogels and enhanced the mechanical properties for the absorption of metal ions.The crosslinked AAm-LSA hydrogels displayed in fig 4 (A-D) the average sizes of spheres is around 10 μm with spherical in shape and smooth surface.As shown by the Fig 4 results, the main advantage of the current technique is that it provided a controlled and uniform distribution of nanoparticles within the hydrogel network.

TEM and DLS
In Fig 5 TEM images of Ag-NCHs specifies that particle size, polydispersity, and location of the silver nanoparticles inside and outside of the hydrogel network.The particles are spherical in shape and constricted size distribution with an average diameter of around 25 nm (Fig 5A & 5B) without any agglomeration.In the case silver nanoparticles surrounded hydrogels showed highly dissemination of nano range silver particles in the hydrogel network.The high resolution image clearly showed that the crystalline nature of Ag-NCHs covered by the hydrogel polymer chain networks.This clearly signifies higher polymer gel networks favor for the Ag-NCHs formation because the feed composition permits the establishment of inter and intramolecular attractions between the hydrogel networks due to less free space in the hydrogel networks.This porosity ultimately helps to controlling the size of the nanoparticles and also provides better stabilization of silver nanoparticles for longer periods.The size of Ag-NCHs are also measured by DLS and the average size of the particles is 24.07 nm (Fig 5D).Size and shape of the Ag-NCHs are imperative for the nanoparticles release on the pathogenic bacteria (Acharya et al., 2009); as a result easy and effective control concluded the carrier size is of great significant.The electron diffraction pattern of Ag-NCHs is evidently visible in

Swelling Studies
In the present study, the swelling capacity of AAm/LSA composed in various formulations as shown in Fig 6A .This is due to presence of more hydrophilic groups such as -NH 2 , -COOH in the crosslinked polymer networks, which assist to improve the swelling.If the monomer content is increased the swelling of the hydrogels also increased due to the increase of hydrophilic groups in gel network (Fig 6B).
This pattern of swelling is reasonable for Ag-NCHs because once the Ag-NCHs are formed inside the polymer networks, the overall porosity of the system increases which allows for more number of water molecules inside the hydrogel network.The other reason may be that the formed nanoparticles have different sizes with different surface charges in the gel networks causing absolute expansion of the networks (Siraj et al., 2013).Increase of crosslinker concentration in the hydrogel synthesis leads to decreased swelling capacities of hydrogels (Fig 6C).The observed fall in swelling ratio is quite obvious because with increasing crosslinker concentration the crosslink density increases to an appreciable extent and does not favor penetration of water molecules into the network and, therefore, the swelling of hydrogel decreases.Another reason for the observed decrease in swelling ratio is quite expected as increase in crosslinker makes the hydrogel more and more compact (Seddiki et al., 2013).

Biological Activity
Composed Ag-NCHs were screened for their antibacterial activity.All these hydrogel composites were possessed variable antibacterial activity against both Grampositive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial activity against Tetracycline.Positive and negative controls produced significantly sized inhibition zones against the tested bacteria.On the basis of maximum inhibitory activity shown against Gram positive-bacteria, Ag-NCHs-2 were found to be most effective against Gram-negative with zone of inhibition fitted in Table 2 with respect to the following concentrations (100, 150, 200, 250, 300 mg/mL).Maximum zone of inhibition 18.14 mm for 250 mg/mL (Fig 7) was found in Escherichia coli and transitional activity was revealed against Gram-positive.The presence of the porous hydrogel accelerates frequent diffusion of liquid media across the hydrogel which could release silver nanoparticles into it (Vimala et al., 2011).The inhibition zone diameter that is produced will specify the susceptibility or resistance of a bacterium to the silver loaded hydrogels.Ag-NCHs retain the chemical stability and appropriate smaller size which make them relaxed to interact with pathogenic bacteria (Rai et al., 2009).
The enhanced activity may be due to the Ag-NCHs particles were smaller size in nano size, enlarged surface area and high probing energy and the positively charged hydrogen on the hydroxyl group of polymer chain has been the potential to associate with the negatively charged phosphates on cell wall of the tested bacteria (Chapot-Chartier et al., 2014;En Huang et al., 2014).Therefore, such nanosized metal particles could effectively bind the substrate on the cell membrane of the microorganisms.

Fig 2 .
Fig 2.UV-visible spectra from 300-600 nm for all formulations of silver nano composite hydrogels.
Fig 3. Plain hydrogels have not exhibited any sharp peaks in XRD.A broad peak at 25 o is due to the polymer network.In the case of Ag-NCHs, sharp peaks are observed at 2θ = 22 o , 28 o , 31 o , 33 o , 37 o , and 47 o .The precise X-ray diffraction patterns indicate the formation of highly crystalline silver nanoparticles (Balaji et al., 2009).The results also indicate that the prepared nanoparticles are face centered cubic structure (Vasireddy et al.,2012) further conformed by electron diffraction pattern of TEM as shown in Fig 5C.
Fig 6D represents the effect of silver ion concentration on the swelling behaviour of the Ag-NCHs.In this study loading of Ag + ions leads to gradually decrease the swelling capacities of hydrogels due to the voids of hydrogel networks are occupied by silver ions.

Table 1 :
Feed compositions for different formulations and swelling kinetic parameters.
Scheme 2. Graphical abstract of formation of SNCHs.

Table 2 :
Diameter zone of inhibition (in mm) by SNCHs-2 against human pathogenic bacteria.