Preparation of Chitosan-Silver Nanoparticles in Nonaqueous Medium under Heating

Chitosan-silver nanoparticles are prepared in nonaqueous medium. In this work, sodium dodecyl sulfate (SDS) was introduced into the dimethylformamide (DMF) solution during silver reduction from solution of its precursor salt AgNO 3, acting as a stabilizing agent to prevent aggregation of silver nanoparticles, while chitosan is used as the solid support to embedded silver particles therein, resulting in chitosan-silver (CS-Ag) nanoparticle as suspension in the medium. The reaction started as homogeneous system which turned into heterogeneous with the formation of particles. The properties of CS-Ag nanoparticles are studied under two different salt concentrations and characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR) and ultraviolet-visible spectroscopy (UV-Vis). Wide particle size distribution of synthesized nanoparticles depicts that concentration of AgNO 3 , which is responsible for the morphology, stability and particle size distribution, should be optimized, suggesting a lower salt concentration is favorable.


INTRODUCTION
There has been a tremendous increase in the number of patients affected by diseases like diabetes and cancer during the last decade. So, it has become the major focus of researchers to synthesize a compound that will be useful for the monitoring of certain elements that are present in excess quantities in the blood while used in the form of biosensors, at the same time being useful for the treatment of diseases like cancer. Chitosan (CS) is a polysaccharide composed of glucosamine and N-acetyl glucosamine linked with a β, 1-4-glycosidic linkage [1]. Chitosan is a biopolymer which is biocompatible and can be degraded by enzymes in human body, the degradation products are nontoxic. Commercial Chitosans are semi-crystalline polymers and crystallinity plays an important role in adsorption efficiency [2]. Chitosan is a polymer which exhibits a broad-spectrum of antimicrobial activity by binding to the negatively charged bacterial cell wall followed by attachment to the DNA, inhibiting its replication [3,4]. For the improvement of bioactivity on chitosan, it is often combined to other bioactive materials, such as drugs.
Silver (Ag) nanoparticles have high therapeutic potential and exhibit good antimicrobial activity. Ag nanoparticles have a wide range of antimicrobial activities and exhibit high performance even at a very low concentration. Ag nanoparticles have been identified to possess good potential for the treatment of cancer [5]. But the major disadvantage of using silver alone is that it is not specific at targeting the cancer cells and also it is toxic to the normal cells when exposed for a longer time when the size of silver is used >20 nm [6], CS-Ag nanocomposite is one of the rare composite materials that is seen to possess a capability of being used as a biosensor as well as in the treatment of cancer as the chitosan present in the nanocomposite is very specific to the cancer cells. It prolongs the action of silver on the affected cells while preventing the normal cell from the effect of silver. One more advantage of this nanocomposite is that it is biodegradable i.e., it can be degraded by the enzymes present in the body making it suitable for the treatment of cancer. Apart from the treatment of cancer, the nanocomposite also possesses good antimicrobial [7] and biosensing activity.
Sodium dodecyl sulfate (SDS) is introduced into the amide solution at the start of reaction or continuously during metal reduction [8]. SDS plays a twofold role it acts as a stabilizing agent to prevent aggregation of metal nanoparticles. In addition, SDS can be adsorbed on the surface of specific crystal faces and serve as shape control agent. In this work, we proposed the synthesis method by reducing AgNO 3 in dimethylformamide (DMF) with chitosan by varying concentration for the preparation of silver nanoparticles. The influence of the salt concentration on the optical properties, structures and morphologies of CS-Ag nanoparticles was characterized by the atomic force microscopy (AFM), ultraviolet-visible spectroscopy (UV-Vis), scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR).

Materials
Prawn shell was collected from Rupsa ferry ghat, Rupsa, Khulna, Bangladesh in Figure 1. Sodium hydroxide pellet, hydrochloric acid and absolute alcohol were obtained from Merc India, acetone was obtained from Merck Germany, potassium bromide was obtained from Sigma ultra USA, DMF from Fisher scientific UK, SDS from Fulka analytical Switzerland, and silver nitrate form Uni-Chem China.

Extraction of chitin from prawn shell
Collected prawn shell was washed with hot water and then dried in an oven at 105 o C for 72 hours. Dried prawn shell was grounded. Deproteinized was carried out with 1N NaOH at boiling temperature for 4 hours in prawn shell:NaOH ratio of 1:16 w/v, followed by neutralization. Residue was demineralized with 1N HCl at boiling temperature for 4 hours in material:HCl ratio of 1:13 w/v. Excess acid was neutralized [9].

Conversion of chitin into chitosan
Deacetylation of chitin was carried out using 40% NaOH (Chitin:NaOH = 1:20, w/w) at boiling temperature for 3.5 hours. After deacetylation solid was separated from alkali and extensively washed with distilled water to remove traces of alkali. Resultant solid was dried in a vacuum oven at 60 o C for 24 hours. The material obtained in this step is known as chitosan. Chitosan was grounded and placed in a desiccator for storage [9].

ILCPA Volume 58
Preparation of chitosan-silver nanoparticles 0.05g of chitosan was dissolved in 20 mL 10N HCl solution and used as stock solution. 5 mL stock solution, 5 mL DMF and 0.05g of SDS were mixed for 30 min using magnetic stirrer to get homogeneous solution. 4.90 mM silver nitrate salt was added in 10 mL DMF and mixed for 30 min using magnetic stirrer to get homogeneous solution. The total volume of solution was 20 mL and final concentration of AgNO 3 was 2.45 mM and these placed in a three neck round bottom flask. Out of three neck of the round bottom flask, one neck was kept for solution upload, the other neck was used for thermometer and the middle one was used for condenser setup. When the effect of concentration of Ag precursor salts was examined, the concentration of AgNO 3 was changed from 2.45 mM to 5 mM for the preparation of CS-Ag nanoparticles. The Ag/SDS molar ratio was kept at the same values as those used at initial condition. The solution temperature was increased from room temperature to 120 o C by heating for 10 min.

AFM observation
Freshly prepared nanoparticles were centrifuged with ethanol, followed by casting onto glass slide for film and subsequent dried in open air dry at room temperature.
International Letters of Chemistry, Physics and Astronomy Vol. 58 3

Effect of precursor salt concentration
The effect of precursor salt concentrations is examined by changing silver nitrate concentrations from 2.45 mM to 5 mM to prepare CS-Ag particles. The molar ratio of Ag salt and SDS was kept constant for the preparation of CS-Ag particles in reaction medium.   Figure 4b shows a mixture of CS and Ag wherein the silver nanoparticles are seen to be enveloped by the chitosan polymer indicated in Figure 4b 10 . In case of 5 mM concentration, the synthesized particles are in the form of aggregates or larger in size than that of 2.45 mM CS-Ag particles in Figure 4c. It is observed that the silver nanoparticles are embedded in a matrix of chitosan. The morphology of the silver nanoparticles was predominantly nearly spherical or plate like in Figure 4c and aggregated into larger irregular structure with no well-defined morphology observed in the micrograph of Figure 4d under heating or storage.  . KBr diluted nanoparticles are washed with alcohol and centrifuged to collect particles, subjected to give the FTIR spectra of chitosan and CS-Ag nanoparticles are depicted in Figure 5. The band at 3411 and 3467 cm -1 correspond to O-H stretching H-bonded alcohols and phenols of CS-Ag nanoparticles and chitosan itself respectively. The peak at 1666 and 1622 cm -1 correspond to N-H bond primary amines of CS-Ag nanoparticles and chitosan itself respectively. The peak at 2835 and 2831 cm -1 correspond to O-H stretch carboxylic acids of CS-Ag nanoparticles and chitosan itself respectively. The peak at 1402 and 1383 cm -1 correspond to C-N stretching of aromatic amine group of CS-Ag nanoparticles and chitosan itself respectively. The other bands are of C-O stretch alcohols and carboxylic acids observed at characteristics spectral shifts. Therefore the synthesized CS-Ag nanoparticles are surrounded by chitosan having functional groups of amines, carboxylic acids, alcohols and esters.

UV-Visible spectroscopy analysis
The UV-Visible spectral measurements were carried out using UV-Visible spectrophotometer (Shidmadzu, UV-1650PC, Japan). UV-Visible absorption spectra recorded is quite sensitive to the formation of silver nanoparticles because of the fact that silver nanoparticles exhibit an intense absorption peak due to surface plasmon resonance (SPR). Figure 6 shows the UV-Vis spectra of silver nanoparticles prepared from two different concentrations of silver nitrate (2.45 mM, and 5 mM) with chitosan (5 mL) for 10 min heating. All spectra exhibit an absorption band in the range of 300-400 nm with a typical plasmon resonance band of CS-Ag nanoparticles. The absorption against wavelength curves at various concentrations is shown in Figure 6. It was noticed that the reduction capacity of chitosan increased with varying concentration of Ag salt. As the salt concentration increases, possibly more and more of hydroxyl groups are converted to carbonyl groups by air oxidation, which in turn reduces more silver ions at a constant chitosan concentration. A single strong peak with a maximum around 295 nm was observed in the UV-Vis spectra, which corresponds to typical SPR of conducting electrons from the surface of silver nanoparticles. The intensity of the absorption of solutions increased with increase in the concentration of AgNO 3 up to 5 mM at constant heating time. Ag nanoparticles with nearly spherical morphology are embedded in the chitosan matrix which arises a small peak at around 380 nm in Figure 6. As the particles increase in size, the absorption peak usually shifts toward the red wavelength [11].

Mechanism
The growth mechanisms of CS-Ag nanostructures under different preparation routes are summarized in Figure 7. In this process a large amount of CS-Ag nanoparticles are prepared. Mechanism in Figure 7 is the grain-rotation induced grain coalescence (GRIGC) and fusion mechanism 12 . According to this model, the rotation of grains among neighboring grains results in a coherent grain grain interface which leads to the coalescence of neighboring grains via the elimination of common grain boundaries, thus forming a larger grain. In our case, single crystal, particles having well defined faces were lost after the formation of large polycrystalline spherical particles. Thus, during GRIGC process fusion of polygonal crystals leading to polycrystalline spherical or plate like particles. In the Ostwald-ripening mechanism, the atoms from one particle undergo dissolution and then they are transferred to another particle. There is a net atomic transport from the particles with sizes smaller than the average value to larger particles. Particles smaller than the average value will shrink or even disappear.

CONCLUSION
Silver nanoparticles were successfully introduced into chitosan using non aqueous thermal reduction method. This was confirmed by the maximum surface resonance peak at 295 nm. Topographical heights of the CS-Ag nanoparticles were revealed to be around 295 nm for most of the particles made from 2.45 mM and 5 mM silver nitrate salt. In SEM micrograph nanoparticles with nearly spherical or plate like morphology are embedded in the chitosan matrix which is arise a small peak at around 380 nm. From FTIR spectra it was revealed that the synthesized nanoparticles are surrounded by chitosans having functional groups of amines, carboxylic acids, alcohols and esters. Wide particle size distribution of nanoparticles synthesized here depicts that the parameter, concentration of AgNO 3 , which is responsible for the morphology and stability, should be optimized, suggesting lower salt concentration is favorable.