Biosynthesis of Nanocomposites Using Leaf Extract

ABSTRACT

Various nanoparticles and nanocomposites have been synthesized using leaf extract and evaluated for their antibacterial activity. This review intends to present biosynthesis of nanocomposites using leaf extract. Here, I have discussed biosynthesis methods of polymer nanocomposites using leaf extract. The potential of nanotechnology and biological science together is enormous. There are many potential antibacterial applications of nanocomposites such as in antimicrobial textiles, food preservation, surface disinfection, burn dressings, safe cosmetics, medical devices, drug carriers, dental fillers and adhesives, water treatment etc. In recent years nanocomposite films have been studied for wound dressing. I have discussed a bionanocomposite film and hydrogel that have application in wound dressing.

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CHAPTER 1: INTRODUCTION
1.1 Nanotechnology

Nanotechnology is a branch of science and technology that deals with matter of size 1-100nm. Since then there has been lot of advancement in nanotechnology. Nanotechnology has applications in almost every field such as electronics, medicine, biomaterials, energy production etc. When a macroscopic material changes to nanomaterials, properties such as electrical, mechanical, optical, catalytic, medicinal, biological etc change. Gold which does not react with other chemicals easily at normal scales acts as a good catalyst when converted to nanoscale.

1.2 Nanoscale materials

Nanoscale materials include materials which posses at least one dimension in the nanometer range i.e. 1-100nm. The key characteristics defining the potential applications of nanoscale materials include the following:

Higher surface area
Higher chemical reactivity
Better catalytic properties
Better adsorption
Variety of chemical synthesis routes.
Natural and synthetic strategies
1.3 Nanocomposites

Nanocomposites are materials made from two or more individual components with properties different from each other, which when combined produce a material with properties completely different from the individual materials. A nanocomposite consists of two or more phases where one phase is monolithic (single crystal) into which the reinforcement are embedded. The monolithic material is known as a matrix. Reinforcement, in a nanocomposite is a nanosized materials embedded into the parent material called matrix. Nanocomposites are broadly classified into three types

a) ceramic-matrix nanocomposites

b) metal-matrix nanocomposites

c) polymer-matrix nanocomposites

d) inter metallic matrix nanocomposites.

CHAPTER 2: BIOSYNTHESIS OF NANOCOMPOSITE USING LEAF EXTRACT

Nanoparticles are being used in many sectors of the economy and it is important to consider the biological and environmental safety of their production. The main methods for nanoparticle synthesis are chemical and physical approaches and these approaches are often expensive and potentially harmful to the environment. Green synthesis approach has been pursued in recent years as an alternative, inexpensive, efficient, and environmentally safe method for synthesizing nanoparticles with specific properties. The main focus is on the role of the natural plant (leaf) extracts involved in the bioreduction and capping of metal salts during the nanocomposite synthesis. Many researchers have reported the biosynthesis of nanoparticles by leaf extracts and their potential applications in various fields.

2.1 Commonly used leaf extracts for synthesis of nanocomposites
Binomial name: Murraya koenigii

Common Name: Curry Tree

Family Name: Rutaceae

Description: Curry tree is native to India and Sri Lanka. The leaves ofMurraya koenigiiare used as an herb in Ayurvedic medicine because of its antioxidant, anti-diabetic, antimicrobial, anti-inflammatory, anti- hypercholesterolemic properties. Curry leaves recently been found to be as a potent antioxidant due to high concentrations of carbazoles, a water soluble heterocyclic compound. Carbazoles found in leaf extract may be responsible for the reduction and stabilization of metal ions. Further research is necessary to explain and extend the reduction mechanism of Murraya koenigii leaf extract for further application.

Binomial Name: Tridax procumbens

Common Name: Coat buttons or Tridax Daisy

Family Name: Asteraceae

Description: Tridax procumbens is a widespread weed and pest plant native to America. The plant has various medicinal properties. Tridax procumbens is rich in alkaloids, flavanoids, carotenoids and tannins. It is used in nanoparticle synthesis as it has high amounts of ketones, amines, phenols, lactones and alkanes which are capable of reducing metal ions.

Binomial Name : Ficus benghalensis

Common Name: Banyan Tree

Family: Moraceae

Description: Banyan tree is a deciduous tree found throughout the forest tract of India, in sub-Himalayan region. Ficus benghalensis is widely used for its medicinal properties. Ficus benghalensis leaf extract has proteins/enzymes which reduce the metal ions and it also contains reducing sugars such as flavanones which provide stability to the nanoparticles.

Binomial Name: Calotropis gigantean

Common Name: Crown Flower

Family Name: Apocynaceae

Description: Calotropis gigantean is a large shrub rich in metabolites responsible for reduction metal ions. Organic compounds like alkaloids, polyphenols, and proteins present in plant extracts are capable of reducing and capping nanoparticles.

Binomial Name: Catharanthus roseus

Common Name: Madagascar periwinkle

Family Name: Apocynaceae

Description: Catharanthus roseus is a medicinal subshurb. Catharanthus roseus contains more than 70 alkaloids.

2.3 Nanocomposites synthesized using leaf extract

A broad spectrum of leaf extracts can be utilized for the biosynthesis of nanoparticles. In this section I have briefly discussed synthesis of two polymer-matrix nanocomposites using leaf extract. In both the examples, reinforcement is silver nanoparticle and the matrix of the nanocomposite is a polymer.

2.3.1 Ag impregnated Microcrystalline Cellulose Bionanocomposite film

Silver nanoparticles are impregnated into microcrystalline cellulose to form a nanocomposite. Curry leaf extract is used for the bioreduction and capping of silver nanoparticles. First 0.001M silver nitrate solution is prepared in 1000 mL of deionised water. 10 g of microcrystalline cellulose is added to the silver nitrate solution and sonicated for 10 minutes. 50 mL of curry leaf broth is added to the mixture and the mixture is stirred for 6 hours. Silver ions are reduced to silver by curry leaf extract. Reduced silver nucleates in to the silver nanoparticles on the microcrystalline fibrils. After 6 hours the mixture is allowed to settle down and excess reaction mixture is decanted. The silver nanoparticles impregnated microcrystalline cellulose is washed with deionized water and ethanol and then dried in oven at 55EsC over night.

The formation of silver nanoparticles is confirmed by UV-vis spectra as the peak is observed at 430 nm. The colour of microcrystalline cellulose is white and after impregnation of silver on it, it changes to yellowish brown.

0.5 g of polylactic acid is dissolved in 20 mL of chloroform with moderate heating and constant stirring for 30 minutes. The dried silver nanoparticle coated microcrystalline powder is added in 5%, 10% and 20% w/w concentration to separate samples. The polylactic acid is stirred with silver impregnated microcrystalline cellulose for a day to allow for dispersion. The mixture is poured to glass Petri dish and left to evaporate. When the chloroform evaporates, the plastic film is removed and collected from Petri dish. Silver impregnated microcrystalline bionanocomposite film is obtained. [6]

2.3.2 Silver/Starch-co-polyacrylamide hydrogel nanocomposite

Gelatinized starch solution is prepared by mixing a known amount of starch powder in 10 mL of deionized water and 1 mL of 0.5 mL of 0.5 M sodium hydroxide solution. The mixture is heated at 90EsC for 10 minutes in a water bath with continuous stirring. A predetermined amount of maleic acid is then added to the gelatinized starch solution. The mixture of gelatinized starch and maleic acid is further heated at 80EsC in a water bath for 4 hours. Then acrylamide is added and stirred for 30 minutes at 50EsC. After that initiator (potassium persulfate or KPS) and crosslinker (methylenebisacrylamide or MBA) is added. Finally, an aqueous solution of tetramethylenediamide (TEMED) is added to the solution and for another 10 minutes same temperature is maintained. The synthesized co-polymeric hydrogel is taken out after the completion of free radical polymerization. Then the synthesized co-polymeric hydrogel is immersed in double distilled water at room temperature for a day to remove excess of unreacted reagents and monomers present in hydrogel network. To remove the residue effectively the double distilled water is refreshed for every 12 hours. At last the hydrogel is dried at ambient temperature for 48 hours.

Precisely weighed dried starch-co-polyacrylamide hydrogel is equilibrated with double distilled water for 48 hours and instantly transferred to a beaker containing 100 mL of 0.005 M silver nitrate solution and then equilibrated for 24 hours. During this process the silver ions are exchanged from solution into free network spaces of co-polymeric hydrogel. To a beaker containing 50 ml Tridax procumbens leaf extract, hydrogel with absorbed silver ions is added and kept for 24 hours. Reduction of silver ions into silver nanoparticles occurs and hydrogel turns into brown colour. The brown colour confirms the formation of silver nanoparticles in hydrogel matrix. [7]

CHAPTER 3: ANTIBACTERIAL ACTIVITY OF NANOCOMPOSITES SYNTHESIZED USING LEAF EXTRACT

Due to the rising concerns of bacterial infections, there is a growing need to develop new and powerful antibacterial agents. Mainly, nanoparticles have been applied in burn dressings, cosmetics, food preservation, medical devices, water treatment etc. There is a wide bioapplication of nanoparticles. It has been recognized that the bactericidal effect of nanoparticles is dependent on their size, size distribution, shape, morphology, surface functionalization, and their stability. Additionally, the use of inorganic nanoparticles as antimicrobial agents has numerous benefits such as enhanced stability and safety in contrast with the organic antimicrobial agents. Green synthesis and functionalization of nanoparticles enhances their antibacterial activity and improves their stability.

In this section antibacterial activity of silver/polymer film and hydrogel is discussed. So the antibacterial activity of nanocomposites is enhanced.

3.1 Antibacterial activity of Ag impregnated Microcrystalline Cellulose Bionanocomposite film

The PLA/MCC sample was tested for antimicrobial activity using Charm disk assay. Firstly an agar plate was seeded with Bacillus stearothermophilus. Then small circular pieces of the films were placed on the seeded agar and incubated. Indicators are present in agar which signifies the status of microbial growth. Yellow colour indicates the microbial growth and purple indicates the inhibition. The initial analysis shows that the film exhibits considerable antibacterial properties. [6]

3.2 Antibacterial activity of Silver/Starch-co-polyacrylamide hydrogel nanocomposite

The antibacterial activity of SNCH was evaluated by disc diffusion technique against gram-positive and gram-negative bacteria such as Bacillus and Escherichia coli. Firstly, nutrient agar medium was prepared by mixing beef extract (3 g), peptone (5 g) and sodium chloride (5 g) in 1000 mL distilled water. The pH of the medium was adjusted to 7. Finally agar (15 g) was added to the prepared solution and then medium was sterilized in an autoclave at a pressure of 15 lbs for 30 minutes at 121EsC. This medium was then transferred into a sterilized glass Petri dish in a laminar air flow chamber. After the media solidified, Escherichia coli and Bacillus culture (50???‡L) was spread on the solid surface of the media. Paper discs (6mm diameter) were soaked in the test compounds (20mg/20mL) overnight. Then these discs were loaded on culture plates. The plates were incubated for 24 hours at 37a??C. The inhibition zone appears around the disc which shows the antibacterial effect of SNCH. [7]

Pure hydrogels are generally inefficient for antibacterial activity. It is seen that smaller the size of silver nanoparticle greater is the antibacterial activity. .The SNCH having low silver nanoparticles concentration still showed excellent antibacterial activity against gram-positive and gram-negative bactericide. This results into inhibition of bacterial cell growth. So SNCH nanocomposites can be used as successful antibacterial agents such as wound-dressing materials. [7]

Modern wound dressing theory, suggests promoting dynamic equilibrium between exudate absorption and optimal surface moisture at the wound surface. In addition, it should be able to exchange gas to provide the wound with adequate oxygen tension.

3.3 Mechanism of antibacterial activity of nanoparticles

Antibacterial activity is a property due to which compounds are capable to kill or slow down the bacterial growth, without causing toxicity to host cells. Such agents are classified as a) bactericidal, which kill bacteria, 2) bacteriostatic, which slow down the bacterial growth. The exact mechanism of nanoparticle toxicity against various types of bacteria is not completely evaluated yet. It is proposed that nanoparticles attach themselves to bacterial membrane by electrostatic interaction and disrupt its integrity. Nanotoxicity is triggered by the initiation of oxidative stress by free radical formation, i.e. ROS, followed by the administration of nanoparticles. The nanoparticle toxicity depends on composition, intrinsic properties, surface modification of the bacterial species and the physical and chemical properties of nanoparticles, indicating the mechanisms to be highly complex.

The antibacterial mechanisms of nanomaterials is not fully elucidated, but the existing concept suggests various combinations of processes that can occur (1) ions are released which is followed by cellular uptake and a cascade of intracellular reactions, (2) extracellular and intracellular generation of ROS and (3) direct interactions between nanoparticles and cell membrane. At sub-micromolar concentrations, ions are internalized and they react with the thiol groups of cellular proteins, which lead to uncoupling of ATP synthesis from respiration, loss of proton motive force, and interference with the phosphate efflux system. At millimolar levels, nanoparticles induce detachment of the cell wall from the cytoplasm, possibly releasing the intracellular content, DNA condensation and loss of replication ability. ROS produces oxidative stress which results in lipid membrane and DNA damage. Finally, nanoparticles increase the cell membrane permeability and, subsequently, penetrate inside cells to induce any one or the entire cascade of effects mentioned above. [9]

CONCLUSION

The most important objective of nanobioscience involves application of nanotools to relevant biological and medical problems and refining these applications. The use of microorganisms and plants for synthesis of metal nanoparticles is of great interest. In contrast to chemical and physical synthesis methods, biological processes for synthesizing nanomaterials can be achieved in aqueous phase in gentle and environment friendly conditions. This approach has become attractive focus in current green nanotechnology. With the help of this approach we can synthesize nanomaterials in less toxic way as it replaces toxic chemical reducing and capping agents. Inorganic nanoparticles naturally possess bacteria-killing properties, but by modifying the inorganic nanoparticles i.e. forming nanocomposites, these properties can be enhanced. In the biomedical field, a synthesis of nanocomposite films and hydrogels by a green process was developed to enhance the inactivation of bacteria in wounds. There are also other potential antibacterial applications of nanocomposites such as in antimicrobial textiles, food preservation, surface disinfection, burn dressings, safe cosmetics, medical devices, drug carriers, dental fillers and adhesives, water treatment etc. Further research on nanocomposites capable of antibacterial activity is necessary for large scale commercial application

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