FIELD OF THE INVENTION

The present invention relates to biological synthesis of Silver nanoparticles by modifying Escherichia coll Besides, the invention also relates to a composition comprising Silver nano particle and method of producing the same.

BACKGROUND OF THE INVENTION

Nanoparticles are considered as the fundamental building blocks of nanotechnology and are considered as the starting materials for subsequent structures in nanotechnology. Solid phase nanoparticles, especially metal nanoparticles have recognized importance in chemical, physical and biological sciences because of their unique properties and applications in wide fields.

Silver nanoparticles (SNPs) are considered to be more effective than silver. The Silver nanoparticles (SNPs) have extensive applications in civil, therapeutic and industrial areas as catalyst, cryogenic superconductor, biosensor, microelectronic and bacteriostatic materials and several other applications. Moreover, Silver nanoparticles have other advantages over silver. It has been observed that silver in ionic form has more harmful effect on skin such as Argyria (discoloration of skin) as compared to non ionic form. Non ionic form of Silver converts into ionic form in a time related mechanism. As compared to silver, nano Silver particles takes more time to convert into ionic form thus delays or does not cause side effects such as Argyria. Also, slower release of silver ions from silver nanoparticles can avoid delivery of excess amount of silver in the predetermined area as compare to the other silver ion based compounds. Besides, the nanoscale size of the nanoparticle implies more surface area, and therefore, requires lower amounts to be effective.

Nano particle are produced by several synthetic techniques that usually employ atomic, molecular, and particulate processing in a vacuum or in a liquid medium (Daniel and Astruc 2004; Yonezawa and Toshima 1995; Link et al. 1999; Silvert et al. 1996; Mizukoshi et al. 1997; Vinodgopalet al. 2006; Anandan et al. 2008; Treguer et al. 1998;Hodak et al. 2000; Chen and Yeh 2001; Mandal et al.2006). The methods that are generally involved in the preparation of silver nanoparticles in prior art include reduction of metal salts with a chemical reducing agent, such as sodium citrate, sodium borohydrate or ethylene glycol (Plyuto et al., 1999; Rivas et al, 2001; Zang et al., 2000). Other methods such as chemical vapor deposition and irradiation are also used. Most of these techniques are capital intensive, inefficient in material use, involve high energy use and are harmful to the environment. Also, conventional synthesis of silver nanoparticles incorporates contaminants that could pose problems in biomedical applications. Hence, there is a need to develop clean, nontoxic, and environmentally friendly and safe process for synthesis of silver nanoparticle size.

Certain prior art process include biosynthesis of silver nanoparticle from microbes such as prokaryotic bacteria. However on the contrary, Silver being a wide range antimicrobial agent (Slawson et al. 1992a, b) is highly toxic against most microbial cells and presented several difficulties in biological synthesis. Certain silver resistant strains such as Pseudomonas stutzeri AG259 have been used in the prior art, but the cultures and maintenance of these strains pose several difficulties and were not fond to be practical.
Hence there is a need for an efficient process and a novel strain for the synthesis of silver nanoparticles.

OBJECT OF THE INVENTION

An object of the invention is to biosynthetically produce silver nanopartices particle in cost effective and environment friendly manner

SUMMARY OF THE INVENTION

The present invention relates to a method of biological synthesis of nano silver particles in cost effective and environment friendly manner. The method disclosed in the present invention employs the use of mutated Escherichia coli (E. coli) for the production of silver nanoparticle. The mutated Escherichia coli exhibit amplified synthesis of silver nanoparticle. The method of synthesizing nanoparticles of silver according to the present invention comprises the following step:

1) mutating E. coli bacterial cells;

2) screening mutated E. coli exhibiting enhanced nitrate reductase activity;

3) culturing screened E. coli; and

4) exposing the cultured cells to a solution of silver nitrate under ambient conditions
37 °C in which bacterial cells produce enzymes and metabolites that reduce silver ions to silver nanoparticles.

5) obtaining silver nano particles.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention discloses a method of biosynthesis of silver nanoparticle in cost effective and environment friendly manner. Wild type Escherichia coli, are highly sensitive to silver metal however, Escherichia coli is the most studied and extensively investigated bacteria is well suited for being employed in the production of silver nanoparticle. The present invention uses the Escherichia coli for synthesis of silver nanoparticle by chemically mutating it. The modified Escherichia coli secretes the enzyme when exposed to stress-full environmental conditions such as exposure to toxic materials (eg: metallic ions) predators and temperature variations. The secretion of enzyme serves as agents for their own survival. The present invention is based, in part, on the surprising finding that on mutating E coli, the Escherichia coli not only becomes resistant to silver ion but it also exhibits amplified synthesis of silver nanoparticle.

The method of synthesizing nanoparticles of silver according to the present invention comprises the following step:

1) mutating Escherichia coli bacterial cells;

2) screening mutated Escherichia coli exhibiting enhanced nitrate reductase activity;

3) culturing screened Escherichia coli; and

4) exposing the cultured cells to a solution of silver nitrate under ambient conditions 37 °C in which bacterial cells produce enzymes and metabolites that reduce silver ions to silver nanoparticles.

5) obtaining silver nanoparticle.

The mutagenesis of the Escherichia coli may be carried out by chemical, physical means, preferably by chemical means.

The chemical mutagenesis of Escherichia coli for the development of desired E. coli mutant strain may be carried out with nitrous acid (HNO2), ethylnitrosourea, Nitrosamines, vinyl chloride Polycyclic aromatic hydrocarbon preferably the mutagen is nitrous acid.

The method of mutagenesis of Escherichia coli may comprise the following steps:

1. subculturing of fresh overnight wild type E. coli culture to a desired OD.

2. washing and spinning the cells;

3. resuspensding the cell pellet in mutagen containing solution;

4. incubating the cells at room temperature;

5. washing the cells with stop buffer, in order to stop the mutation;

6. plating the washed cell;

7. Screening Escherichia coli cell for higher synthesis of nitrate reductase;

8. Confirming the type of mutant in the gene encoding nitrate reductase enzyme by
DNA sequencing.

The Wild type E. coli culture may be sub-cultured in LB, TB and SOC medium.

Preferably the E. coli may be sub cultured in 5 ml of LB medium.

The washing of the cells in step 2 may be carried out with sodium acetate buffer (pH 4.3) that favors the removal of cell debris without affecting the production of enzyme Nitrate reductase. Sodium citrate buffer (pH 3.5) may also be used. Preferably the buffer is sodium acetate buffer (pH 4.3)

The strength of the buffer may be from 75 mM to 200 mM, preferably strength of buffer is100mM.

The mutagen in step 3 may be nitrous, sulphurous, acetic or citric, preferably the mutagen is nitrous acid.

The cells in step 4 may be incubated for a period of 5 to 50 mins, preferably for 10 to 30 min, more preferably the period of incubation is 10 min.

Screening of the mutant E. coli that produces higher nitrate reductase enzyme may be carried out by the nitrate reductase assay.

The mutation in the gene encoding nitrate reductase enzyme is checked by DNA sequencing. This step is important to confirm that the mutation is in the ds-acting elements but not in the nitrate reductase protein coding region.

The E. coli mutants which showed the 5 fold enhanced nitrate reductase activity were used in the present invention for the preparation of Silver nanoparticles.

The selected E. coli mutants are prepared for the synthesis of the silver nanoparticles. The bacteria may be prepared by growing the bacterial cells in the Luria Berthani (LB) broth containing 0.1 to 0.5 % of glucose and 20 mM to 50 mM Sodium nitrate at ambient temperature with continuous mixing at 150 rotations per minute (rpm) for 72 hrs.

Preferably the concentration of glucose is 0.1 % and Sodium nitrate is 20 mM.

The ambient temperature may be 37 °C, 40 °C, 50 °C or 60 °C. Preferably the ambient temperature is 37 °C.

The biomass containing bacterial cell is separated by centrifuging the broth at 5000 rpm for 5 min. Post wash, the pellet was washed with sterile distilled water three times and is used as the biomass for the synthesis of silver nanoparticles.

For synthesis of the silver nanoparticle, the wet biomass of bacteria is mixed with 50-150 ml, preferably 75-125 ml, most preferably, 100 ml of aqueous solution of silver nitrate (AgN03) of strength 0.5mM-5mM, preferably, 0.5mM-1.5mM, most preferably 1 mM silver nitrate (AgN03) at ambient condition for 50-150 hrs, preferably, for 120 hrs.

The above method helps in size controlled synthesis of silver nanoparticles by the chemically mutated E. coli that has mutation in the cis acting region of nitrate reductase enzyme. During this process silver nanoparticles are produced from silver ions by the extracellular enzymes produced by the E. coli bacterial biomass.

The produced silver nanoparticles may have a diameter between I nm to 100 nm concentrating on 10 - 2 nm, preferably 15-20 nm.

Thus, the Silver nanoparticle produced biosynthetically by chemically mutated
Escherichia coli are smaller in size, the small sized Silver nanoparticle are more effective against microbes that prevail in the wound environment.

The present invention also provides an antimicrobial composition of the silver nanoparticles. The antimicrobial composition of the silver nanoparticle may comprise therapeutically effective amount of silver nanoparticle along with pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a polymer, a semi gelling agent, a preservative, a humectant and an isotonicity agent or any other pharmaceutically acceptable excipient.

The polymer may be selected from the group consisting of Alginic acid, carrageenam, chitosan, pectinic acid and polymers derived from monomers such as vinyl chloride, vinyl alcohol, vinyl pyrrolidone, furan, acrylonitrile, vinyl acetate, methyl acrylate, methyl methacrylate, styrene, vinyl ethyl ether, vinyl propyl ether, acrylamide, ethylene, propylene, acrylic acid, methacrylic acid, maleic anhydride, salts of any of the aforementioned acids and mixtures thereof; polyvinyl chloride; polypropylene; acrylic/maleic copolymers; sodium polyacrylate; polyvinyl pyrrolidone; glucomannan and optionally another natural polysaccharide with a polyhydric alcohol. Preferably the polymer is polyacrylamide.


The amount of polymer may be present in the range of 0.1 % to 5 %.

The tonicity modifier may be selected from the group consisting of glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose etc. Preferably the tonicity modifier is sodium chloride.

The amount of tonicity modifier may be present in the range of 0.2% - 0.3%.

The preservative may be selected from the group comprising ethanol, benzoic acid and the sodium or potassium salts thereof, sorbic acid and the sodium or potassium salts thereof, methyl paraben and propyl paraben, chlorobutanol, benzyl alcohol, phenylethanol, methyl-, ethyl-, propyl- or butyl-p-hydroxybenzoates, disodium edetate, phenol, m-cresol, p-chloro-m-cresol, those selected from the group of the PHB esters, e.g. mixtures of PHB-methyl with PHB-propylesters, quaternary ammonium compounds such as benzalkonium chloride, thiomersal, phenyl-mercury salts such as nitrates, borates.

Preferably the preservative is methyl paraben and propyl paraben.

The amount of preservative may be present in the range of 0.02 % w/w to 0.5 %, preferably 0.4%.

The gelling agent may be selected from the group comprising guar gum, algae extract (alginates, carrageenates, gelose), polysaccharides (xanthan gum, arabic gum, tragacanth gum), starches, pectins, cellulose derivatives, in particular those obtained by esterification or by etherification of cellulose, and acrylic derivatives such as carbomers, polycarbophiles and acrylates. Preferably the gelling agent may be carbomer.

The amount of gelling agent may be present in the range of 1.00 % w/w to 2.00 % w/w, and the preferably value is 1.00 % w/w.

The formulation of the present invention may be present in various pharmaceutical dosage forms. The dosage forms of the present invention may be processed into suitable dosage forms for topical application such as creams, powder and emulsions.

The pharmaceutical dosage forms of silver nano particles formulation is designed such that there is sustained release of silver nano particle. The sustained release of silver nano particle render release of silver nano-particles at regular intervals of time.

In an aspect, the present Invention provides a method for producing the formulation that exhibit sustained release of silver nano particle. The method comprising the steps of:

i. swelling of the polymer in water to obtain a slurry;

ii. adding of silvernano particle solution to the slurry of step I;

iii. preparing the slurry II by adding a gelling agent, a tonicity agent in appropriate solvent;

iv. preparing the slurry III by dissolving at least one preservative in heated solvent;

v. adding slurry II and slurry III to obtain a resultant mix;

vi. adding resultant mix of step v to the slurry of step I;

vii. addition of isopropyl alcohol and making the volume to obtain a mixture; and

viii. homogenizing the mixture of step VII to obtain a homogenized gel formation.

The polymer at step 1 may be selected from the group consisting of Alginic acid, carrageenam, chitosan, pectinic acid and polymers derived from monomers such as vinyl chloride, vinyl alcohol, vinyl pyrrolidone, furan, acrylonitrile, vinyl acetate, methyl acrylate, methyl methacrylate, styrene, vinyl ethyl ether, vinyl propyl ether, acrylamide, ethylene, propylene, acrylic acid, methacrylic acid, maleic anhydride, salts of any of the aforementioned acids and mixtures thereof; polyvinyl chloride; polypropylene; acrylic/maleic copolymers; sodium polyacrylate; polyvinyl pyrrolidone; glucomannan and optionally another natural polysaccharide with a polyhydric alcohol. Preferably the polymer is polyacrylamide.

The amount of polymer may be present in the range of 0.1 % to 5 %.

The gelling agent at step III may be selected from the group comprising guar gum, algae extract (alginates, carrageenates, gelose), polysaccharides (xanthan gum, arabic gum, tragacanth gum), starches, pectins, cellulose derivatives, in particular those obtained by esterification or by etherification of cellulose, and acrylic derivatives such as carbomers, polycarbophiles and acrylates. Preferably the gelling agent may be carbomer.

The amount of gelling agent may be present in the range of 1.00 % w/w to 2.00 % w/w, and the preferably value is 1.00 % w/w.

The tonicity agent at step III may be selected from the group consisting of glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose etc.

Preferably the tonicity modifier is sodium chloride.

The amount of tonicity modifier may be present in the range of 0.2% - 0.3%.

The appropriate solvent at step III may be selected from the group comprising methanol, ethanol, heptane, hexane glycerol and other environmentally preferable solvents. Preferably the solvent is glycerol.

The preservative at step IV may be selected from the group comprising ethanol, benzoic acid and the sodium or potassium salts thereof, sorbic acid and the sodium or potassium salts thereof, methyl paraben and propyl paraben, chlorobutanol, benzyl alcohol, phenylethanol, methyl-, ethyl-, propyl- or butyl-p-hydroxybenzoates, disodium edetate, phenol, m-cresol, p-chloro-m-cresol, those selected from the group of the PHB esters, e.g. mixtures of PHB-methyl with PHB-propylesters, quaternary ammonium compounds such as benzalkonium chloride, thiomersal, phenyl-mercury salts such as nitrates, borates.

Preferably the preservative is methyl paraben and propyl paraben.

The amount of preservative may be present in the range of 0.02 % w/w to 0.5 %, preferably 0.4%.

The present invention also provides a method for converting the toxic metal ions to non-toxic metal nanoparticles through the catalytic effect of the enzyme nitrate reductase of the bacteria.

Without being limited by theory, it is submitted that the Escherichia coli of the present invention is rendered resistant to silver on chemical mutagenesis. This is a surprising and novel finding since Escherichia coli is highly sensitive to silver ion. It is believed that the chemical mutation in Escherichia coli with HN02, may result in mutation in cis acting region of the gene, encoding Nitrate reductase enzyme. The HN02,exerts its effects on nucleic acids by the deamination of the amino groups of adenine, cytosine and guanine residues (Kotaka and Baldwin, 1964) that leads to A-T—»G-C. The accumulation of the mutation in cis acting region of the gene encoding Nitrate reductase enzyme may lead to the development of a mutated Escherichia coli that exhibit amplified production of the nitrate reductase enzyme.

The mutation in cis acting region of the gene may drive the up regulation of the expression of the gene encoding Nitrate reductase enzyme.

While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

Example 1

Development of mutated E. coli

Wild type E. coli strain was mutated by Chemical mutagenesis. Chemical mutagenesis was carried out by exposing Wild type E. coli strain to HNO2. Posts to HNO2 treatment, E. coli were screened for mutated strains that possess higher nitrate reductase activity (Fig. 1). The method of mutagenesis of Escherichia coli may comprise the following steps:

1. subculturing of fresh overnight Wild type E. coli culture in 5 ml of LB medium until it reached a density of 2-3X109 cells/ml;

2. washing the spun down cell with 100 mM sodium acetate buffer (pH 4.3);

3. resuspension of cell pellet in 1 ml of 50 mM fresh nitrous acid solution (as described by Miller, 1972);

4. incubating the cells at room temperature for 10 min;

5. washing the cells with 10 vol. of cold IX M9 medium to stop the reaction;

6. plating the washed cell on LB agar plates;

7. Screening the Escherichia coli cell which exhibit higher synthesis of nitrate reductase;

8. Confirming the type of mutant in the gene encoding nitrate reductase enzyme by DNA sequencing. This step is important to confirm that the mutation is in the exacting elements but not in the nitrate reductase protein coding region;

Around 500 colonies were screened for the nitrate reductase assay as per the procedure mentioned in the Example 3. Among them, 20 colonies showed enhanced (2-5 fold) nitrate reductase activity compared to the Wild type E. coli.

The E. coli mutants which showed the 5 fold enhanced nitrate reductase activity were used in the present invention for the preparation of Silver nanoparticles.

Example 2

Preparation of bacteria E. coli for synthesis of silver nanoparticle

Various strains of chemically mutated E. coli were used for experimentation. The bacterial inoculates were cultured in Luria Berthani (LB) Agar media at 37 °C in sterile petriplates. For preparation of bacteria E. coli cells to synthesis the silver nanoparticles, the bacterial cells were grown in 250 ml flasks each containing 100 ml of liquid media of LB with 0.1 to 0.5 % of glucose and 20 mM to 50 mM Sodium nitrate. The flasks were incubated at various temperatures 37 °C, 40 °C, 50 °C and 60 °C (Table 1) each with continuous mixing at 150 rotations per minute (rpm) for 72 hrs. The bacterial cells were separated by centrifuging the broth at 5000 rpm for 5 min and the pellet was washed with sterile distilled water three times and was used as the biomass for the synthesis of silver nanoparticles. The growth of the bacteria and the nitrate reductase enzyme levels was checked in each case and is tabulated in (Table 1). The method for checking the enzyme level is provided in Example 3.

Example 3

Nitrate reductase assay

The culture supernatants were assayed for optimal nitrate reductase activity by adding supernatant to the mixture of reagent A (25 mM Potassium phosphate buffer with 10 mM Potassium nitrate and 0.05 mM ethylenediaminetetraacetic acid, pH 7.3 at 30 °C) and reagent B (2.0 mM P-nicotinamide adenine dinucleotide) 0.01 ml in the 2 ml labeled microtubes. A standard solution of 1.45 mM Sodium Nitrite in 50 ml of reagent A. For control, the blank i.e., reagent A 1.90 ml and for the standards, standard solution 1.80 ml. was added in the tubes. Followed by addition of the standard solution and the test enzyme solution, the mixture was incubated at 30 °C for 2 mins. The reaction was stopped by adding reagent D (58 mm sulfanilamide solution) 1.0 ml to each of the tube followed by addition of reagent E (0.77 mM N-(l-naphthyl) ethylenediamine dihydrochloride solution). The reaction mixture was mixed and was incubated for 10 mins at 25 °C. The absorbance of the final solution was checked at 540 nm for the test, blank and standards in UV- Vis spectrometer as shown in Table 1.

c . The growth pattern of the chemically mutated E. coli and the enzyme activity of nitrate reductase were measured at different time period, as shown in Figure 1.

Table 1: Nitrate reductase enzyme activity at different temperature of incubation after 72 hrs.

As evident, from the Table 1 the optimum temperature for Escherichia coli for the synthesis of nitrate reductase enzyme is 37 °C. Also, the enzyme activity was observed to be more at 72 hrs of the incubation.

Example 4

Synthesis of silver nanoparticles

The silver nanoparticles were synthesized by the mutated E. coli by addition of 100 ml aqueous solution of 1 mM silver nitrate (AgNC^) to 10 g of the wet biomass containing the selected mutated bacterial strains. The mixture was later placed in a 100 rpm rotating shaker at 40 °C for 120 hrs which leads to production of nitrate reductase enzyme. Later on the nitrate reductase enzyme renders the size controlled synthesis of silver nanoparticles from silver ions.

Example 5

Analysis of silver nanoparticles

Silver nanoparticles were characterized by spectroscopy techniques such as UV-Vis light spectroscopy, Atomic Absorbance Spectroscopy (AAS) and Transmission Electron Microscopy and particle size analyzer.

The formation of the silver nanoparticle was monitored routinely by measurement of the absorption of the solution by UV-Vis spectra. The quantification of the amount of silver nanoparticle produced biosynthetically is determined by correlating the absorption spectra of test sample with the absorption spectra of the standard solution of the known amount of silver nanoparticle Figure 2.

The UV-Vis spectrum recorded for the E. coli reaction vessel at different wavelengths at different period of time, shown in Fig. 3 and Fig 4. As evident, there was no appreciable change in the net magnitude of the UV-Vis absorbance of the reaction product after 72 hrs although the reaction was allowed to proceed for one month (Fig 4). This implicates that the silver nanoparticles produced by the E coli present in the solution are extremely stable even after a month of reaction, with no evidence of aggregation of particles.

For Atomic Absorbance Spectroscopy (AAS) the sample and the standard were incubated with concentrated Nitric acid until clear solution appears, and this solution was subjected to the Atomic Absorbance . The strong surface Plasmon resonance is centered at about 405 -420 nm is characteristic of nanosilver. The spectra also clearly show the increase in the intensity of the silver as the reaction time proceeds, indicating the formation of increased number of silver nanoparticles with time in the solution.

The TEM was carried out to determine the shape of the silver nano particle. Samples were prepared for TEM by placing a drop of the centrifuged suspension supernatant on carbon-coated copper grids (40 um x 40 urn mesh size) and allowed water to evaporate.

TEM observations were performed on an 1200 EX (Joel, Japan) at an accelerated voltage of 120 V. The images of silver nanoparticles by TEM were found to be in the size range 1-100 nm (Fig. 5(a)). As observed in the image, there is separation between silver nanoparticles in the TEM images. The separation is due to the presence of proteins in biologically synthesized silver nanoparticles and would explain UV-Vis spectroscopy measurements, which is a characteristic of well dispersed nanoparticles when compared with that of the mechanically synthesized silver colloidal particles as shown in Fig. 5 (b) are clumped due to lack of differentiating protein phase. As noticed in the TEM image the silver nanoparticles are crystalline, which is also confirmed by the selected area Surface Plasmon Resonance (SPR) pattern recorded from one of the nanoparticles in the aggregates.

The results of TEM has showed that the chemically mutated E. coli produces size controlled silver nanoparticles with its highly efficient extracellular enzymes and other metabolites.

The particle size analysis was further confirmed with Zeta particle sizer (Fig. 6). The range of silver nanoparticles present in the sample can be elucidated with this method. A histogram of silver nanoparticle sizes was obtained in which the average silver nanoparticle size is 1 to 100 nm.

Table 2: A AS method of quantification of biosynthesized silver nanoparticles in formulations.

Example 6

Preparation of formulation of silver nano particle.

The formulation of silver nano particle was prepared as described below:

1.00 % w/w to 2.00 % w/w of Polyacrylamide was allowed to swell in known quantity of purified water for an hour under constant stirring. The 0.001 % w/w to 1 % w/w of silver nanoparticle solution was added to the polyacrylamide and incubated for lhr. At the same time, slurry was prepared by dispersing 0.1 % to 5 % w/w carbomer in 10 % w/w to 15 % w/w of glycerol. To this slurry, a predetermined amount of Sodium chloride dissolved in known quantity of purified water was added. Another, solution was prepared by adding known quantity 0.02 % w/w to 0.5 % of propyl paraben and methyl paraben in heated purified water at 70 °C. The propyl paraben and methyl paraben were dissolved with proper stirring to get a clear solution of parabens. Then the carbomer slurry was added to the above prepared Paraben solution slowly with constant stirring. The resultant mixture was added to the polyacrylamide and silver nano particle mixture slowly with continuous stirring followed by the addition of Isopropyl alcohol. The volume was made up by addition of purified water and the final gel formulation was homogenized using homogenizer.

The Formulation of biosynthesized silver nanoparticles

The composition of the pharmaceutical formulation comprising

(1) 0.001 % w/w to 1 % w/w of biologically synthesized silver

(2) 1.00 % w/w to 2.00 % w/w of gel forming agent (polymer) like polyacrylamide

(3) 0.1 % to 5 % w/w semi-gelling agent like carbomer

(4) 0.02 % w/w to 0.5 % of preservatives (optional) like methyl paraben and propyl paraben

(5) 10 % w/w to 15 % w/w humectant like glycerol

(6) Other pharmaceutical^ acceptable inert isotonicity adjusting agents like sodium chloride

Example 7

Anti-microbial efficiency of biosynthesized silver nanoparticle

The anti-microbial activity of the formulation was determined by the extent of decrease in the growth of the microorganisms such as Pseudomonas aeruginosa ATCC 9027 or MTCC 1688 (Table 3 (a)), Staphylococcus aureus ATCC 6538 or MTCC-737 (Table 3 (b)), Candida albicans ATCC 10231 or MTCC-227 (Table 3 (c)) and Aspergillus niger ATCC-16404 (Table 3 (d)) by silver nanoparticle formulation as comparision with various silver salts like silver nitrate, silver chloride, silver acetate and silver sulphadiazine and was discussed further through tables below. For testing the inhibitory action of the silver nano particle, a fresh bacterial culture of 100 ml with optical density (O.D.) 1.0 was cultured. To 3 ml of this culture, 1.0 gram of the test biosynthesized silver nanoparticle formulation sample was added and incubated for 5 hrs. For comparison of efficacy with other salt, the same procedure is repeated with solution of various silver salts of concentration 1 mg/ml. For analysis, the treated bacterial culture were diluted and plated on sterile petriplate with microbial agar media and incubated for a week. A negative control without any formulation or silver salts has to be maintained.

Table 3 (a): Comparative inhibitory action of various Silver salts and Silver Nanoparticle Formulation on Pseudomonas aeruginosa ATCC 9027.

Note: An inoculum of Pseudomonas aeruginosa ATCC 9027 having 0.79 x 106CFU/ml (Optical density 1.01 at 600 nm) was used for the above experiment.

Table 3 (b): Comparative inhibitory action of various Silver salts and Silver Nanoparticle Formulation on Staphylococcus aureus ATCC 6538.

Note: An inoculum of Staphylococcus aureus ATCC 6538 having 0.80 x 106CFU/ml (Optical density 1.00 at 600 nm) was used for the above experiment.

Table 3 (c): Comparative inhibitory action of various Silver salts and Silver Nanoparticle Formulation on Candida albicans ATCC 10231.

Note: An inoculum of Staphylococcus aureus ATCC 6538 having 0.85 x 106CFU/ml (Optical density 1.00 at 600 nm) was used for the above experiment.

Table 3 (d): Comparative inhibitory action of various Silver salts and Silver Nanoparticle Formulation on Aspergillus niger ATCC 16404.

Note: An inoculum of Aspergillus niger ATCC 16404 having 0.85 x 106CFU/ml (Optical density 1.00 at 600 nm) was used for the above experiment.

Example 8

Test for sustained release of silver nanoparticles

10 g of silver nanoparticles formulation in 500 ml of saline was taken and mixed uniformly in container A. A semi-permeable membrane that is similar to the cell membrane was attached to the beaker and was connected to another beaker (B) which does not contain any silver nanoparticle. The set up was left for 72 hrs for the silver nanoparticles to check the controlled release activity of the cross-linking polymer based formulation. The concentration of silver nanoparticle in beaker B was analyzed every 1 hr.

The concentration of silver nanoparticle in the first hour was equivalent to the concentration of silver nanoparticle analyzed in the seventy-secondth hour but started to decreased after eighty-fourth hour onwards (Table 4), which explains the sustained release activity of the polymer used after 72 hrs.
Table 4: The table showing the results of amount of silver nanoparticles released at regular intervals of time during sustained release study of silver nanoparticles from polymer.

We Claim:

1. A method for synthesis of silver nanoparticles by modified E coli wherein the method comprise the steps of:

i. mutating Escherichia coli bacterial cells;

ii. screening mutated Escherichia coli exhibiting enhanced nitrate reductase activity;

iii. culturing screened Escherichia coli; and

iv. exposing the cultured cells to a solution of silver nitrate under ambient conditions in which bacterial cells produce enzymes and metabolites that reduce silver ions to silver nanoparticles.

v. obtaining silver nano particle.

2. The method as claimed in claim 1 wherein the mutation of E. coli is achieved by treating the E. coli cells either chemically or by radiation, preferably chemically.

3. The method of claim 1 and 2 , wherein the chemical mutation is achieved by treating the E. coli cells with chemical mutagen selected from the group comprising nitrous acid, sulphurous acid, acetic acid or citric acid, preferably the mutagen is nitrous acid.

4. The method of claim 1, wherein the diameter of the biologically synthesized silver nanoparticles is in the range of 1 to 100 nm.

5. A composition comprising therapeutically effective amount of silver nanoparticles as prepared by method of claim 1 along with pharmaceutically acceptable excipient.

6. The composition as claimed in claim5 wherein the pharmaceutically acceptable excipients is selected from the group comprising a polymer, a semi gelling agent, a preservative, a humectant and an isotonicity agent or any other pharmaceutically acceptable excipient.

7. The composition as claimed in claim5 wherein the polymer is selected from the group comprising Alginic acid, carrageenan, chitosan, pectinic acid and polymers derived from monomers such as vinyl chloride, vinyl alcohol, vinyl pyrrolidone, furan, acrylonitrile, vinyl acetate, methyl acrylate, methyl methacrylate, styrene, vinyl ethyl ether, vinyl propyl ether, acrylamide, ethylene, propylene, acrylic acid, methacrylic acid, maleic anhydride, salts of any of the aforementioned acids and mixtures thereof; polyvinyl chloride; polypropylene; acrylic/maleic copolymers; sodium polyacrylate; polyvinyl pyrrolidone; glucomannan and optionally another natural polysaccharide with a polyhydric alcohol, preferably the polymer is polyacrylamide.

8. The composition as claimed in claim5 wherein the amount of polymer is in the range of 0.1% to 5%.

9. The composition as claimed in claim5 wherein the tonicity modifier is selected from the group comprising glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose, preferably, the tonicity modifier is sodium chloride.

10. The composition as claimed in claim5 wherein the amount of tonicity modifier is present in the range of 0.2% - 0.3%.

11. The composition as claimed in claim5 wherein the preservative is selected from the group comprising ethanol, benzoic acid and the sodium or potassium salts thereof, sorbic acid and the sodium or potassium salts thereof, methyl paraben and propyl paraben, chlorobutanol, benzyl alcohol, phenylethanol, methyl-, ethyl-, propyl- or butyl-p-hydroxybenzoates, disodium edetate, phenol, m-cresol, p-chloro-m-cresol, those selected from the group of the PHB esters, e.g. mixtures of PHB-methyl with PHB-propylesters, quaternary ammonium compounds such as benzalkonium chloride, thiomersal, phenyl-mercury salts such as nitrates, borates, preferably the preservative is methyl paraben and propyl paraben.

12. The composition as claimed in claim5 wherein the amount of preservative is present in the range of 0.02 % w/w to 0.5 %, preferably 0.4%.

13. The composition as claimed in claim5 wherein the gelling agent is selected from the group comprising guar gum, algae extract (alginates, carrageenates, gelose), polysaccharides (xanthan gum, arabic gum, tragacanth gum), starches, pectins, cellulose derivatives, in particular those obtained by esterification or by etherification of cellulose, and acrylic derivatives such as carbomers, polycarbophiles and acrylates, preferably the gelling agent is carbomer.

14. The composition as claimed in claim 5 wherein the amount of gelling agent is present in the range of 1.00 % w/w to 2.00 % w/w, preferably 1.00 % w/w.

15. The composition as claimed in claim 5 wherein the composition is processed into
suitable dosage forms selected from the group comprising creams, powder and emulsions.

16. The composition as claimed in claim 5 wherein the size of biologically synthesized silver nanoparticles is in range of 1 - 100 nm, preferably in the range of 2-75nm, more preferably in the range of 5-50 nm.

17. The composition as claimed in claim 5 wherein, the composition exhibit sustained release activity of silver nanoparticles having a mass of about 2 % w/w of the total mass of the formulation.

18 A method for producing the formulation that exhibit sustained release of silver nano particle as claimed in claim 17, comprising the steps of:

i. swelling of the polymer in water to obtain a slurry;

ii. adding of silvernano particle solution to the slurry of step I;

iii. preparing the slurry II by adding a gelling agent, a tonicity agent in appropriate solvent;

iv. preparing the slurry III by dissolving at least one preservative in heated solvent;

v. adding slurry II and slurry III to obtain a resultant mix;

vi. adding resultant mix of step v to the slurry of step I;

vii. addition of isopropyl alcohol and making the volume to obtain a mixture; and

viii. homogenizing the mixture of step VII to obtain a homogenized gel formation.

19 The method as claimed in claim 18 wherein the polymer is selected from the group comprising alginic acid, carrageenam, chitosan, pectinic acid, polymers derived from monomers such as vinyl chloride, vinyl alcohol, vinyl pyrrolidone, furan, acrylonitrile, vinyl acetate, methyl acrylate, methyl methacrylate, styrene, vinyl ethyl ether, vinyl propyl ether, acrylamide, ethylene, propylene, acrylic acid, methacrylic acid, maleic anhydride, salts of any of the aforementioned acids and mixtures thereof; polyvinyl chloride; polypropylene; acrylic/maleic copolymers; sodium polyacrylate; polyvinyl pyrrolidone; glucomannan and optionally another natural polysaccharide with a polyhydric alcohol; preferably the polymer is polyacrylamide.

20 The method as claimed in claim 18 wherein the polymer is present in the range of 0.1 % to 5 %.

21 The method as claimed in claim 18 wherein the gelling agent as claimed in claim 18 is selected from the group comprising guar gum, algae extract (alginates, carrageenates, gelose), polysaccharides (xanthan gum, arabic gum, tragacanth gum), starches, pectins, cellulose derivatives, in particular those obtained by esterification or by

etherification of cellulose, and acrylic derivatives such as carbomers, polycarbophiles and acrylates; preferably the gelling agent may be carbomer.

22 The method as claimed in claim 18 wherein the gelling agent is present in the range of 1.00 % w/w to 2.00 % w/w, preferably in the range of 1.00 % w/w.

23 The method as claimed in claim 18 wherein the tonicity agent is selected from the group comprising glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose; preferably the tonicity modifier is sodium chloride.

24 The method as claimed in claim 18 wherein the tonicity agent is present in the range of0.2%-0.3%.

25 The method as claimed in claim 18 wherein the appropriate solvent is selected from the group comprising methanol, ethanol, heptane, hexane glycerol and other environmentally preferable solvents; preferably the solvent is glycerol.

26 The method as claimed in claim 18 wherein the preservative is selected from the group comprising ethanol, benzoic acid and the sodium or potassium salts thereof, sorbic acid and the sodium or potassium salts thereof, methyl paraben and propyl paraben, chlorobutanol, benzyl alcohol, phenylethanol, methyl-, ethyl-, propyl- or butyl-p-hydroxybenzoates, disodium edetate, phenol, m-cresol, p-chloro-m-cresol, those selected from the group of the PHB esters, mixtures of PHB-methyl with PHB-propylesters, quaternary ammonium compounds such as benzalkonium chloride, thiomersal, phenyl-mercury salts such as nitrates, borates; preferably the preservative is methyl paraben and propyl paraben.

27 The method as claimed in claim 18 wherein the preservative is present in the range of 0.02 % w/w to 0.5 %, preferably 0.4%.