Field of the Invention
The present invention relates to a process for the preparation of nanosilver nanohydrogel (nSnG) based antimicrobial agent having functional groups so that it can modify the surface of any biomaterial. More precisely, the present invention relates to a process for the preparation of nanoSilver-nano-hydrogel based antimicrobial agent by Gamma irradiation by inverse emulsion process.
Background of the Invention
Preparation of nano Silver is a very common process and lots of methods are available, like Chemical reduction of Silver From its salt.
Inorganic reducing agent-
Sodium Borohydride is the most frequently used one. The other agents are hydrazine hydrate, sodium triacetoxy borohydride, ascorbic acid, trisodium citrate, hydrogenation at 80°C or higher temperature, dimethylamine borane (DMAB), sodium formaldehyde sulfoxylate (SFS), corona discharged ions, sodium hypophosphite , sodium hydroxide and sodium cyanoborohydride.
Organic reducing agent-
Glucose, Southern Live Oak {Quercus virginiana), Southern Magnolia {Magnolia grandiflora), Kudzu (Pueraria lobata) and Loblolly Pine (Pinns taeda) extracts, Capcicum annuum L, Vitamin- E langmuir monolayer, N,N-dimethylformamide (DMF), formamide.
Polymeric reducing agent
Polyethylene glycol the most popular polymer for reducing Silver. Following substances are also being used like, S-PEEK by its sulphonate group, polyhydrosiloxane, poly(ethylene glycol) methacrylate (PEGMA), gelator i.e. the conjugate of cholic acid and polyethylene glycol 400, acrylamide (AM) and methacrylic acid (MAA) copolymer at pH-8, polyethyleneimine, amidationed polyethyleneimine (PEI), polyglycerol N,N,N',N'-tetramethyl-p-phenylene diamine. Some researchers have claimed to use living substances like fungus to produce silver nano particle, in reduced form by converting
the NADP+ to NADPH like Phoma sp.3.2883, Alfa Sprout, Fusarium oxysporum strains. Yin et al. has been used electrochemical method for Synthesis of Silver Nanoparticles in the Protection of poly(N-vinylpyrrolidone).
Non-Chemical reduction of Silver from its salt:
Non-chemical method of reducing Silver includes γ-irradiation; ultrasonic irradiation method ; UV-irradiation, thermal decomposition method, laser irradiation, freezing-thawning method, light, electron irradiation. The second important parameter is to stabilize the silver nano particles. This can be done mainly by polymers like PVA, PVP, Polystyrene, PAAm, polyacrylic acid & stabilizers like AOT, Span ™80, Tween ™80 etc. are being used, instead of this Ti02, Daxad 19 (sodium salt of a' high-molecular-weight naphthalene sulfonate formaldehyde condensate), sodium dodecyl sulfate,"green" biosurfactant i.e. rhamnolipid are also being used for the stabilization of the silver nano particles.
In most cases, AgNO3 or silver acetate is being used as the source-of Silver in the experiment bath. But in few cases pure silver plates are being used as source. In general in laser ablation technique or in corona discharge technique this type of source form is being used.
Kriwet et al. have prepared nano & micro particles of poly(AcrylicAcid) of size 80—150 nm & l-5um using free radical inverse emulsion polymerization method with cross linker PEG Zhang et al. have been utilized the AOT microemulsion system for preparation & characterization of silver nano particles. Xia et al. have reported about Acrylamide (AM) and methacrylic acid (MAA) copolymer microgels prepared by a reverse suspension polymerization technique. The microgels are used as templates for the preparation of silver-poly (acrylamide-co-methacrylic acid) [Ag-P(AM-co-MAA)] composite microspheres. They have obviously used the chemical reduction method.
There are other studies by Xia. et al. also on nano silver production. Where the reduction methods (Formaldehyde reduced microgels is being reported as of smooth surface structure instead of zigzag one as reported when NaBH4 is the reducing agent
due to time difference in the reduction procedure as the first is first diffused in the micelle & the when pH increased to 8 then reduction started so no surface morphology change takes place, where the second stars the reduction as it comes in contact with the micelle so it ultimately changes the surface topology) employed have a significant effect upon the surface structures of the microspheres Pavlyukhina et al. has been reported about Silver nanoparticles prepared by reducing AgNO3 with sodium hypophosphite in water-in-oil micro emulsions in the presence of a mixture of ethoxylated alkyl phenols as a surface active substance (SAS) with an Al203 support. The average size of these Ag particles is 70A. The proposed reduction mechanism of Silver Nitrate is as follows.
(1)
(2) (Formula Removed )
The reactions and chemical intermediates that precede in the formation of metallic clusters in solution, due to the possibility of controlling cluster sizes during the early stages of nucleation processes has been discussed for silver by Mercado et al. Silver clusters represent an excellent system to work for the development of experimental methodology for particle size growth because the intermediates involved in the synthesis of metallic silver particles from the reduction of the silver cation, Ag+, in basic solution that are well known.
Ag+ cations are reduced in basic solution to form single Ag atoms according to: (Formula Removed ) (I)
Silver atoms react with silver ions to form the silver dimmer cation, Ag2+, according to:
(Formula Removed ) (2)
The silver dimmer can couple to other dimmers to form a double charged tetramer, Ag42+, according to:
(Formula Removed ) Further reactions of silver metal atoms and the dimmer and tetramer cations, result in the formation of small silver clusters (Agn). Large, metallic, silver clusters exhibit peak at 380 nm in UV absorption experiments due to the excitation of a plasmon. The silver dimmer and tetramer exhibit UV absorption peaks at 310 and 275 nm, respectively. Small, non-metallic silver clusters have absorption peaks between 350 and 300 nm. Ohde et al. have prepared Nanometer-sized silver metal particles by chemical reduction of Ag+ ions dissolved in the water core of water in supercritical fluid carbon dioxide microemulsion. Sodium cyanoborohydride and N,N,N,N'-tetramethyl-p-phenylene-diamine are effective reducing agents used in this case. Ohde et al. have reported about the preparation of silverhalide nanoparticles made by a water-in-C02 microemulsion in supercritical carbon dioxide. Here they have prepared two separate emulsion for silver & halide ion & then mixed them with each other. Sun et. al. have prepared perfluoropolyetheramonium carboxylate (PFPE-NH4) stabilized water-in-C02 microemulsion to dissolve silver nitrate salt for the RESOLV (Rapid Expansion of a Supercritical Solution into a Liquid Solvent) process. The solution of reverse micelles with an aqueous silver nitrate core in C02 was rapidly expanded into an ethanol solution of sodium borohydride (NaBH4) as a reducing agent at room-temperature & ultimately forms silver nanopartioles of size 2 and 2-3.5 nm.
Dai et al. have approached for preparing Silver nano particle with di-(2-ethylhexyl) phosphoric acid (D2HPA) as the mobile carrier in membrane phase, ascorbic acid (Vd) as reducing agent in inner phase and cyclohexane as the-membrane of CEAs. Petit et al have reported about a method to prepare Silver nanosized crystallites synthesized in AOT reverse micelles by using a functionalized surfactant, silver sulfosuccinate (Ag(AOT)), as reactant. This results in a high concentration of monodisperse and stable nanosized particles. By using "green" biosurfactant, rhamnolipid Xie et al. have prepared nano Silver particles in reverse micelle.process. As oil phase in inverse microemulsion technique different hydrocarbons are being used, like- paraffin, Dodecane, hexane, n-heptane, n-bunol. As our aim is also to utilize less amount of chemicals so we are inclined on the application of γ-irradiation. Now nanoparticles can be formed by this
method is being evidenced by different workers results. Like, Chen et al. has prepared Silver nanoparticles of 4-5 nm at 100 kGy radiation dose in acetic water & with chitosan, Liu et al. has reported about the nanocomposties of stable nanosilver particles embedded in polyacrylonitrile matrix synthesized by γ-irradiation, in which the monomer acrylonitrile (either in the DMF or in very dilute aqueous solution) was polymerized and the silver ions (Source is AgNO3) were reduced simultaneously to form composites in situ. There is strong interaction between silver ions with —CN groups of polyacrylonitrile as reported by them.
Krkljes et al depicted a novel and convenient way of preparation of colloidal silver stabilized by polymer. In this case water insoluble PVA films filled with Ag nanoparticles were prepared by a y -irradiation. In this method at first cross linking of PVA was performed & in the second step Ag+ ions were reduced in PVA hydrogel using electron transfer reactions from radical species formed in water radiolysis. The decomposition path of the cross linked PVA matrix was affected by the presence of Thermal properties of the Ag-PVA nanocomposites were a function of the preparation route. The Ag-PVA nanocomposite prepared only with polymer radicals showed a higher temperature of the onset of thermal degradation. All Ag-PVA nanocomposites showed lower temperatures of the onset of thermo oxidative degradation and increased rates of degradation compared with the neat cross linked PVA matrix.
The reduction of Ag cation is a two stage process, ie
(i) Direct reduction by irradiation or by the produced radicals. This actually a kind of nucleation
(ii) The second stage is further reduction of left metal ions where the previously prepared particles or atoms act as the catalyst. But this effect is also restricted because of restricted mobility of the reducing elements in the hydrogels.
The radiation-induced Ag+ ion reduction was followed by cross linking of the PVA chains. PVA was found to be a very efficient stabilizer to prevent aggregation of Ag clusters. The
clusters produced in the hydrogel matrix were expected to be smaller than the pore size (2-20 nm) of the gels used in the study. These Ag clusters were unable to reduce methyl viologen (MV2+) chloride and were stable in air.
The above cited prior art shows that most of the technologies are based on a high chemical consuming methods along with which they are not actually preparing a nanoparticle of hydrogel which inside in itself incorporates the nanosilver particle which are in nano size, they are antimicrobial but not harmful for the human cells as covered by the hydrogel along with that having some functionality so that they can modify any biomaterials. As they are in particulate structure so they are easy to apply not like the film.
Accordingly, the object of the present invention is to obviate the aforesaid drawback of the prior art and to provide a process of preparation of nanosilver-nanohydrogel based antimicrobial agent.
Further object of the present invention is to provide nanosilver-nanohydrogel (nSnG) which is low chemical consuming fast preparation process of an antimicrobial material based on nano silver nano hydrogels (nSnG) exhibiting chemical functional groups so that the material becomes interactive with biomaterials.
Another objective is to provide a simple method of developing nanosilver functional coating material so as to provide modification of any biomaterial surface by the present antimicrobial agent.
Summary of the invention
In order to achieve the aforesaid objects the present invention provides a process for the preparation of nanoSilver-nanoHydrdgel based antimicrobial agent by Gamma irradiation, with a dose of 0.15 to 0.2. kGy/hour for 10 minutes to 24 hours by invert emulsion process. The water in oil emulsion consists water along with hydrogel forming monomer Acrylamide and Water soluble Silver Salt Silver Nitrate and the oil phase i.e.
heptane consists of Surfactant AOT. This is followed by the separation process consisting chelation by saline water along with dissolution by methyl ethyl ketone.
The water percentage of total emulsion is varied from 1% to 20% (v/v) and the effect on the emulsion stability and the particle size is studied. Further, the hydrogel forming monomer silver salt concentration is also being varied from 2:1 to 1:2 including 1:1 (w/w) and the effect on the particle size is studied.
The concentration of surfactant is varied from is also being varied from 1% to 20% w/v and the effect on the emulsion stability and, the particle size is studied.
The gamma irradiation time is varied from 10 minutes to 24 hours and the effect on the particle size was studied.
Detailed description of the Invention:
Hereinafter, a process of preparation of nanosilver-nanohydrogel (nSnG) based antimicrobial agent according to the present invention will be described in detail with reference to the accompanying examples.
A process of preparation of nanosilver-nanohydrogel (nSnG) based antimicrobial agent according to the present invention broadly comprises the steps of preparation of w/o inverse emulsion; where the water in oil emulsion consists of water along with hydrogel forming monomer (Acrylamide) and Water soluble Silver salt (Silver Nitrate) and the oil phase i.e. Heptane consists of Surfactant (AOT). This inverse emulsion is being prepared at normal room temperature, at high stirring in magnetic stirrer in sealed beaker. Then the inverse emulsion is being kept under gamma irradiation.
The separation of nSnG is being carried out by pouring the Saline water (Sodium Chloride solution) then the separated particles are being washed in methyethyl ketone to remove remaining surfactant.
In one embodiment of the present invention the water percentage of total emulsion is varied from 1% to 20% (v/v) and the effect on the emulsion stability and the particle size is studied.
In another embodiment of the present invention the hydrogel forming monomer percentage of total emulsion is varied from 10% to 40% (w/v) and the effect on the emulsion stability and the particle size is studied.
In further embodiment of the present invention the water soluble silver salt percentage of total emulsion is varied from 1% to 20% (w/v) and the effect on the emulsion stability and the particle size is studied.
In yet another embodiment of the present invention the oil phase percentage of total emulsion is varied from 80% to 99% (v/v) and the effect on the emulsion stability and the particle size is studied.
In yet another embodiment of the present invention the ratio of hydrogel forming monomer and silver salt concentration is also being varied from 2:1 to 1:2 including 1:1 (w/w) and the effect on the particle size is studied.
In further embodiment of the present invention the concentration of surfactant is also being varied from 1% to 20% (w/v) and the effect on the emulsion stability and, the particle size is studied.
In yet another embodiment of the present invention the gamma irradiation, a dose of 0.15 to 0.20 kGy/hour, preferably 0.16kGy/hour, time is varied from 10 minutes to 24 hours and the effect on the particle size was studied.
In further embodiment the chemical composition is analyzed by the Furiour Transform Infra Red Spectroscopy (FTIR) and Energy Dispersive X-ray analysis (EDX) study.
In yet another embodiment the polymer silver by weight ratio is calculated by the Thermo Gravimetric analysis (TQA).
The actual structure of the nSnG is studied by the Transmission Electron Microscope (TEM).
The particle size is studied by the Transmission Electron Microscope (TEM) for dried particle size and particle size analyzer for swelled size.
The nano silver size is estimated by the Transmission Electron Microscope (TEM) study and the UV-Visible spectroscopy.
In another embodiment the separation yield has been studied for different saline water concentration from 1% to 20% (w/v).
Present invention is thus directed to the nanostructure composite which is antimicrobial due to nano silver along with biocompatible due to the hydrogel nano layer coating. The hydrogel also provides physiochemical bond so that the silver cannot leach out from the nano particles. Also the hydrogels provide functional groups so that they can be easily attached on any biomaterial surface. More over due to their very low size the dose rate can be minutely controlled. One additional feature is these nSnG can be aligned under high voltage difference according to the path of the electron beam. These may have use in electronic circuit formation.
The following are the key features of the process:
(I) Low chemical consuming
(II) Low time consuming, takes only few hours
(III) Normal temperature process with a very high range
(IV) highly stable emulsion along with stable particles
(V) Particle size of 4-100 nm
(VI) As covered by the hydrogel coating, so that nSnG is not harmful for handling
(VII) Self organizing nanohydrogel structures
(VIII) Antimicrobial in nature
The following examples are given by the way of illustration and therefore should not be constructed to limit the scope of the invention.
Example I
1% (v/v) water having 20% (w/v) AAm and 20% (w/v) of AgNO3 in 99% Heptane having 8% (w/v) AOT shows cloud point at -10°C. This inverse emulsion irradiated for 2 hours results average particle size of 6.93 nm with S.D 0.98.
Example 2
3% (v/v) water having 20% (w/v) AAm and 20 % (w/v) of AgNO3 in 97% Heptane having 8% (w/v) AOT shows cloud point at -5°C. This inverse emulsion irradiated for 2 hours results average particle size of 190.32 nm with S.D 19.49.
Example 3
20% (v/v) water having 20% (w/v) AAm and 20 % (wlv) of AgNO3 in 80% Heptane having 8% (w/v) AOT shows cloud point at 60 °C. This inverse emulsion irradiated for 2 hours results with no particle as in normal temperature the emulsion breaks.
Example 4
2% (v/v) water having 20% (w/v) AArn and 20 % (w/v) of AgNO3 in 98% Heptane having 1% (w/v) AOT shows cloud point at 94 °C. This inverse emulsion irradiated for 2 hours results with no particle as in normal temperature the emulsion breaks.
Example 5
2% (v/v) water having 20% (wlv) AAm and 20 % (\yl_v) of AgNO3 in 98% heptane having 8% (w/v) AOT shows cloud point at -7°C. This inverse emulsion irradiated for 2 hours results average particle size of 10.78 nm with S.D 2.36.
Example 6
2% (v/v) water having 20% (w/v) AAm and 20% (w/v) of AgNO3 in 98% heptane having 20% (w/v) AOT shows cloud point at -7°c This inverse emulsion irradiated for 2 hours results average particle size of 10.448 nm with S.D 5.565.
Example 7
2% (v/v) water having % (w/v) AAm and 20 % (w/v) of AgNO3 in 98% Heptane having 8% (w/v) AOT shows cloud point at -7°C. This inverse emulsion irradiated for 2 hours results average particle size of 6 nm with S.D 1.76.
Example 8
2% (v/v) water having 40% (w/v) AAm and 20 % (w/v) of AgNO3 in 98% Heptane having 8% (w/v) AOT shows cloud point at (-7) °C. This inverse emulsion irradiated for 2 hours results average particle size of 9.24 nm with S.D 1.81.
Example 9
2% (v/v) water having 20% (w/v) AAm and 20 % (w/v) of AgNO3 in 98% Heptane having 8% (w/v) AOT shows cloud point at (-7) °C. This inverse emulsion irradiated for 10 minutes results average particle size of23.0I nm with S.D 3.
Example 10
2% (v/v) water having 20% (w/v) AAm and 20 % (w/v) of AgNO3 in 98% Heptane having 8% (w/v) AOT shows cloud point at (-7) °C. This inverse emulsion irradiated for 720 minutes results average particle size of 13.30 nm with SD 7.01.

We claim:
1. A process of preparation of nanoSilver-nanoHydrogel (nSnG) based antimicrobial agent comprising of:
• preparation of water in oil inverse emulsion comprising water with hydrogel forming monomer, silver salt and oil phase;
• keeping said inverse emulsion under gamma irradiation with a dose rate of 0.15-0.20 kGy/hour;
• separating nanoSilver-nanoHydrogel obtained from the irradiation of said inverse emulsion with gamma radiation by chelation by saline water;
• dissolution of the said separated particles in a ketone to obtain the nanoSilver-nanoHydrogel (nSnG).

2. A process as claimed in claim 1, wherein said emulsion is prepared from 1-20%v/v water and 10-40% w/v hydro gel forming monomer and l-20%w/v water soluble silver salt and 80-99% v/v oil phase having 1-20% w/v surfactant.
3. A process as claimed in claim 1, wherein said hydrogel forming monomer is preferably acrylamide.

3. A process as claimed in claim 1, wherein said water soluble silver salt is preferably silver nitrate.
4. A process as claimed in claim 1, wherein said hydro gel forming monomer: silver salt concentration is in a ratio of 2:1 to 1:2 including 1:1.
5. A process as claimed in claim 1, wherein said oil phase is heptane.
6. A process as claimed in claim 1, wherein surfactant is AOT.
7. A process as claimed in claim 1, wherein said inverse emulsion is kept under
gamma irradiation with a dose rate of 0.16 kGy/hour and exposure time 10 to
720 minutes.
8. A process as claimed in claim 1, wherein the inverse emulsion is prepared by
stirring at 1100 r.p.m.
9. A process as claimed in claim 1, wherein said dissolution is done in methyl ethyl
ketone.
10. Particles of nanoSilver nanoHydrogel as and when prepared by the process as
claimed in any of the preceding claims.
11. Particles of nanoSilver nanoHydrogel as claimed in claim 10, wherein the size of
said nanoSilver nanoZHydrogel particle is 0-100nm.
12. A process of preparation of nanoSilver nanoHydrogel substantially as herein
described with reference to the foregoing examples.
13. Particles of nanoSilver nanoHydrogel prepared substantially as herein described
with reference to the foregoing examples.