Description:TECHNICAL FIELD
The present invention is related to fibroin protein. The present invention specifically provides a method for obtaining nanodispersion of fibroin. The nanodispersion obtained by the method of present invention can be advantageously adopted in biomedical applications because of its stability with ß sheets and ease of processability. The method of obtaining the nanodispersion is economical, environmentally safe and is industrially scalable.

BACKGROUND AND PRIOR ART
Fibroin is a water insoluble protein of general structure of formula I

Formula -I
The fibroin forms an important part of silk produced by various insects including silk moth. The rigid structure of the fibroin renders silk in textile industry, parallelly its similarity with ß- keratin provides advantages in biomedicine. In the recent past, fibroin has been explored for adoption in various biomedical applications like for example in woundcare, tissue engineering, implantable devices, cosmetics.
The water insoluble characteristics of the fibroin is a challenge, which is being addressed by various researchers. The patent document CN102417733 titled “Preparation method of silk fibroin nanospheres” discuss about a method of preparation of fibroin nanospheres. The method is not ecofriendly and expensive owing to the usage of multiple solvents, high temperature and poor yield. In another patent document US4233212 titled “Process for producing a fine powder of silk fibroin” a method of preparing silk powder is disclosed, the method adopts expensive inorganic salt solutions and the steps adopted are not feasible for large scale preparation of fibroin powder.
The patent document CN101736430 titled “Method for preparing silk fibroin nano-fibre with skin-care effect” provides a cumbersome method involving high temperature and inorganic salts, the final product is coupled with vitamin C. The product is specific for skin care effects and cannot be adopted in implants.
The huge demand for adopting fibroin in biomedical applications necessitates to develop an industry scalable method which provides high purity fibroin of nanoscale, cost effectively. The present invention aims to provide a method which provides nanodispersion of fibroin by a simple industry scalable method.
SUMMARY OF INVENTION
Accordingly the present invention provides a method for the preparation of nanodispersion of fibroin comprising steps of-
degumming waste cocoons to remove sericin washing and drying,
hydrolyzing the degummed and dried fibroin in acidic medium and drying,
milling the dried and hydrolyzed fibroin to size of less than 1000micron by cutter,
milling the cutter milled fibroin by attritor milling, and
Adding aqueous solution of buffer to the attritor milled fibroin and repeating the attritor milling and adjusting the pH to 5-8.5 to obtain the fibroin nanodispersion.

Nanodispersion of fibroin obtained by abovesaid method, wherein the fibroin is of particle size ranging from 30 nm to 200 nm. The nanodispersion can be adopted in biomedical applications as it is free of residual impurities and stable because of intact ß sheet structure.
BRIEF DESCRIPTION OF FIGURES
The features of the present invention can be understood in detail with the aid of
appended figures. It is to be noted however, that the appended figures illustrate only
typical embodiments of this invention and are therefore not to be considered limiting
of its scope for the invention.
Figure 1: illustrates the fibroin and particle size at each step of the process. The figures 1(i) and (ii) illustrate the degummed fibroin. The figures 1(iii) and (iv) illustrate the acid hydrolyzed fibroin; the figures 1(v) and 1(vi) illustrate the fibroin obtained after the cutter milling, the figure 1(vii) and figure 1(viii) illustrate the fibroin obtained after dry attritor milling and figure 1(ix) and figure 1(x) illustrate the particle size of the fibroin nanodispersion.
Figure 2: illustrate the size distribution of fibroin in the nanodispersion.
Figure 3: illustrate the FTIR of fibroin powder processed in different ways; Silk fibroin nano dispersion (SFND) indicate the FTIR of nanodispersion of fibroin of present invention and regenerated silk fibroin solution (RSFS) respectively.
DETAILED DESCRIPTION OF INVENTION
The foregoing description of the invention has been presented for the purpose of illustration. The embodiments described are not intended to be exhaustive or to limit the invention to the precise form disclosed, as many alternative variations and sequences are possible in light of this disclosure for a person skilled in the art in view of the figures and description. Additional features and advantages of the disclosure will be described hereinafter which form the focus of the description of the disclosure. It may further be noted that as used herein, the singular “a” “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by a person skilled in the art.
Definition:
Nanodispersion: Dispersion of fibroin in water, wherein the size of the fibroin particles is ranging from 30nm to 200nm.
The present disclosures relates a method for the preparation of fibroin as a nanodispersion. The method comprises acts of degumming waste silk cocoons, hydrolysis, cutter milling sequentially followed by dry and wet attrition milling to obtain the nanodispersion of the fibroin.
In an embodiment of present invention, the method of preparation of the fibroin nano dispersion comprises the steps-
degumming waste cocoons by heating or autoclaving in aqueous medium with or without sodium bicarbonate solution, washing and drying to wash out the sericin protein,
hydrolyzing the degummed and dried fibroin in acidic medium and drying,
milling the dried and hydrolysed fibroin to size of less than 1000 micron by cutter mill,
milling the cutter milled fibroin by dry attritor milling, and
adding aqueous solution of buffer to the dry attritor milled fibroin and repeating the milling and adjusting the pH to 5.0-8.5 to obtain the fibroin nano dispersion.
The figures 1(i)-(x) illustrates fibroin and particle size at each of the abovementioned steps. The figures 1(i) and (ii) illustrate the degummed fibroin. The figures 1(iii) and (iv) illustrate the acid hydrolyzed fibroin; the figures 1(v) and 1(vi) illustrate the fibroin obtained after the cutter milling, the figure 1(vii) and figure 1(viii) illustrate the fibroin obtained after dry attritor milling and figure 1(ix) and figure 1(x) illustrate the particle size of the fibroin nanodispersion.
In an embodiment of present invention, the degumming of the silk cocoon is carried out by heating at a temperature ranging from 115? to 125? in aqueous medium.
In another embodiment of present invention, the degumming of the fibroin is carried out by heating at 121? in aqueous medium under pressure.
In another embodiment of present invention, the sodium bicarbonate solution is 0.2% sodium bicarbonate solution in water solvent.
In an embodiment of the invention, the hydrolysis of the degummed fibroin is carried out in an acidic medium adopting an acid selected from a group comprising Sulphuric acid, hydrochloric acid, Formic acid, Phosphoric acid Nitric acid.
In an embodiment of the invention, the hydrolysis of the degummed fibroin is carried out in sulphuric acid.
In an embodiment of the invention, the hydrolysis is carried out for a period ranging from 30 to 75minutes.
In an embodiment of the invention, the hydrolysis is carried out for a period ranging from 60 minutes.
In an embodiment of the invention, the hydrolysis is carried out heating at a temperature ranging from 90? to 125?.
In another embodiment of present invention, the hydrolysis of the fibroin is carried out by heating at 90?.
In another embodiment of present invention, the milled fibroin is air dried and followed drying in an oven.
In another embodiment of present invention, the milling of dried fibroin is carried out by a cutter mill repeating the milling cycles upto 20 times.
In another embodiment of present invention , the attritor milling is carried out with Yttrium coated Zirconium balls of 5mm.
In still another embodiment of present invention, the attritor milling is carried out for a period ranging from 20hr to 48hr in wet or dry state.
In still another embodiment of present invention, the dry attritor milling is carried out for a period of 24hr.
In still another embodiment of present invention, the attritor milling is carried out for a period of 10hr.
In an embodiment of present invention, the attritor milled fibroin of less than 1000 micron is subjected to wet attritor milling by adding tris hydrochloride buffer in water and milling with Zirconium balls of size varying from 1mm to 10mm to obtain a dispersion.
In an embodiment of present invention, the wet attritor milled fibroin dispersion is subjected to milling for a period ranging from about 15hr to 40hr.
In another embodiment of present invention, pH of the fibroin nanodispersion is adjusted to a value ranging from 5.0-8.5.
In another embodiment of present invention, the buffer of the fibroin nanodispersion is selected from a group comprising Tris buffer of Tris HCl, Sodium bicarbonate buffer, Phosphate buffer a combination of KH2PO4 and K2HPO4,Ammonium buffer of Ammonium Sulfate, Citrate buffer – a combination of citric acid and sodium Citrate and the like.
The present invention provides nanodispersion of fibroin wherein the size of the fibroin is ranging from 30nm to 200nm.
In an embodiment of present invention, the concentration of fibroin ranges from 0.5% to 15% where it contains greater than 99% of dry mass of silk fibroin.
In an embodiment of present invention the viscosity of the nanodispersion of fibroin ranges from 1000 mPas to 40000 mPas at room temperature- 20? to 40?.
In an embodiment of present invention the nanodispersion of fibroin comprises majorly intact ß- sheets to provide stability. As indicated in figure 3, the reconstituted Silk Fibroin (RSFS) which is widely adopted for different application has IR band ranging 1640-1650 cm-1 and corresponds to a-Helical secondary structures, whereas silk fibroin nano-dispersion (SFND) of present invention has IR band 1615-1625 cm-1which corresponds to ß-sheets representing crystalline nature of nanodispersion.
In an embodiment the nanodispersion of fibroin of present invention can be adopted in pharmaceutical, cosmetic and nutraceutical products along with necessary excipients.
In an embodiment the nanodispersion of fibroin can be adopted as an ointment, cream nanodispersion, gel, film, powder and the like along with other active and inactive ingredients and/or excipients.
EXPERIMENTAL
The commercially available waste cocoons are obtained from silk breeding center Reshamandi, Bengaluru, Karnataka and National Silkworm Seed Processing Organization, Bengaluru, Karnataka.
Scanning electron microscopy (Hitachi - S-4300 FESEM) is adopted to evaluate the change in fibre structure after each processing with size range of fibres. After the preparation of nanodispersion of silk fibroin the particle size distribution is measured using dynamic light scattering. The viscosity of nanodisperion is measured using rotating viscometer at room temperature at 20? to 40?. The concentration of Silk fibroin is measured using residue by drying method in hot air oven and the same is validated in Kjeldahl method.
Method of preparation of nanodispersion of fibroin
General method:
1kg of waste silk cocoons of Bombyx Mori is cut and autoclaved at a temperature ranging from 115°C to 125°C in sodium bicarbonate solution for degumming. The degummed cocoons are washed repeatedly and dried. The dried cocoons is suspended in 0.1 % sulphuric acid and autoclaved at 100?, washed repeatedly till the solution is neutral and dried. The dried cocoons are milled by cutter milling to a particle size of less than 1000 microns , followed by dry attritor milling using Yttrium coated Zirconium balls of size ranging 3 mm to 10 mm for about 20hr to 28hr and later by wet attritor milling adopting tris hydrochloride solution and milling for about 24 hr-50hr and neutralizing using a buffer to obtain nano dispersion of fibroin.
Example 1:
1kg of waste cocoons is cut and boiled at 100°C in 0.2% bicarbonate solution. Then the cocoons are washed repeatedly and dried. The dried cocoons are taken in 10 litre of water containing 100 ml of concentrated sulphuric acid and autoclaved for 3 hr at 100 °C. Later it is washed repeatedly till it become neutral and dried. The dried cocoons are subjected to cutter milling for about 20 cycles till the particle size of the cocoons is less than 1000 micron. The cutter milled fibroin particles are milled by attritor milling with 2500 grams of Yttrium coated Zirconium balls of 5 mm size in an attritor mill for 24hr. The attritor milled fibroin powder is added a solution of tris hydrochloride buffer pH 8.5 in water, and subjected to attritor milling again with Yttrium coated Zirconium balls of 10 mm size and milled for 48 hours to obtain the nanofibroin dispersion.
Example 2:
1 kg of cut cocoons are autoclaved at 121°C and 15 lb pressure in 20 lit 0.2% of sodium biocarbonate medium followed by repeated washing with distilled water and dried. 1 kg of dried degummed cocoon is taken in a medium of 10 litre of water with 200 mL of Sulphuric acid, and autoclaved at 121 °C temperature at 15 lb pressure for 2 hour. The hydrolysed fibres are washed repetitively till it became neutral and dried. The hydrolyzed cocoons are subjected to multiple cycles of cutter milling to beget fibroin of size, less than 1000 microns. Said milled fibres are dry milled with Yttrium coated Zirconium balls of 3 mm/5mm/10mm in the w/w ratio of 2-%/50%/30% size for 48 hours. Then 20 litres of bicarbonate buffer of pH 8 is added and wet milled for 24 hours.
Example 3:
1 kg of cut cocoons were autoclaved at 121°C and 15 lb pressure in 20 lt purified water followed by repeated washing with distilled water and dried. 1 kg of dried degummed cocoon is taken in a medium of 10 litre of water with 500 mL of formic acid and heated at 90 °C temperature for 4 hour. The hydrolysed fibres are washed repetitively till it become neutral and dried. The hydrolyzed cocoons are subjected to multiple cycles of cutter milling to get the size less than 1000 microns. The primary milled fibres were dry milled with Yttrium coated Zirconium balls of 3 mm/5mm/10mm size for 48 hours Then 20 litres of Citrate buffer of pH 5 is added and wet milled for 24 hours.
Example 4:
1 kg of cut cocoons are heated at 100°C in 20 lt of purified water followed by washed repeatedly with distilled water and dried. 1 kg of dried degummed cocoon is taken in a medium of 10 litre of water with 50 mL of Sulphuric acid and autoclaved at 121 °C temperature at 15 lb pressure for 4 hour. The hydrolysed fibres were washed repetitively till it become neutral and dried. The hydrolyzed cocoons are subjected to multiple cycles of cutter milling to get the size less than 1000 microns. The primary milled fibres are wet milled with Yttrium coated Zirconium balls of 3 mm/5mm/10mm size for 48 hours bicarbonate buffer pH 8.
Example 5:
1 kg of cut cocoons are autoclaved at 121°C and 15 lb pressure in 20 lt of 0.2% of sodium biocarbonate solution followed by repeated washing with distilled water and dried. 1 kg of dried degummed cocoon is taken in a medium of 10 litre of water with 100 mL of Sulphuric acid, and boiled for 4 hours at 90 °C temperature. The hydrolysed fibres are washed repetitively till it become neutral and dried. The hydrolysed cocoons were subjected to multiple cycles of cutter milling to get the size less than 1000 microns. The primary milled fibres are dry milled with Yttrium coated Zirconium balls of 3 mm/5mm/10mm size for 48 hours Then 20 litres of bicarbonate butter of pH 8 is added and wet milled for 24 hours.
Analysis of nano dispersion of fibroin on purity.
The particle size distribution is measured using dynamic light scattering ( figure 2). As indicated the size distribution is found to be between 40-200 nm with a Z-average of 86.27 nm the overall distribution is found to be optimal with a Poly dispersity index (PDI) of 0.176.
Analysis of nano dispersion of fibroin on beta sheets
Regenerated Silk Fibroin Solution (RSFS) which is widely adopted for different application has IR band ranging 1640-1650 cm-1 which corresponds to a-Helical secondary structures, whereas Silk Fibroin Nano dispersion (SFND) of present invention has IR band 1615-1625 cm-1 (figure 3)which corresponds to ß-sheets representing crystalline nature of nanodispersion.
Information on the other characteristics like stability and viscosity
The nanodispersion is stable and its pH, viscosity does not alter upon storage at room temperature, ranging from 20? to 40? upto two years. However the nanodispersion in its current form needs preservatives for stabilization against microbial contamination. The use of preservatives can be minimized by storing the nanodispersion at 4°C. Viscosity of nanodispersion depends on the concentration of nanofibroin in the nanodispersion. A nanodispersion of fibroin with concentration of nanofibroin ranging from 5-15% w/w, is having viscosity of 1000-40000 mPas at room temperature- 20 ? ranging from 40?.
, Claims:WE CLAIM
A method of obtaining nanodispersion of fibroin, said method comprising acts of –
degumming waste cocoons to remove sericin washing and drying,
hydrolyzing the degummed and dried fibroin in acidic medium and drying,
milling the dried and hydrolyzed fibroin to size of less than 1000micron by cutter mill,
milling the cutter milled fibroin by attritor milling, and
adding aqueous buffer solution of pH ranging from 5.0-8.5 to the attritor milled fibroin and repeating the attritor milling to obtain the fibroin nanodispersion.
The method as claimed in claim 1, wherein the fibroin nanodispersion is of particles of size ranging from 30nm to 200nmand concentration of fibroin ranging 0.5-15% w/v.
The method as claimed in claim 1, wherein the nanodispersion of fibroin comprises ß sheets and viscosity ranging from 1000-40000 mPas.
The method as claimed in claim 1, wherein the degumming of the silk cocoon is carried out by heating at a temperature ranging from 115? to 125? with or without pressure.
The method as claimed in claim 1, wherein the degumming is carried out in aqueous medium either in the presence or absence of sodium bicarbonate.
The method as claimed in claim 1, wherein the hydrolyzing the degummed fibroin is carried out with an acid selected from a group comprising Sulphuric acid, Hydrochloric acid, Formic acid, Phosphoric acid Nitric acid.
The method as claimed in claim 1 and 6, wherein the hydrolysis is carried out for a period ranging from 30 to 75minutes.
The method as claimed in claim 1, wherein the milling of dried fibroin is carried out by a cutter mill repeating the milling cycles upto 20times.
The method as claimed in claim 1, wherein the attritor milling is carried out with Yttrium coated Zirconium balls of 3-10mm.
The method as claimed in claim 1, wherein the attritor milling is carried out for a period ranging from 20hr to 48hr in dry or wet state.
The method as claimed in claim 1, wherein the attritor milled fibroin is subjected to wet attritor milling by adding tris hydrochloride solution in water and milling with Zirconium balls of size varying from 3mm to 10mm to obtain a dispersion.
The method as claimed in claim 1, the attritor milled fibroin dispersion is subjected to wet attritor milling for a period ranging from 24hr to 48hr.
The method as claimed in claim 1, wherein the pH of the fibroin nanodispersion is adjusted by adopting a buffer selected from a group comprising Tris buffer, Sodium bicarbonate buffer, Phosphate buffer, and Ammonium buffer.
Nanodispersion of fibroin obtained by method claimed in claim 1, wherein the fibroin is of particle size ranging from 30nm to 200nm.