Field of Invention:
This invention relates to field of medicinal particularly to an improved medicinal device for wound healing by using Silk-protein based bilaminate, used for dressing wound including wound created by burn injuries, skin donor sites, primarily made of crystalline and non-crystalline fibroin, sericin, cross-linking components and anti-bacterial/ anti-inflammatory agents and a method for preparing the same.
Prior Art:
At present pig-skin, chitin, purified collagen sheets etc are used, as a wound dressing which also includes film made of solubilized fibroin, with or without sericin, collagen and chitosan. The collagen based film being in use appears to show inconsistency in nature and wherein the property of collagen depending on the source may lead to high hydrophylicity leading to swelling, possible side effects like bovine spongeform, encephalopathy and mineralization.
As a medical material originating from a protein fibre, silk is under use for a long time especially because of its excellent biocompatibility.
A permeable membrane that comprises crystalline and water insoluble fibroin has been described in Japanese Patents publication nos, 63-246,169 and 1-118,545.
A water insoluble fibroin material described in Japanese Patent publication No,l-118,545 has various uses such as artificial skins, wigs, sweat clothes because of excellent vapour permeability, improved transparency and mechanical strength and desirable affinity to the human body.
Another Japanese patent publication no. 2-233,128 describes that a crystalline substance containing and water insoluble membrane prepared from fibroin and sericin exhibits very good oxygen permeability, mechanical strength, desirable biocompatibility and high stability to the human body and thereby finds use in making contact lenses, artificial skins, blood bags etc.,

On the other hand, Japanese Patent publication no. 56-10,156 describes that a porous membrane is prepared by blending a water-soluble compound such as ethylene glycol, poly alkaline glycol and glycerin to sericin, which exhibits appropriate water absorption properties and vapour permeability required for skin protecting material and improved flexibility.
In these materials containing water insoluble fibroin, the extent of crystallinity is necessarily more than 10% preferably more than 15% so as to keep better mechanical strength. At the same time water content is maintained between 10-60% by weight.
However, these conventional membranes exhibit insufficient flexibility and water absorption properties to use as a wound dressing material for healing a skin defect region, while in the case of porous membranes, the infection controllability is also in question.
This limitation is partially addressed in US Patent Publication No,6,175,053 that talk about amorphous film of crystallinity below 10% containing fibroin and sericin as main components. In order to further improve the healing effect to a wounded region, various healing agents or remedies like disinfectants, cure-accelerating agents were incorporated into the film.
The products based on solubilized fibroin alone along with other additives may not achieve required mechanical strength and such a formulation involve exhaustive processing complexities to achieve complete removal of mineral contaminants, which makes scale up process very tedious. Further more, enhancement of water absorption capacity also would risk chances of moisture absorption by the film from the atmosphere,
Object of the invention (Problem & Solution):
The Principle object of the invention is to develop a silk-protein based bilaminate film for wound healing and method of preparing the same using properties offered both by natural mostly crystalline and amorphous

regenerated silk proteins and other necessary adjuvant. The necessary properties required ideally in the wound dressing like resistance to infection, absorption of wound exudates, acceleration of epithelial cell regeneration, easy exchange of vapors and resistance for moisture absorption from atmosphere and secondary infection preventing components.
Another other object of invention is to provide method to prepare a bilaminate film which has optimum mechanical strength, differential water absorption characteristics, incorporating antibacterial agents and epithelial growth factors.
Novel features of the invention:
A bilaminate film, having least water absorption from the surface exposed to atmosphere when it is covered on the wound and moderate water absorption from the surface in contact with the wound. The surface in contact with the wound also would exhibit antimicrobial properties and help in acceleration of epithelial cell regeneration in terms of enhanced growth rate of fibroblast and keratinocyte cells.
A bilaminate wound covering material made basically from two different types of silk proteins having variation in hydrophylicity. The layer coming in contact with the wound will have capacity to absorb exudates from the wound and at the same time provide suitable epithelial growth factors and conditions for speedy wound healing. The outer layer would provide mechanical strength to the film allows adequate passage and exchange of gaseous material and inhibits entry of moisture from atmosphere when applied on to the wound.
Statement of Invention:
Accordingly the invention provides, a silk-protein based bilaminate film for wound healing and method of preparing the same, consisting of dressing materials used for skin covering and repairing, primarily made of crystalline

and non-crystalline fibroin, sericin, cross-linking components and anti-bacterial/anti-inflammatory agents and a method for preparing the same along with following Properties and Functions.
1. Biocompatibility with the skin with no irritation or allergic symptoms
2. Sufficient mechanical strength so that film does not get torn but still maintains some flexibility.
3. Breathable allowing exchange of gaseous material.
4. Quick absorption of exudates from the wound at the same time resist absorption of moisture from the atmosphere.
5. Have one or two antibacterial component in the formulation that would prevent any secondary infection from setting in.
6. Have an epithelial growth factor in the formulation that would aid better skin fibroblast and keratinocyte proliferation and hence help in quicker wound repair
There has been developed various products keeping in mind one or more of the above factors in mind by different researchers but there is no product having all the above properties.
The product developed being a bilaminate having contrasting property in water absorption between the two layers the present invention scores over other alternatives available at present. The products currently available in market and described in various patents have uniform property at both the surfaces leading to equivalence in water absorption property, which may lead to chances of either infection occurring or least absorption of fluid from the wound depending on the composition.

Detailed description of the invention:
The present invention provides a novel silk protein bases bilaminate film for
wound dressing and treating dermal wounds including wound created by
burn injuries, skin donar sites, primarily made of native crystalline fibroin
and native non-allergenic component of sericin and added low crystalline
fibroin cross linking components and and anti-bacterial /anti-inflamatory
agents of herbal, synthetic or biotechnology origin and sericin of required
molecular weight in some cases. The bilaminate layer with two layers one
exclusively made of crystalline fibroin film one of the side is coated with
cellulosic polymeric material to enhance protection to wound from the
possibility of secondary infection from setting in. The native fibroin film itself
acts as hydrophobic surface disallowing the moisture from entering into the
wound through the bilaminate film. This layer made of native fibroin and
native non-allergenic component of sericin and any other coating material
described earlier and this layer will not come in direct contact with the wound
as it forms the exterior surface of the bilaminate wound dressing system,
Where as the interior layer of the bilaminate wound dressing system is made
of blend film constituted by solubilized fibroin, co-polymer like gelatin,
solubilizer and glidents like glycerin and polysorbates, cross-linking agents
like formaldehyde or glutaraldeyde or salts of alginic acids,
antibacterial/anti-inflammatory agents in the form of herbal extracts of
Centella asiatica or Alovera or Curcoma olanga or Berberis aristata or
Calotropis procera or Garcinia morella or Terminalia arjuna or Terminalia
chebula or Azadirachta indica or Acacia catechu or Andrographis paniculata
or Asparagus racemosus or Embelia ribes or Moringa olifera or Ocimum
sanctum or Morus indica or Morus alba. This interior film may also include
antibiotics of either synthetic origin or bio-technological origin like
Mupirocinn or Silver sulphadiazine or Neomycin. This interior layer contain
sericin of required molecular weight that would help in enhancing epithelial
growth action of solubilized fibroin already present and enhancing the
hydrophilicity of the interior layer.

The proposed invention ensures mechanical strength to the wound dressing and minimizes secondary infection in the form of exterior crystalline fibroin laminate, exudates absorption capability, epithelial growth stimulators, anti-bacterial and anti-inflammatory activities in the form of interior laminate. The ratio of fibroin, sericin of required type and gelatin is standardized while designing interior laminate in such a way that it has got moderate hydrophylicity. Further, this bilaminate wound dressing would allow gaseous exchange and peeling off of the dressing after cure of wound would give negligible pain. Overall this invention would provide a bio-material bilaminate wound dressing with no risk of allerginicity, toxic side-effects and speedy regeneration of epithelial cells at the wounded area.
The bilaminate film of thickness around 0,15 to 0.5mm, more specifically 0.18 to 0.23mm made of bio-material like planar silk sheet consisting mostly of crystalline fibroin coated with a blend film made of soluble fibroin, sericin, gelatin, cross-linking agents, water repelling components, glidents in an appropriate proportion to give desired property to the bilaminate film. High mechanical strength, adequate air permeability, relatively higher water absorption capability of the surface coming in contact with wound and presence of wound healing agents derived either from herbal or synthetic or biotechnological origin constitutes desired property of the bilaminate film. The size of the bilaminate film would vary from a maximum of 36 X 36 cm to a minimum of 3 X 3 cm giving an option of covering various sizes of wounds specially caused by burn injuries.
A typical formulation and bilaminate film making protocol is provided here under;
Natural or woven silk sheet available in the market of required size is selected and peeled, if required to have layers of size 0.02 to 015mm thickness, more specifically between 0.088 to 0.125 mm thickness, dewaxed by passing it through organic solvent like hexane or chloroform or carbon tetra chloride for

time ranging from 10 minutes to 60 minutes. After evaporating all the solvent residues the silk sheet is sandwiched between a stainless steel mesh set up and kept in an autoclave and steamed for 25-90 minutes more specifically 30-55 minutes to remove allergenic sericin fractions. This material is kept ready for lamination at a later stage.
On the other hand the blend film is prepared by selecting fibroin solution available in the market having a molecular weight in the range of 25-250KDa, desalted using transmembrane pressure hallow fibre system, if required. The final concentration of this fibroin solution is brought to around 5-12% either by suitable evaporation techniques or by dilution techniques. 50-80ml of this solution is mixed with 4-14ml of polysorbate of suitable molecular size and 2-7ml glycerol and 6-15g of 25% gelatin aqueous solution and made up with water of pharmaceutical grade to 100ml. The gelatin used is of pharmaceutical grade and of required grade to provide suitable strength to the film. 0.005-0.2% of cross linking agents like glyceraldehyde or glutaraldehyde or salts of alginic acid or zero length polymer is added followed by one of the active principles to an extent of 2% from the range of herbal extract of Centella asiatica or Alovera or Curcuma olanga or Berberis aristata or Calotropis procera or Garcinia morella or Terminalia arjuna or Terminalia chebula or Azadirachta indica or Acacia catechu or Andrographis paniculata or Asparagus racemosus or Embelia ribes or Moringa olifera or Ocimum sanctum or Morus indica or Morus alba or antibiotics from either synthetic origin or bio-technological origin like Mupirocin or Silver sulphadiazine or Neomycin is added to the above solution and stirred to get uniform mix.
The processed silk sheet obtained in the first step is used as a base and the solution prepared in the next step is uniformly coated on to it. The ratio of the solution prepared above to the surface area of the film is maintained in the range of 100ml: 5000-20,000sqcm, The resultant film is dried in a current of filtered air maintained between 20-65 deg centigrade, more specifically in the range of 30-45 degree centigrade until it reaches a residual moisture level of

less than 10%. To the other surface of the base film 5-18% of a suitably selected cellulosic polymers like hydroxy propyl methyl cellulose, hydroxy propyl ethyl cellulose or in general any hydroxy alkyl celluose is coated and taken through a similar drying cycle.
A typical film produced has following properties:
a. Air permeability -14.2 m3/m2/min
b. Breaking load - 680g per 50mmX 8mm film
c. Elongation - up to 20%
d. Water absorbance - Interior side (coming in contact with wound) - 0.15
min/ drop of water and exterior side 2.27 min/drop of water.
e. No irritation observed when film was placed up to 24 hours on wound
created in a rabbit model.
Example
Step 1:
Flat silk sheet containing silk protein sericin and Fibroin in the undegraded native form is procured from the market and suitably cut into the required size say 10 cm X10 cm. The silk sheet obtained has to be properly dewaxed by dipping in organic solvents such as di ethyl ether or chloroform and dried until the traces of organic solvents are removed completely. The water soluble lower molecular weight sericin can be removed by suitably autoclaving the silk sheet.
Step 2:
The Fibroin solution can be prepared by dissolving the fibroin fibers degummed by various techniques such alkali, enzyme , urea etc more specifically the silk cocoons can be degummed by boiled the cocoons in 0.05% Na2C03 at a temperature 80 Degree Centigrade for 1 hour.

The resulting cocoon fibers has to be completely air dried and can be dissolved in various alkaline earth metals such as LiBr or LiSCN or CaCb or MgCh or the like with or without organic solvents such as ethyl alcohol or methyl alcohol etc. More specifically the 10 grams of cocoon fibers can be dissolved by boiling in 100 ml of CaCl2 : Water: Ethanol in the molar ratio of 1:8:2. The solution thus obtained has to be purified to remove the salts and organic solvents either by conventional dialysis bags or by the application of transmembrane pressure or by advanced chromatographic separation techniques and has to be suitably concentrated to obtained the desired fibroin concentration preferably 10 %.
Step 3:
The purified and concentrated fibroin solution obtain by the above method has to be blended with polymers such as PVA or gelatin etc. A typical formulation is as follows
Fibroin solution (10%) :50 ml
Polymer : 5 gram
Humectant :5 gram
Surfactant :5 gram
Cross Unking agent -.O.lgram
Herbal extract : 0.5 grams
Water : 100ml (QS)
Step 4:
The silk protein blend film obtained by the above method has to be coated on the other side with polymer solution such as Methyl cellulose etc and dried suitably.

Patent References:
a. JP2006183231A2: Composite fiber
b. JF2005218460A2: Face mask and production method thereof
c. US20040219630A1; Production of functional polypeptides originating
from silk protein and use thereof
d. US20016175053B1: Wound dressing material containing silk fibroin
and sericin as main component and method for preparing same
e. JP2003171875A2: Sericin fibroin-adhered cellulose article and method
for producing the same.
f. WO03022909A1: Method for the preparation of silk fibroin hydrogels
g. WO0234885A1: Sericin-containing material, process for producing the
same and method of using the same.
h. W09857676A1: Wound covering material containing silk fibroin and silk sericin as the main components and process for producing the same
i. US005951506A: Wound covering material
j. US4818291: Silk-fibroin and human-fibrinogen adhesive composition