DESCRIPTION
Field of the Invention
The invention relates to a process for the formulation of a polymer-silk nanocomposite
composition comprising at least the following steps: preparing first nanocrystalline silk from
degummed silk fibres, preparing a physical mixture of nanocrystalline silk in a suitable
medium, melt compounding to obtain proper dispersion of nanocrystalline silk into the polymer
melt, extrusion at certain temperature and screw rotation to obtain the nanocomposite. The
nanocomposite is sustainable, thermally-stable biomaterial which is hydrophobic and
compatible with a wide range of synthetic and natural polymers. Nanocrystalline silk-PLA
nanocomposites have enhanced water vapour barrier and mechanical properties relative to
PLA, and are potentially biocompatible and recyclable. Nanocrystalline silk-PLA
nanocomposites can be suspended in many organic solvents or dried to form a solid substrate
that can be processed using conventional polymer processing techniques to develop three
dimensional structures.
Background of the Invention
It is difficult to imagine the current world without plastics. Products range from baby bottles
to packaging materials to components in aircraft. While polyolefins have been the basis of
almost all commercial plastic development, questions have been raised about their long-term
usage due to concerns over recycling, health and environmental responsibility—e.g., the need
to use chemicals such as bisphenol A (BPA), an endocrine disruptor, in the manufacture of
polycarbonate plastics and epoxy resins.
From the past few decades, 'composite materials' have been prepared by adding minerals to
all kinds of plastics.1 These materials when compared to bulk materials have improved

mechanical and thermal properties. Now with the ability to synthesize and fabricate
nanomaterials a new class of materials has come into picture which has superior properties
required for high performance applications. The possible applications for such materials are
packaging, automobiles, portable electronic devices, etc.
Now research is focussed on producing nanoparticle-filled polymer composites in which
nanoparticle has any one dimension comparable to polymer chain dimensions, has a high aspect
ratio and excellent dispersibility. Few nanoparticles have now been extensively researched to
improve mechanical properties, electrical and thermal conductivity such as carbon nanotubes
(CNT), carbon black, alumina, silica, inorganic clays, etc.
Silk is composed of fibroin and sericin, with fibroin accounting for about 75% of the total
weight. Silk fibroin is a natural protein, mainly composed of glycine, alanine, serine and other
amino acids, which has good biocompatibility, biodegradability and low toxicity and has broad
application prospects in biological and medical fields. Fibroin helps in lowering blood
cholesterol levels, promoting insulin secretion, lowering blood pressure and prevention of
Alzheimer's disease, and has anti-bacterial and other physiological functions.
Significant efforts have been made to develop bioplastics and biomaterials from renewable
resources. Nanocrystalline silk is highly crystalline silk isolated from mulberry or non-
mulberry silk. Polylactic acid (PLA) is a thermoplastic, aliphatic polyester made up of lactic
acid (2-hydroxy propionic acid) building blocks. PLA is derived from renewable plant sources,
such as starch and sugar, and has potential applications in medicine, engineering and food and
beverage packaging. The degradation of PLA can be achieved through the hydrolysis of the
ester bonds without the need for enzymatic treatment. However, PLA has certain limitations

compared to polyolefins, particularly during processing, which has greatly limited its use. PLA
is essentially hygroscopic and possesses low thermal resistance. Nano-scale reinforcement can
be used to enhance the rheological, mechanical and physical properties of polymers, thereby
improving their processability, functionality and end-use performance. Nanocrystalline silk
can provide high performance reinforcement owing to its large specific surface area, high
strength and high surface reactivity. To achieve this, two critical conditions are required: (i)
excellent dispersion of the nanocrystalline silk within the polymer matrix, and (ii) perfect
compatibility between the two in order to produce excellent interfacial mechanics. Resulting
. flexible barrier packages can enclose various types of contents that are rather sensitive such as
electronic, medical materials, eatables food and drinks.
In view of silk/PLA nanocomposite water vapour barrier films, no prior art is available. One
patent, CN101053670A, discusses about blending of the two materials in a common solvent
and then casting films. Here silk was the main component (90-99%) while the rest is polymer.
Reported moisture permeability is 94.7-113.2 g/m2.h. Patent WO2011008842A2 reports
electrospun silk material systems for wound healing using silk fibroin/ polyethylene oxide
blended materials, and the resulting materials are suitable for biomedical applications such as
wound dressings. Here oxygen transmission rate of the silk mats is more than 15460
cm3/m2/day; and water vapour transmission rate of the silk mats is more than 1934 g/m2 /day.2
Similar water vapour transmission rate data were obtained using different combinations of silk-
chitosan (2500 g/m2 /day)3, silk-collagen (1013.8 g/m2/day)4, silk-PLA(835 g/m2 /day )5.
Another report of blending silk sericin with whey protein isolate gives a reduction of ~2 folds,
from 22.2 to 12.2 g mm/m2day kPa, as the sericin concentration increased from 0 to 0.1%, and
this decrease slowed down from 0.1 to 0.4%.6

By using silk fibre as reinforcement for biodegradable polymer, the mechanical properties do
show a substantial change. Cheung et al. demonstrated that the use of silk fibre to reinforce
PLA can increase its elastic modulus and ductility by 40% and 53 %, respectively. Lee et al.,
have shown similar results by adding silk fibres to another biodegradable polymer,
poly(butylene succinate).
Brief Description of the Invention
The present invention provides nanocrystalline silk fibroin nanoparticles with well-defined
disk-like morphology and diameter in the range of 20-200 nm which can be seen in figure 1.
The invention also provides a polylactic acid and synthesized nanocrystalline silk fibroin
composite film, which is made with PLA as the main raw material. The composite film contains
95 to 99.5% PLA, the rest being nanocrystalline silk fibroin. The invention has the advantages
that the prepared composite film is non-toxic, biocompatible, biodegradable, has good
mechanical properties, UTS of 62.2-58.0 MPa, is semicrystalline, has a thickness of 100±0.1
urn, and a water vapour transmission rate of 7.9-11.4 gm.mm/m2.day.
The invention also provides a method for preparing polylactic acid and nanocrystalline silk
fibroin composite film. The polylactic acid is dissolved in chloroform solvent, to which
nanocrystalline silk fibroin chloroform solution is added. Now the polylactic acid and
nanocrystalline silk fibroin solution of a certain proportion, stir, pour into a petri dish and then
transferred to the drying temperature prepared polylactic acid and nanocrystalline silk fibroin
composite film. The polylactic acid is dissolved in chloroform solvent, polylactic acid and
chloroform solvent mass ratio of 0.3 to 0.5:10 volume, polylactic acid and nanocrystalline silk
fibroin in a ratio in which the quantity of nanocrystalline silk fibroin and polylactic acid ratio
is 0.5 to 10:100.

It is therefore an object of the present invention to provide a nanocomposite with an improved
and/or higher water vapour barrier properties. It is also an object of the present invention to
provide a process for preparing a nanocomposite with improved water vapour barrier
properties. These objects can be met in any combination by the present nanocomposite and the
process for their production. By selecting the appropriate composite combination, high barrier
properties can be easily achieved using standard extrusion processes.
An aspect of the invention provides a nanocomposite comprising a polymer matrix consisting
of a biodegradable polymer, say polylactic acid and at least 0.05% by weight of nanocrystalline
silk, relative to the total weight of the nanocomposite. According to a second aspect, the
invention provides formed articles comprising the nanocomposite according to the first aspect
of the invention. According to a third aspect, the invention encompasses a process for preparing
the nanocomposite according to the first aspect of the invention, comprising the steps of:
(a) providing a biodegradable polymer, wherein the polymer has preferably narrow molecular
weight distribution;
(b) providing at least 0.05% by weight of nanocrystalline silk, relative to the total weight of the
nanocomposite; and
(c) blending the provided nanocrystalline silk with the provided polymer to obtain the
nanocomposite according to the first aspect of the invention.


FIG. 2 is a scanning electron microscope image at 50000x magnification of a nanocomposite
comprising 0.5% by weight of nanocrystalline silk and polylactic acid.


FIG. 3 is an atomic force microscope image (5x5 urn) of a nanocomposite comprising 0.5% by
weight of nanocrystalline silk and polylacii; acid.
Detailed Description of the Invention
The invention relates to a process for the preparation of a material with improved water vapour
barrier properties. The invention further relates to the material with improved water vapour
barrier properties and its uses. Materials with good barrier properties find use in the packaging
industry so as to protect the materials and aricles (together "contents") inside the packaging
against unwanted influences from the outside A material aimed for packaging should protect
the contents inside against liquids, vapours and/oi gases in the atmosphere on the outside of
the package. Depending on the sensitivity of the contents the requirements put on the barrier
properties of the packaging material are differeni. Soms types of contents are sufficiently
protected by a packaging material with low or medium harrier properties, while others need
high barrier properties to keep an acceptable qmality over a sufficient period of time.

Disclosed herein are flexible water vapour barrier packages composed of materials that are free
of virgin, petroleum-based compounds. The flexible water vapour barrier package is a
nanocomposite that is 100% bio-based. This nanocomposite can be extruded. Ink/screen
printing can optionally be deposited on either side of the extruded material. This flexible water
vapour barrier package is able to enclose various classes of contents that are rather sensitive
such as electronic, medical materials, eatables (food and drinks) like fruit juice, beer,
carbonated soft-drinks and food-stuff that needs to stay crispy for example chips and cereals,
personal care products such as wipes, shampoo, conditioner, skin lotion, shave lotion, liquid
soap, bar soap, toothpaste, and detergent.
To obtain materials with improved water vapour barrier properties sometimes a layer of metal
or metal-oxide or polyvinylidene chloride (PVDC) is applied. However the metal layers that
are used are non-transparent, cause environmental concern as they make recycling difficult and
make the contents non- microwaveable. Metal oxide layers are easily damaged, expensive and
are difficult to produce reliably. PVDC type of barrier films cause environmental concerns
because of their chlorine content. Therefore there is a need for other and/or better materials
with excellent water vapour barrier properties. A way to improve barrier properties is described
herein in which "polymer-silk nano-composite" means composite with polymer and silk
particles wherein the silk has at least one of its dimensions, such as length, width or thickness,
in the nanometre range.
With "silk" is meant the wide range of continuous filaments spun by several species of
Lepidoptera and Arthropoda. Examples of silks suitable for use in the present invention are
wild variety and mulberry silks. Silk used in the present invention has been modified wherein
amorphous portion is removed by acid hydrolysis. More preferably Antheraea assama silk is

used in the present invention, because with A. assama the final film appearance and barrier
properties are better. Although, in principle, all kinds of nano-sized silk are suitable for use in
the present invention, it was found that silks with a certain particle shape give better results in
improving the barrier properties. Therefore a silk with average particle size of less than 200
nm is preferred and silk with average particle size of -50 nm is more preferred.
The nanocrystalline silk can be mixed with suitable medium under normal shear or under high
shear conditions. When using a high shear process, preferably a homogenization process is
used in which the silk mixture is prepared typically at a shear rate of 10,000 - 20,000 rpm.
When a normal shear mixing process is used the shear rate is generally in the range of 50-1000
rpm. A suitable range for the solid content of the nanosized silk can be 0.1-10 wt%, and
preferably 1.0-5 wt%.
Polymer-silk composite mixture can also be obtained by polymerization of lactide in
combination with silk. Mixing can occur by melt extrusion as well as solution evaporation
technique. During the mixing step the viscosity of the combination can increase. The
polymerization is stopped when the characteristics for the desired polymer are reached.
The use of polymer-nanocrystalline silk composite composition according to the present
invention improves especially the barrier properties against water vapour. A substrate coated
with a polymer-nanocrystalline silk according to the present invention has a water vapour
permeation that is lower than the unfilled polymeric film. This improvement can easily be
reached in a one-step process. The polymer-nanocrystalline silk can advantageously be used in
blends with polymers. They can also be used in coating all kinds of surfaces.

Silk Fibroin Degumming
To obtain degummed silk fibres, cocoons of A. assama were first cleaned to remove eggs and
plant debris and then boiled twice using 0.5% (w/w) sodium carbonate (Na2CO3) at 98 °C for
30 min. The water soluble sericin is removed from the cocoon pieces while the water-insoluble
fibroin forms a mass of fibres. The fibre then washed multiple times in de-ionized water to
remove salts. Later degummed fibre was dried overnight at 60 °C in hot air oven to remove
water.
Chemical Hydrolysis- Sonication setup
The dried Muga fibre was cut into small fibrils using high speed universal disintegrator. The
nanocrystalline silk was produced by adding fibrils into a round bottom flask containing
aqueous sulfuric acid solution (60 wt%) at 45 °C. The contents were stirred for 2 hours at a
high speed (~500 rpm) using mechanical stirrer and subjected to sonication. The temperature
was maintained by continuous circulation of water.8 Upon completion of reaction, the
hydrolysis was stopped by addition of chilled de-ionized water. The hydrolyzed material was
washed multiple times with de-ionized water followed by centrifugation (three cycles, each at
12,000 rpm for 15 min each) and then dialyzed under running tap water for 72 h to ensure
removal of free acid and a final pH of ~7. Insufficiently hydrolyzed fractions were removed by
filtration. The nanocrystalline silk suspension in deionized water was then freeze-dried after
quench freezing with liquid nitrogen to obtain dried nanocrystalline silk powder.
Nanocrystal (Size, Morphology and Molecular Conformation).
The size and surface morphology of silk nanocrystals were assessed after suspension of dried
powder in ultrapure water using transmission electron microscopy (TEM) with a JEM-2100
system (JEOL, USA) and atomic force microscopy (AFM) in tapping mode with an Agilent
AFM 5500 system (Agilent Technologies, USA). For TEM, Freshly prepared water suspension

of silk nanocrystals was dropped onto a 200 mesh carbon-coated copper grid (Pacific grid, SF,
USA) and allowed a short incubation period (5 min) at room temperature. In order to investigate
the crystalline structure in more detail, selected area electron diffraction (SAED) was
performed on the same TEM instrument.
The solid state 13C NMR spectroscopy for fabricated nanocrystalline silk was performed using
Bruker Avance 500 MHz spectrometer. The chemical shifts near 20 ppm are attributed to the
Ala Cβ which corresponds to a P-sheet conformation; peaks at 46.9 ppm, 42 ppm, 63.8 ppm
and 170.4 ppm correspond to the Ala Cα , Gly Cα , Ser Cp and Ala C=0 respectively. These
peaks are the characteristics of P-sheet conformation of the isolated nanocrystalline silk.
Preparation ofsilk-PLA nanocomposite
Nanocomposites were produced by melt-compounding of PLA (grade 2003D purchased from
Nature Works, USA, having D-Lactic content -4%) with nanocrystalline silk at 195 °C with a
recycle time of 1 min at 100 rpm using a twin screw extruder (Haake MINILAB II, Thermo
Scientific Corporation, Germany).


Preparation of nanocomposite films by solvent casting
The extrudate obtained from melt compounding of PLA and nanocrystalline silk was cut into
small pieces and measured quantities were added to chloroform and subjected to magnetic
stirring. After a period of 24 hours, the stirring was stopped and the flask was subjected to
vacuum to break the small air bubbles produced during stirring. Thereafter this liquid was
poured onto a Teflon petriplate which was kept for drying in a fume hood. Upon drying the
film was peeled off from the petriplate and conditioned under vacuum at 38°C for a period of
72 hours. Upon conditioning these films were tested for water vapour barrier properties.
Preparation of 3-D structures of nanocomposite using injection moulding
The melt from the extruder was collected into the cylinder collector of injection moulding after
1 min to 10 min of recycle at 195-210 °C. The cylinder temperature was maintained at 190-
205°C. The material was pushed with the help of high pressure of 500-700 bar into the mold
maintained at 80-110 °C. The post pressure was maintained in the range of 500-700 bar for 5-
60 sec). The 3-D dumbbells were prepared of dimensions as per ISO standard. These dumbbells
were tested for mechanical properties analysis using universal tensile testing machine.
In a preferred embodiment, the melt flow index (MFI) of the nanocomposite is at most 20 g/10
min, for example at most 16 g/10 min, for example at most 12 g/10 min, preferably at most 9
g/10 min, with the MFI being measured by the ASTM D-1238 standard at a temperature of
190° C and a load of 2.16 kg.
In an embodiment, the nanocomposite has a density of from 1.260 to 1.290 g/cm3, as
determined with the ISO 1183 standard.

In a preferred embodiment of the invention, the material has a weight average molecular weight
(Mw) of at least 60,000 Da, preferably at least 100,000 Da, more preferably at least 200,000 Da
as determined by size exclusion chromatography (SEC).
In a preferred embodiment of the invention, the polymer has a polydispersity index of at least
2.00, preferably of at least 1.5, more preferably of at least 1; The polydispersity index is defined
as the ratio of the weight average molecular weight (Mw) to the number average molecular
weight (Mn).
The present invention is illustrated with the following non-limiting examples. In all examples,
the wt% nanocrystalline silk given refers to the amount of nanocrystalline silk as percentage
of the total weight of the solid polymer and nanocrystalline silk.
EXAMPLES
Example 1 Silk degumming
10 g of starting silk material was added to 400 ml of deionized water and 0.5% (w/w) of
anhydrous sodium carbonate was added at a temperature of 98 °C into the water bath. The
mixture was cooked for 30 minutes, and then cleaned with distilled water; this process was
repeated twice. The cooked silk was soaked in distilled water for 12 hours, shifted to the rotary
dryer for 10 minutes, and the resulting material was placed in a drying oven at 60 °C for 24
hours.

Example 2 Chemical hydrolysis under magnetic stirring
2 g of the degummed silk fibrils of Example 1 was added to 20 ml sulfuric acid solution
(concentration 64 wt%) where the material-to-acid solution ratio was kept to 1:25. The
hydrolysis was carried out for 2 hours at a temperature of 50 °C wherein the contents were
stirred using a magnetic stirrer (~500 rpm). Upon completion of reaction the contents were
diluted with 100 ml of chilled distilled water, and then the hydrolyzed solution was transferred
to centrifuge tube and centrifuged 3 times at a speed of 12,000 rpm for 15 min. Then the
solution was transferred to 12,000 MWCO dialysis bag, dialyzed for 72 hours against tap water
to a solution of pH = 7 to obtain nanocrystalline silk fibroin suspension. The particle size of
the nanocrystalline silk fibroin was in the range of 200-500 nm.
Example 3 Chemical hydrolysis under mechanical stirring
2 g of the degummed silk fibrils of Example 1 was added to 20 ml sulfuric acid solution
(concentration 60 wt%) where the material-to-acid solution ratio was kept to 1:10. The
hydrolysis was carried out for 2 hours at a temperature of 45 °C wherein the contents were
stirred using a mechanical stirrer (-900 rpm). Upon completion of reaction the contents were
diluted with 100 ml of chilled distilled water, and then the hydrolyzed solution was transferred
to centrifuge tube and centrifuged 3 times at a speed of 12,000 rpm for 15 min. Then the .
solution was transferred to 12,000 MWCO dialysis bag, dialyzed for 72 hours against tap water
to a solution of pH = 7 to obtain nanocrystalline silk fibroin suspension. The particle size of
the nanocrystalline silk fibroin was in the range of 150-200 nm.
Example 4 Chemical hydrolysis under mechanical stirring and sonication
2 g of the degummed silk fibrils of Example 1 was added to 20 ml sulfuric acid solution
(concentration 60 wt%) where the material-to-acid solution ratio was kept to 1:10. The

hydrolysis was carried out for 2 hours at a temperature of 45 °C wherein the contents were
stirred at a high speed (-900 rpm) using a mechanical stirrer and subjected to sonication. The
temperature was maintained by continuous circulation of water in a sonication bath. Upon
completion of reaction the contents were diluted with 100 ml of chilled distilled water, and
then the hydrolyzed solution was transferred to centrifuge tube and centrifuged 3 times at a
speed of 12000 rpm. Then the solution was transferred to 12,000 MWCO dialysis bag, dialyzed
for 72 hours against tap water to a solution of pH = 7 to obtain nanocrystalline silk fibroin
suspension. The particle size of the nanocrystalline silk fibroin was in the range of 50-100 nm.

Example 5
Nanocomposites were produced by melt-compounding of PL A (having D-Lactic content ~4%)
with fabricated nanocrystalline silk at 195 °C with a recycle time of 1 min at 100 rpm using a
twin screw extruder (Haake MINILAB II, Thermo Scientific Corporation, Germany). The
extrudate obtained was later cut into small pieces and measured quantities were added to
chloroform and subjected to magnetic stirring. After a period of 24 hours, the stirring was
stopped and the flask was subjected to vacuum to break the small air bubbles produced during

stirring. Thereafter this liquid was poured onto a Teflon petriplate which was kept for drying
in a fume hood. Upon drying, the film was peeled off from the petriplate and conditioned under
vacuum at 38°C for a period of 72 hours. After conditioning, these films were tested for water
vapour barrier properties. The present invention is illustrated with the following non-limiting
examples in Table 2. In all examples, the wt% nanocrystalline silk given refers to the amount
of nanocrystalline silk as percentage of the total weight of the solid polymer and
nanocrystalline silk.


60 sec.
Example 6
Nanocomposites were produced by melt-compounding of PLA (having D-Lactic content ~4%)
with fabricated nanocrystalline silk 195 °C with a recycle time of 1 min at 100 rpm using a

twin screw extruder (Haake MINILAB II, Thermo Scientific Corporation, Germany). The
extrudates was then collected in a cylinder at 200 °C and placed inside injection moulding
machine (Minijet) where mould temperature was set to 90 °C. The obtained dumbbells were
tested for mechanical properties using universal tensile testing machine. The present invention
is illustrated with the following non-limiting examples in Table 3. In all examples, the wt%
nanocrystalline silk given refers to the amount of nanocrystalline silk as percentage of the total
weight of the solid polymer and nanocrystalline silk.
Table 3: Comparison of mechanical properties of PLA and PLA-silk nanocomposites.


Example 7
Surface hydrophobicity is a very important property for PLA-based materials used in
applications such as food packaging films exposed to high humidity conditions. Surface
interactions between a liquid drop and a PLA substrate cause the liquid to spread and the liquid
cohesive forces cause the drop to dewet. The contact angle is determined by the competition
between these two forces and is generally used to determine wettability. Nanocrystalline silk
is highly hydrophobic material having a water contact angle as high as -130°. The
incorporation of nanocrystalline silk into the PLA matrix leads to an increase in the water
contact angle of the PLA substrate surface (~78°). That is, as the nanocrystalline silk
concentration increased from zero to 0.5%, the measured CA increased from ~78.3 ° to ~82.1°.
Then, as the nanocrystalline silk concentration continued to increase to ~5%, the water contact
angle further increased to ~90°. Better hydrophobicity (with higher water contact angle) may
induce reduced water contact/interaction which would promote dewetting of the composite
film surface and could also help in improvement of water vapour barrier properties thus
resulting in better packaging material.

WE CLAIM:
1. a nanocrystalline silk and polyester based composites formulation comprising
(i) crystalline part of silk fibroin containing hydrophobic amino groups (A) and polyester
including poly (lactic acid) (B) in the weight ratio of 1:99 to 50:50 as additive for reducing
water vapour permeability and improvement in mechanical properties of cast films
(ii) when diluted in synthetic hydrophobic polyester comprising of backbone of
heteroatoms.
2. The formulation as claimed in claim 1 can be a melt extruded film.
3. The films as claimed in claim 1 yield higher contact angle.
4. The process as claimed in claim 1 wherein nanocrystalline silk can be dispersed in
synthetic hydrophobic polymer films by using solution casting process or melt extrusion
process.
5. The process as claimed in claim 1 wherein nanocrystalline silk can adopt shapes including
spherical, planar, rod and in combination thereof
6. The process as claimed in claim 1 wherein A-B can be dispersed in synthetic hydrophobic
polymer films in the range of 20 nanometre to 10 micrometre particle size.
7. The poly (lactic acid)-silk nanocomposite films as claimed in claim 1 yield higher
Young's modulus.
8. The process as claimed in claim 1 where 0.5 weight percentage to 20 weight percentage
dispersion of A-B in synthetic hydrophobic polymers shows reduction in water vapour
permeability compared to its pristine synthetic hydrophobic polymers.

9. The process as claimed in claim 1 wherein melt compounded poly lactic acid
with nano-silk based film shows improved thermal, mechanical properties than
solution casted poly lactic acid films.
10. nano-silk claimed in claim 1 prepared by breaking crystalline and amorphous
part through acid hydrolysis yield disc diameter as low as 30 nanometre.