A stable sericin biopolymer composition and preparation method thereof

Abstract

The invention relates to a stable sericin biopolymer composition comprising a sericin, a cross-linking agent and optionally a catalyst. The stable sericin biopolymer composition is prepared by freezing-thawing process. The process of cross-linking sericin using the freeze- thaw cycle does not require curing at high temperature or under strong pH thus preventing the damage to the sericin proteins. The stable sericin biopolymer is used for preparation of cost effective sericin substrate platform such as gels, films, scaffolds and electrospun films for 3D cell culture and other biomedical applications.

Description

TITLE OF THE INVENTION: A Stable Sericin Biopolymer Composition and Preparation Method ThereofCROSS-REFERENCE TO RELATED APPLICATION:

[0001] The present application claims priority from the Complete Application No. IN 202441043965 filed on June 06, 2024, the full disclosure of which is hereby incorporated by reference herein.FIELD OF THE INVENTION

[0002] The present invention relates to a stable sericin biopolymer composition and its method of preparation. The stable sericin biopolymer composition of the present invention is used for preparing sericin substrate platforms such as gels, films, scaffolds and electrospun films for 3D cell culture and other applications.BACKGROUND OF THE INVENTION

[0003] Traditionally, 2D in-vitro cell culture system has been used for cancer research and other cell based applications. However, this model cannot be translated into in vivo animal studies or clinical trials to ideally represent physiological conditions. Also, it fails to mimic the natural microenvironment due to lack of cell-cell and cell-matrix interaction and communications. To overcome these limitations, 3D cell culture systems are increasingly developed in research and have become essential for tumor research, tissue engineering and basic biology research. 3D cell culture has received much attention in the field due to its ability to mimic tissue structure and function.

[0004] In vitro 3D cell culture has evolved as a transition step that bridges the gap between the 2D in-vitro and in-vivo animal studies which are crucial steps in drug discovery, vaccinedevelopment, pharmaceuticals, drug dosage studies, toxicology assays, and cancer research and to understand the host pathogen interactions.

[0005] 3D cell culture or spheroids growth are the dense 3D aggregates of cells that more or less accurately recapitulate cells in the native environment. 3D cells exhibit extensive cellcell adhesion, retain their endogenous extracellular matrix and have properties that closely mimic their in-vivo tissue counterparts. As cells grow in 3D pattern in the living system, the 3D cell culture also show similar characteristics to cells and organs of the human body establishing cell-cell and cell- extracellular matrix (ECM) interactions. Given their similarities with in-vivo systems, 3D cell culture models are expected to become an optimized and efficient biotechnology platform for studying human physiology and host pathogen interactions.

[0006] Culturing cells in 3D ensures natural cell polarity where growth is not limited to a single plane. The surface area over which the cells adhere and cell-cell communication takes place will be enhanced. As the cells grow in close proximity with ECM, the accessibility to the local concentrations of important cytokines, enzymes, growth factors are greater than the cells growing in 2D monolayers. 3D cell growth have greater control over cell behavior and function due to the accessibility of ECM ligands to spatially oriented cells that afford simultaneous stimulation of several signal transduction pathways.

[0007] Substrates that serve as ideal 3D microenvironment include polymer scaffolds, hydrogels, porous cellulose beads, electro spun scaffolds, 3D printed scaffolds and decellularized tissues. These substrates mimic the in-vitro microenvironment, sufficiently porous substrate are being developed that provide spatial freedom to allow the movement of cells as well as nutrient transportation. In addition, these substrates facilitate cell adhesion ligands and growth factors are incorporated to bestow adhesive and proliferative propertiesto these substrates to recreate the natural environment (Schuurman, 2013: Liao, 2006). Currently, the substrates used for growing cells in 3D models are broadly classified into 2 forms scaffold and non-scaffold forms. Spheroids or 3D cell culture can be cultured in different techniques such as hanging drop method, gel casting, fabricated micro plates and using microfluidic approaches. Though, hanging drop method is easily doable they are forced to form aggregates and most of the spheroid formed is of the same size which may not mimic the cell as in physiological environment. Moreover, all these substrates or platforms have limited applications and are cell or tissue specific. Though, a few 3D cell culture platforms are available, they are expensive with limited availability.

[0008] Currently available substrates are very expensive and required careful storage and handling procedures. Collagen I, obtained from rat tail is a fibrous protein that forms a rope like structure providing tensile strength to extracellular matrix (ECM) where it plays a key role in cell growth, differentiation, attachment and cell migration. Type I collagen is most commonly found in the skin, bone, tendons and other connective tissues. Collagen obtained from rat tail can be prepared as a clear gel providing a 3D matrix or substrate that can support the growth of variety of cell types (Gibco, Life technologies Cat # A10483-01).

[0009] Another substrate most commonly used for the 3D matrix is the Corning Matrigel basement matrix, which is a soluble basement membrane extract of the Engelbreth-Holm- swarm (EHS) mouse tumor that gels at room temperature to form genuine reconstituted basement membrane. The major component of corning matrigel matrix are laminin (60%), collagen IV (30%), entactin (8%) and heparin sulfate proteoglycan (Corning matrigel basement membrane matrix, 5ml vial, catalog # 356234)

[0010] Currently, hydrogels like collagen and extracellular matrix, other polymeric scaffolds, hanging drop method and fabricated micro plates are used for growing 3D cellmodels. Though these techniques are ideal for 3D growth of cells, they are expensive, requires cold storage and have limited shelf life, assay specific limitations and might not be universally acceptable for all 3D cell culture studies for different applications. Due to the growing demand of 3D based cell culture models, we need a system that not only promotes quick spheroid growth but also ensures the credibility, reproducibility and stability of the product to cater to the diverse needs of researchers and technological applications at reasonable costs.

[0011] There is arising requirement of 3D cell models in Biotech and Pharmaceutical industry for drug discovery and development, vaccine development, cancer research, 3D bio printing and others. Also, regulatory agencies are pushing for in-vitro models than the dependence on in vivo animal models. Research community focusing on 3D cell culture technology are in desperate need for a proper model to accurately predict cell growth. The demand for such 3D models is expected to grow many folds in the near future. However, currently available materials / substrates that can support 3D cell culture are very few and some of them are being obtained from animal sources and there is a challenge to meet the global demand for these substrates. Currently, no indigenous product is available in the Indian market and researchers have to rely on expensive commercially available substrates which has hindered the progress in this field. Available products such as Coming Matrigel Matrix and Collagen obtained from animal source have to be stored in cold conditions; raw materials are not easily available and products may not be available in larger volumes.

[0012] The biopolymer sericin, produced from the silk glands of silk insect is a splicing alternative product of genes Seri, Ser2 and Ser3. Due to this, the protein has high molecular heterogeneity of 20 to 400 kDa, with variations even on amino acid molar percentage. After being secreted from the silk glands, the sericin finds its place in silk cocoons accounting for 25 to 30% of cocoon weight. The protein is a water soluble glycoprotein and characterizedby the presence of 18 amino acids with strong polar side groups (Hydroxyl, carboxyl and amino groups) with high content of serine, aspartic acid and glycine resulting in hydrophilic nature of the protein. With wide physicochemical properties of sericin, this protein has proven potential to be a biocompatible material for biomedical applications (Kunz, 2016).

[0013] Sericin is inherently soluble in water and hence does not have the stability under aqueous conditions whereas scaffolds (films, fibers, gels, freeze dried structures) intended for tissue engineering, 3D cell culture and other applications have to be stable.

[0014] The proposed invention addresses many of the shortcomings of the current products in the market thus scaling up the feasibility for commercialization, economic and reliability aspects.OBJECT OF THE INVENTION

[0015] The main object of the invention is to develop a stable sericin biopolymer composition with enhanced stability in aqueous conditions or physiological conditions.

[0016] It is another object of the invention to provide a method of preparation of the stable sericin biopolymer composition by process.

[0017] It is yet another object of the invention to use freezing-thawing

[0018] The stable sericin for preparation of cost effective sericin substrate platform such as gels, films, scaffolds and electrospun films for 3D cell culture and other applications.

[0019] Other objects of the invention will be apparent from the description of the invention herein below.SUMMARY OF THE INVENTION

[0020] The invention relates to a stable sericin biopolymer composition comprising sericin, a cross-linking agent and optionally a catalyst. The cross-linking agent is citric acid or malic acid or butanetetracarboxylic acid or ascorbic acid or cysteine or catechol. The catalyst is sodium hypophosphite or sodium phosphate.

[0021] The invention also relates to the method of preparation of stable sericin biopolymer composition prepared by freezing-thawing process. The stable sericin biopolymer is used for preparation of cost effective substrate platform such as gels, films, scaffolds and electrospun films for 3D cell culture and other applications.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] For a better understanding of the invention, few embodiments are described below with reference to the accompanying figures, purely by way of example and non-limiting in which:Fig. 1 shows schematic representation of the process of sericin extraction from cocoons and its ability to support spheroid or 3D cell growth comprising: (A)- Extraction of protein components from silk cocoons; (B)- Reformulating the silk protein polymer to increase the stability; (C)- Develop different substrate platforms (gels, films, scaffolds and electrospun films) of silk protein; (D)- Evaluating the ability of the substrate platforms to support 3D cell growth of normal and cancer cells; (E)- Examples of gels prepared from sericin supporting the growth of Liver cancer cell lines (HepG2) as spheroids or 3D cell culture models; (F)- Examples of gels prepared from sericin supporting the growth of skin keratinocytes (HaCat) cells as spheroids or 3D cell culture models;Fig. 2 shows scaffolds of various sizes obtained by varying the sericin freeze-thaw conditions and lyophilization parameters;Fig. 3 depicts FTIR analysis showing the presence of functional groups of sericin substrates;Fig. 4 shows compression strength of four different concentrations of sericin with four different crosslinking agents of ascorbic acid (AA), malic acid (MA), citric acid (CA) and butanetetracarboxylic acid (BTCA);Fig. 5 shows a) frozen sericin with different concentrations of crosslinkers and b) after sericin subjected to freeze thaw cycle from frozen (-20°C) to room temperature. Only one concentration of the five different concentrations tested was able to withstand the freeze thaw cycle and was stable to form gel again at room temperature;Fig. 6 depicts sericin scaffolds formed through the freeze-thaw cycle (3 cycles) and later lyophilized to form 3D structures. Although solid scaffolds were formed, they disintegrated when immersed in water. The middle white scaffold is the stable chitosan scaffold, which was used as a positive control;Fig. 7 depicts sericin scaffolds stable in water even after 7 days at 37°C after optimizing the concentrations of crosslinkers, crosslinking temperature and the binding agent;Fig. 8 shows mouse fibroblast cells growing on the sericin films under two different magnification (A and B); andFig. 9 shows pre-crosslinked scaffolds (A and B) and scaffolds with pre and post crosslinking before and after immersion in water (C and D).DESCRIPTION OF THE INVENTION

[0023] For the better understanding of the objects, technology and advantages of the present invention, the instant invention will be further explained in detail with respect to embodiments and accompanying figures as given above. It should be understood that the specific embodiments described herein are only to be used for explaining the present invention but not used to limit the present invention.

[0024] The biopolymer sericin, produced from the silk glands of silk insect is a splicing alternative product of genes Seri, Ser2 and Ser3. Due to this the protein has high molecular heterogeneity of 20 to 400 kDa, with variations on amino acid molar percentage. After secreted from the silk glands the sericin finds its place in silk cocoons accounting for 25 to 30% of cocoon weight. The protein is water soluble glycoprotein and characterized by the presence of 18 amino acids with strong polar side groups (Hydroxyl, carboxyl and amino groups) with high content of serine, aspartic acid and glycine resulting in hydrophilic nature of the protein. With the wide physicochemical properties of sericin, this protein has proven to be a potential biocompatible material for biomedical applications.

[0025] Sericin from textile industry waste, if reused could have scientific and commercial value. Previously, numerous researches have shown potential of sericin for biomedical applications. One of the drawbacks could be batch to batch variations given the extraction methods and the silk worm lineage, which can lead to variations in molecular weight and amino acid concentration of sericin. Given the presence of highly hydrophobic amino acids and its antioxidant properties, the protein finds its applications in food and cosmetic industry.

[0026] In cell culture applications, sericin has been shown to be used in culture medium and in cry opreservation of cell lines, vehicle for drug delivery etc. With all these sericin has proven to be used an effective and important biomaterial for diverse applications.

[0027] Sericin is inherently soluble in water and hence does not have the stability under aqueous conditions. Whereas substrates (gels, films, scaffolds, and electrospun films) intended for tissue engineering, 3D cell culture and other applications have to be stable. This is limiting the use of sericin in 3D cell culture and other applications.

[0028] Cross-linking agent can be used for making protein and bio polymeric materials stable to aqueous conditions. However, cross-linking also requires the material to be immersed in the solution or exposed to vapors. This requires that the materials to be crosslinked have minimum stability upon which further improvement will be possible. Also, cross-linking using carboxylic acids needs high temperature (120-175°C) curing for the cross-linking to occur. Without high temperature treatment, cross-linking with carboxylic acids is not possible. Some reports are available on wet cross-linking of proteins using carboxylic acids but here the material has to be immersed in the cross-linking solution. All the above described methods demand prolonged exposure to crosslinkers and harsh crosslinkers, which may not be suitable for cell culture applications.

[0029] Unfortunately, materials developed from sericin cannot undergo either the dry or wet cross-linking due to their inherently weak mechanical properties and aqueous stability. When immersed in cross-linking solutions, sericin based biopolymer gel dissolve immediately. When exposed to high vapors, the sericin based materials become moist and collapse. Even high temperature treatment of sericin causes it to be brittle.

[0030] Accordingly, currently there are no means of preparing sericin biopolymer or gel with the aqueous stability and mechanical properties required for cell culture and other medical applications.

[0031] The present inventor has identified that a series of freeze-thaw cycles in the presence of the cross-linking chemicals at lower concentrations which is able to improve the aqueous stability and mechanical properties of sericin.

[0032] Accordingly, the present invention relates to a stable sericin biopolymer composition with enhanced stability in aqueous conditions, the composition comprises a sericin, a cross-linking agent and optionally a catalyst.

[0033] In a non-limiting embodiment, the cross-linking agent is selected from carboxylic acid, citric acid, malic acid, butanetetracarboxylic acid, ascorbic acid, cysteine, catechol and mixture thereof.

[0034] In a non-limiting embodiment, the catalyst is selected from sodium hypophosphite and sodium phosphate.

[0035] In certain embodiments, such as gels, the sericin biopolymer composition is prepared by freezing-thawing process.

[0036] In a non-limiting embodiment, the concentration of sericin is 2-10 % (wt / wt) and cross-linking agent is 5-20 % (wt / wt) in the composition as in films, scaffolds ad electrospun films.

[0037] In a non-limiting embodiment, cross-linking agents are added into the sericin solution and the solution is later placed in a freezer at temperature between -8°C and -20 °C for 2 to 24 hours. During this time, the sericin solution freezes and forms a semi- solid substance as in films, scaffolds and electrospun films and gets crosslinked to a certain extent.

[0038] Later, the frozen sericin is thawed by allowing it to relax under room temperature for 1 - 5 hours and heating at 40- 60°C for 5-45 min. Due to the pre-crosslinking in the first freezing cycle, the sericin maintains its shape even at room temperature. After the sericin has attained room temperature, it is once again placed in the freezer at temperature between -8°Cand -20 °C for 2 to 24 hours. The frozen sericin is again thawed at room temperature and this freeze-thaw cycle is repeated 2 to 5 times depending on the extent of crosslinking and stability of the scaffolds desired.

[0039] Multiple and excess exposure to the freeze-thaw cycle will increase the crosslinking and make the sericin unable to be processed into various forms. By controlling the amount of cross-linking agent and the freeze-thaw cycle, the sericin can be obtained with desired strength and stability.

[0040] Further, the pre-crosslinked sericin can be used to cast into films, extruded into fibers or lyophilized to form 3D scaffolds. The pre-crosslinked sericin can also be electrospun into micro and nanofibers in the form of a 2D or 3D structure.

[0041] Advantages of freeze thaw cycle: This process of crosslinking sericin using the freeze-thaw cycle does not require curing at high temperature or under strong pHs. This prevents the damage to the sericin proteins when the conventional heating and curing is done for carboxylic crosslinking. Also, the freeze thaw cycle allows the sericin peptides to slowly agglomerate and form bridges using the crosslinking agent. The network structure of the proteins is increased and stable scaffolds are obtained. Hence, sericin is able to be converted into various shapes and sizes required for 3D cell culture and other applications.

[0042] An aspect of the invention relates to a method of preparing stable sericin biopolymer composition, the method comprises: a. extracting and preparing sericin solution from silkworm cocoons; b. adding cross -linking agents to the sericin solution; c. optionally, adding a catalyst to the above solution;d. allowing the above obtained solution mixture to solidify at room temperature to obtain gel; e. freezing the gel at temperature between -8°C and -20 °C to obtain frozen gel; f. thawing the frozen gel under room temperature and heating at 40- 60°C to obtain sericin solution; g. allowing the solution to solidify at room temperature to obtain gel; h. freezing the gel at temperature between -8°C and -20 °C to obtain frozen gel; i. optionally, repeating the freezing - thawing cycles 2- 5 times, to obtain stable sericin biopolymer composition.

[0043] In a non-limiting embodiment, the silkworm cocoons are procured from Retail market in Bangalore and from the Central Silk Board, Bangalore, Karnataka.

[0044] In a non-limiting embodiment, the cross-linking agent is citric acid or malic acid or butanetetracarboxylic acid or ascorbic acid or cysteine or catechol or mixture thereof and the catalyst is sodium hypophosphite or sodium phosphate.

[0045] In a non-limiting embodiment, the concentration of sericin and cross-linking agent used in the method of preparation of stable sericin biopolymer composition is 2-10 % (wt / wt) and 5-20 % (wt / wt) respectively.

[0046] In a non-limiting embodiment, in the method of preparation of stable sericin biopolymer, in the step d, the solution is allowed at room temperature for 2 to 8 hours.

[0047] In a non-limiting embodiment, in the method of preparation of a stable sericin biopolymer, in the step e, the freezing is performed for 2 to 24 hours.

[0048] In a non-limiting embodiment, in the process of preparation of stable sericin biopolymer, in the step f, thawing is performed for 1 to 5hrs and heating for 5-45 mins.

[0049] In an embodiment, the process of preparing stable sericin biopolymer composition comprises following steps:

[0050] Step 1 - Protein extraction: Degumming the silk cocoons / fibers in water by adding 1:20 to 1:30 or the required ratio of cocoons in water. The cocoons are then heated to above 90°C either in open air boiling or under pressure in an autoclave. Heating can be done by conduction, convection or any other means possible. Heating is done for about 20 minutes to 1 hour depending on the temperature. After heating / autoclaving, the sericin is released into the water. The degummed cocoons / fibers are separated and the solution containing sericin is collected. The amount of sericin in the solution varies anywhere between 0.5 and 4% depending on the species of silk, time, temperature of treatment and amount of water used. The sericin in solution is directly used to form the substrates or freeze dried or the water is evaporated by suitable means to collect the dry sericin powder.

[0051] The cocoons are procured from three different species to ensure that there is no species to species and batch variations of sericin properties. The sericin from cocoons is extracted using hot water extraction. The extracted protein are quantified and characterized by measuring the total protein content and the molecular weight of the protein bands are resolved by sodium dodecyl sulphate Polyacrylamide gel electrophoresis (SDS-PAGE).

[0052] Step 2- Addition of crosslinking agent and catalyst: Sericin solution obtained in step 1 is either used directly to make the gels or made into powdered sericin by evaporating water and is dissolved in desired quantity to get the appropriate concentration for preparing the gels. To the sericin solution, crosslinkers including but not limited to carboxylic acids such as, citric acid, malic acid, and butanetetracarboxylic acid or ascorbic acid or cysteine orcatechol are added. Several enzymes such as proteases can also be used as the crosslinking agent. The concentration of the catalyst is varied between (2-5%) based on the weight of the sericin. Optionally, a catalyst such as sodium hypophosphite, sodium phosphate is added and the concentration of the catalyst is between 10 to 50% on weight of the cross-linking agent. Alternatively, the pH of the sericin solution containing the crosslinking is adjusted between 7 and 13 when the pH of the solution itself acts as a catalyst.

[0053] Step 3- Freeze thaw cycle (preprocessing of sericin for effective crosslinking and to attain stable gels): The mixture of protein, catalyst and the cross linker is allowed to solidify at room temperature (takes 2 to 8 hrs depending on the concentration of the sericin, crosslinking agent, catalyst, pH etc). The gel is then placed in a freezer with temperature between -8 °C and -20 °C freezer for 2 to 24 hours. During this time, the sericin solution freezes and forms a semi-solid substance similar to gel and gets crosslinked to a certain extent. Next, the frozen sericin is thawed by allowing it to relax under room temperature for about 1 to 5hrs. Later, the gel is heated between 40 and 60°C for 10 to 30 minutes to return to the sericin solution. The solution is again allowed to solidify under ambient conditions (2 to 8hrs) and frozen at -8°C to -20 °C freezer for 2 to 24 hours. This freeze-thaw cycle is repeated 2 to 5 times depending on the extent of crosslinking and stability of the scaffolds desired. After the freeze-thaw cycles, the sericin solution turned into a gel is stable and holds it shape when inverted in a glass bottle of test tube.

[0054] The stable sericin biopolymer or sericin biopolymer gel obtained is tested for the ability to support cell attachment, growth, proliferation and spheroid formation using standard procedures as in Fig 8.

[0055] The sericin obtained after the multiple freeze thaw cycles is considered for preparing the various sericin substrate platforms (films, scaffolds and electrospun films) for3D cell culture.

[0056] The obtained stable sericin biopolymer or sericin biopolymer composition is used for preparation of films, electrospunfibres, 3D scaffolds as below.

[0057] Preparation of films: The pre-crosslinked sericin obtained after the freeze-thaw cycle as described in step 3 above is used to prepare the films. The sericin solution is poured onto glass plates or suitable platform and the water is allowed to evaporate between 25 and 50°C by drying under room temperature or in an oven. The concentration of sericin and amount of cross-linking agent used during the freeze-thaw cycle affects the ability of the solution to form the film and the properties of the film. If necessary, in addition to crosslinking agent used during step 3, plasticizers such as glycerol or sorbitol (1 to 10% w / w) are added into the sericin solution before pouring into film casting plates. Once dried, the films are placed in an oven at 150-180 °C and heated for 5 to 60 minutes for the crosslinking reaction to occur. Films are then taken out and they now have the mechanical properties and desired stability for the cell culture studies.

[0058] Preparation of 3D scaffolds: Sericin solution obtained in step 3 is poured into containers having desired shape and size. Additional cross-linking agent, catalyst, plasticizers are added into the solution. The solution is now placed in a freezer between -15 and -80°C for about 8 hours. Later, the frozen solution is placed in a lyophilizer where all the water is removed and scaffolds are formed. The lyophilization temperature and time (4 to 72 hours) are varied to obtain scaffolds with the desired shape and structure. The scaffolds obtained are allowed to equilibrate at room temperature for 8 to 24 hours and later placed in an oven and heated at 150 to 180 °C for 5 to 60 minutes for the crosslinking reaction to occur. After cross-linking the scaffolds will be ready to be used for the 3D cell culture. (Fig 2)

[0059] Preparation of electrospun fibers: Sericin solution obtained from Step 3 is added with cross-linking agent, catalysts and salts such as sodium chloride (2 to 10%) to increase the conductivity of the solution. This solution is placed in a syringe and attached to a needleand the syringe and needle are then placed in a syringe pump connected to a high voltage supply. Electrospinning is done by varying the voltage 5kV to 30 kV and syringe speed (0.5 to 3 ml per minute). Fibers formed are collected either on a stationary target or a drum rotating between 5 and lOOrpm. Fibers are deposited (electrospun) for 2 to 6 hours depending on the thickness, porosity of the scaffolds desired. The fibers in the form of films or sheets are removed the electrospinning unit and placed in an oven at heated temperatures between 150 and 180 °C for the crosslinking to occur. The crosslinked electrospun fibers are now ready for 3D cell culture.

[0060] Testing and characterization of different sericin scaffolds for their ability to support 3D cell growth: The schematics of the work flow is described in Fig 1. Sericin substrates (films, scaffolds, electrospun structures) obtained as described above were tested for their swell ability and ability to absorb moisture. The swelling index and moisture content varied considerably depending on the sericin concentration, type of process used for making the scaffold and the cross-linking agent type and its concentration.

[0061] The swelling index and moisture content of the substrates obtained for 3D cell culture developed using the steps described above was dependent on the amount of sericin (SS) concentration, type and amount of cross-linking agent and the form of sericin and the results are shown in the table below.Table 1AA: ascorbic acid; MA: Malic acid; BTCA: Butanetetracarboxylic acid; CA: Citric acid

[0062] FTIRanalysis: The sericin substrates obtained were studied for their chemical changes using FTIR. FTIR analysis shows the presence of functional groups. In the figure 3below, we observed the peaks at 1050 Cm'1& 1404 Cm'1shows C-0 & C-O-H stretch in the fingerprint region. Peaks at 1544 Cm'1shows N-H bending and 1646 Cm'1shows C=O asymmetric bonding in double bond region. Peak at 3286 Cm'1shows O-H in single bond stretch region. As observed, 2SS / 10MA & 4SS / 10MA showed almost same peaks in which both the scaffolds had Sericin & Malic acid. The peak in Fig. 3 shows the presence of glycerol, sericin, and the cross-linking agent.

[0063] Measurement of Compression strength: Sericin at four different concentrations (2 to 10%) were subjected to two freeze-thaw cycles with 4 different crosslinking agents (10% Ascorbic acid (AA), 5% malic acid (MA), 15 % citric acid (CA) and 20% butanetetracarboxylic acid (BTCA). Stable gels were obtained but the strength and stability depended on the amount of sericin and type of cross-linking agent. As seen from Figure 4, gels obtained using 4% sericin and malic acid or 8% sericin and BTCA provided considerably stable gels with better stability compared to the others.

[0064] The sericin gels obtained by the process of the present invention i.e. after crosslinking through the freeze-thaw process are stable for more than 90 days when kept at 37°Cand in CO2 Incubator. Such stability is required for tissue engineering, 3D cell cultureand other medical applications. Further, cells grown on the gels under laboratory conditions by incubating them in CO2 incubator at 37°C with 95% humidity. This environment mimics the natural human body conditions where the cells multiply and grow.

[0065] The sericin scaffolds developed through the invention are considerably cheaper than existing scaffolds for 3D cell culture applications. For example, Polystyrene scaffold cost Rs 1800 / insert whereas, the present invention scaffold would potentially cost Rs 800 / sample i.e. almost 2.25 times lesser than the cost of commercially available product.

[0066] The present invention scaffold is cheaper than product available in the market, for instance, the product is 2 times lesser than spheroid medium and 17 times lesser than peptide based scaffold.

[0067] The above description of the invention, together with the below accompanying examples should not be construed as limiting the invention because those skilled in the art to which this invention pertains will be able to devise other forms thereof within the ambit of the appended claims.Examples

[0068] Example 1- Protein extraction example: Degum the silk cocoons / fibers in water by adding 1:20 to 1:30 or the required ratio of cocoons in water. The cocoons are then heated to above 90°C either in open air boiling or under pressure in an autoclave. Heating can be done by conduction, convection or any other means possible. Heating is done for about 20 minutes to 1 hour depending on the temperature used. After heating / autoclaving, the sericin are released into the water. The degummed cocoons / fibers are separated and the solution containing sericin is collected. The amount of sericin in the solution varies anywhere between 0.5 and 4% depending on the species of silk, time, temperature of treatment andamount of water. The sericin in solution is directly used to form the substrates or freeze dried or the water is evaporated by suitable means to collect the dry sericin powder

[0069] Example 2: Sericin (2%) was dissolved in water and citric acid (10%) based on weight of sericin was added and the mixture was placed in a -20°C freezer for 8 hours. The sericin was observed to form a stable gel, but when allowed to equilibrate at room temperature then the gel was dispersed or collapsed into solution except only one of the concentration tested. (Fig. 5)

[0070] Example 3: Sericin (2%) was dissolved in water and citric acid (10%) based on weight of sericin and catalyst sodium hypophosphite was added and the mixture was placed in a -20°C freezer for 8 hours. The pH of the solution was adjusted to 7 using sodium hydroxide solution. The sericin was observed to form a stable gel, but when allowed to equilibrate at room temperature it did not retain its shape. The gel was further freeze dried to form a scaffold through freeze drying (lyophilization). Scaffolds with 3D structure were obtained but when placed in cell culture media it was dispersed or disintegrated within few minutes suggesting that the above concentration of either protein or crosslinker doesn’t allow the scaffolds to be water stable and further optimization of protein and crosslinkers are necessary(Fig.6).

[0071] Example 4: Sericin extracted from the cocoons which had a concentration of about 4%. To this solution, citric acid (10%) and sodium hypophosphitecatalyst (5% on weight of cross-linking agent) were added to the solution and the solution was placed in a -20 °Cfreezer for 8 hours. Later, the sericin was thawed to room temperature to observe the stability. The stable sericin was subjected to freezing and thawing again for 3 cycles. The sericin scaffold obtained was highly stable even after exposure to 37°C for 7 days. The form of the scaffold depended on the type of container used for the freeze-thaw cycle. Sericin solution placed inmulti-well plates formed circular scaffolds in the form suitable for cell culture and tissue engineering. (Fig 7)

[0072] Example 5: Cross-linking agent malic acid (15%) was added along with sodium hypophosphite catalyst into the sericin solution and the freeze-thaw cycle was repeated 5 times to obtain stable sericin. The stable sericin was dispersed in water along with 1-10% malic acid as cross-linking agent and catalyst. The sericin solution was cast into molds to form 2D films. The films were dried at room temperature and later subjected to high temperature (150°C for 30 min) curing. The films had excellent mechanical properties and were also stable in aqueous conditions for up to 15 days. The films were also able to support the growth and proliferation of cells. The mechanical properties of sericin films formed after different freeze thaw cycles and containing 1 to 10% cross-linker agents are shown in the below table.Table 2

[0073] Example 6: Preparation of sericin scaffolds: Stable sericin scaffolds obtained through the freeze-thaw cycle were seeded with mouse fibroblast cells and the cells were cultured for up to 10 days. The scaffolds were stable and also supported the attachment and growth of cells.

[0074] Example 7: Preparation of electrospun films: Sericin pre-crosslinked with citric acid was taken and the solution was electrospun into fibers. The electrospun mat obtained was heated at 50°C for further crosslinking to occur and provide required stability. Thiselectrospun structure was suitable for cell growth and showed cell spreading but no spheroid formation were observed.

[0075] Example 8: Preparation of sericin scaffold: Sericin pre-crosslinked with malic acid through 8 freeze-thaw cycles was placed in a lyophilizer and the sericin was formed into a 3D scaffold suitable for cell culture. This scaffold was stable even at 50°C and was used to culture cells. The scaffold supported cell growth in 3D and microscopic examination showed the formation of spheroids.

[0076] Example 9: About 10% ascorbic acid was added to 5% sericin solution and the mixture was placed in a freezer for 8 hours. After thawing, it was found that the sericin had formed a stable structure which was not dispersing or dissolving in water. The scaffold was seeded with cells and placed in an incubator for 10 days. Gradual growth of cells and formation of spheroids after the 6thday was observed.

[0077] Example 10: Sericin solution crosslinked with 10% citric acid through 3 freezethaw cycles had limited stability. This sericin solution with an additional 10% cross-linking agent was poured onto glass plates coated with Teflon. The plates were placed at -4°C for 6 hours. Further cross-linking would occur and the solution was formed into thick swollen film. Upon drying at room temperature or in an oven at 30°C, a stable solid film was obtained. This film was seeded with mouse fibroblast cells and cultured for 7 days. Though, growth of cells was evident, cells growing as spheroid models were not reported or noticed.

[0078] Example 11: Dry sericin powder was dissolved in water and about 10% citric acid and sodium hypophosphite catalyst were added into the solution and dissolved. The solution was exposed to 5 freeze-thaw cycles when a stable scaffold was obtained. The scaffold was able to support growth of cells and formation of spheroids was observed.

[0079] Example 12: Sericin was combined with 5% butanetetracarboxylic acid (BTCA) and freeze-thawed for 5 cycles with each cycle being for 12 hours. Later stable sericin in the form of gel was added with 10% BTCA and the mixture was placed in a lyophilizer to form 3D scaffolds. The gels obtained were converted into 3D scaffolds suitable for cell culture. However, the scaffolds with only pre-crosslinking disintegrated when immersed in water, whereas those with both pre and post crosslinking were stable in water even after 7 days. (Fig 9).

[0080] While the invention has been described in connection with an illustrative embodiment, it is not intended to limit the scope of the present disclosure to the particular form set forth, but on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the scope of the present disclosure.

Claims

CLAIMS1. A stable sericin biopolymer composition comprising a sericin, a cross-linking agent and optionally a catalyst, wherein the cross-linking agent is selected from citric acid, malic acid, butanetetracarboxylic acid, ascorbic acid, cysteine, catechol and the catalyst is selected from sodium hypophosphite and sodium phosphate.

2. The stable sericin biopolymer composition as claimed in claim 1, wherein the composition is prepared by freezing-thawing process.

3. The stable sericin biopolymer composition as claimed in claim 1, wherein the concentration of sericin is 2-10 % (wt / wt) and cross-linking agent is 5-20 % (wt / wt).

4. A method of preparing stable sericin biopolymer composition, wherein the method comprising steps of: a. extracting and preparing sericin solution from silkworm cocoons; b. adding cross -linking agents to the sericin solution; c. optionally, adding a catalyst to the above solution; d. allowing the above obtained solution mixture to solidify at room temperature to obtain gel; e. freezing the gel at temperature between - 8°C and -20 °C to obtain frozen gel; f. thawing the frozen gel under room temperature and heating at 40 - 60 °C to obtain sericin solution; g. allowing the solution to solidify at room temperature to obtain gel; h. freezing the gel at temperature between -8°C and -20 °C; and i. optionally, repeating the freezing - thawing cycles 2- 5 times to obtain stable sericin biopolymer composition.

5. The method as claimed in claim 4, wherein the cross-linking agent is citric acid or malic acid or butanetetracarboxylic acid or ascorbic acid or cysteine or catechol and the catalyst is sodium hypophosphite or sodium phosphate.

6. The method as claimed in claim 4, wherein the concentration of sericin is 2-10 % (wt / wt) and cross-linking agent is 5-20 % (wt / wt).

7. The method as claimed in claim 4, wherein in the step d, the solution is allowed to solidify at room temperature for 2 to 8 hours.

8. The method as claimed in claim 4, wherein in the step e, freezing is done for 2 to 24 hours.

9. The method as claimed in claim 4, wherein in the step f, thawing is done for 1 to 5 hrs and heating for 5-45 mins.

10. A method of preparing sericin substrate platform selected from gels, films, scaffolds and electrospun films for 3D cell culture, using the sericin composition as claimed in claim 1.

11. A method of preparing sericin substrate platform selected from gels, films, scaffolds and electrospun films for 3D cell culture, using the sericin obtained by method as claimed in claim 4.

12. A method of preparing sericin films from stable sericin biopolymer, comprising steps of: a. pouring the stable sericin biopolymer composition obtained in claim 4, onto glass plates or suitable platform and allowing water to evaporate between 25 and 50°C by drying under room temperature or in an oven; b. adding plasticizers such as glycerol or sorbitol (1 to 10% w / w) into the sericin solution and pouring into film casting plates; and c. drying and placing the films in an oven at 150-180 °C and heating for 5 to 60 minutes for the crosslinking reaction to occur and obtaining sericin films.

13. A method of preparing sericin scaffolds from stable sericin biopolymer, comprising steps of: a. pouring the stable sericin biopolymer composition obtained in claim 4 into containers having desired shape and size; b. adding additional cross-linking agent, catalyst, and plasticizers in to the above solution; c. freezing the above solution between -15 and -80°C for about 8 hours; d. placing the frozen solution in a lyophilizer, for removing water and formation of scaffolds; e. allowing obtained scaffolds to equilibrate at room temperature for 8 to 24 hours; and f. placing in oven and heating at 150 to 180 °C for 5 to 60 minutes for the crosslinking reaction to occur, and obtaining sericin scaffolds.

14. A method of preparing sericin electrospun fibers from stable sericin biopolymer, comprising steps of: a. adding additional cross-linking agent, catalyst and salts such as sodium chloride (2 to 10%) to stable sericin biopolymer composition obtained in claim 4; b. performing electrospinning at a voltage of 5 kV to 30 kV in an electrospinning unit; c. collecting the fibers formed either on a stationary target or a drum rotating between 5 and 100 rpm; d. depositing the electronspun fibers for 2 to 6 hours depending on the thickness, porosity of the scaffolds desired; ande. removing the fibers in the form of films or sheets from electrospinning unit and placing in an oven at heated temperatures between 150 and 180 °C for the crosslinking reaction to occur, and obtaining sericin electrospun fibers.

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