WO2021205471A1 - Composition, injectable hydrogel and methods thereof - Google Patents

Abstract

The present disclosure relates to a composition comprising: (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200. The present disclosure relates to an injectable hydrogel comprising the composition and at least one therapeutic agent. The present disclosure also relates to process of preparing the composition, injectable hydrogel and methods thereof.

Description

COMPOSITION, INJECTABLE HYDROGEL AND METHODS

THEREOF

FIELD OF INVENTION [001] The present disclosure broadly relates to silk fibroin-based materials and particularly refers to a composition and an injectable hydrogel for biomedical applications.

BACKGROUND OF INVENTION [002] Diabetes is a chronic disease affecting over 400 million people worldwide.

Inadequate production of insulin due to loss of beta cells or insulin resistance within the body imbalances the glucose homeostasis, result in abrupt increase of blood glucose level. Diabetes is a metabolic disorder and is typically characterized by the elevated physiological blood glucose levels, a major causative factor for severe long-term complications such as eye damage, cardiovascular diseases, chronic kidney dysfunction, and neurodegenerative diseases. The disease condition arises from the failure of pancreatic b-cells to produce sufficient insulin (Type I, T1DM), a peptide hormone that regulates cellular glucose receptor activity to maintain the blood glucose homeostasis (D.R. Owens, B. Zinman, G.B. Bolli, Insulins Today and Beyond, Lancet 358 (2001) 739-746). On the other hand, liver hepatocytes, muscle and adipocyte cells become resistant to insulin (Type II, T2DM), hampering the normal mechanism of glycogenesis (Pillai, Qmaihanu, and Ramesh Panchagnula. "Insulin therapies -past, present and future." Drug discovery today 6.20 (2001): 1056-1061). The bi-guanidine drugs are used as a first-line medication for T2DM as they potentially suppress hepatic glucose production and induce the phosphorylation of insulin- sensitive glucose transporter type 4 (GLUT4) enhancer factor to increase the glucose uptake (Veiseh, Qrnid, et al. "Managing diabetes with nanomedicine: challenges and opportunities.” Nature Reviews Drug Discovery 14.1 (2015): 45-57). However, T1DM and the advanced stage of T2DM conditions requires external administration (subcutaneous) of insulin to the body, once or multiple times a day. The multiple subcutaneous insulin injections are associated with pain, local tissue necrosis, infection, nerve damage and locally concentrated insulin amyloidosis responsible for inability to achieve physiological glucose homeostasis which can be overcome with controlled and sustained insulin delivery.

[003] In recent times, researchers have focused on the development of self- regulated injectable hydrogel-based insulin delivery systems for continuous delivery of insulin. Attractive features of hydrogels such as porous cross-linked three-dimensional network structure, diverse stimuli-responsive chemical compositions and optimum mechano-physical properties have made these soft materials as potential candidates for innumerable biomaterial applications. The porous morphology allows encapsulation of various drug molecules within the hydrogel matrix. The encapsulate drugs are subsequently released depending on the diffusion coefficient of drugs and pore size of the gel-network. Additionally, the injectable hydrogels have an advantage of minimally invasive delivery without surgical implantation and infection risk.

[004] US 10137199B2 discloses a method of making hydrogel comprising carboxymethylated hyaluronan (CMHA-S) and gelatin thiolated polymers and a therapeutic agent. US8206774B2 reveals a method for producing a modified silk composition, comprising a diazonium salt with a silk polymer solution to form a modified silk mixture.

[005] Although literature discloses injectable hydrogel-based drug delivery systems for sustained drug release, most of such prior compositions are associated with drawbacks such as long gelation period, poor hydrogel stability, high immunogenic properties, poor drug compatibility, low biocompatibility and biodegradability and poor retention of injectable property. Thus, there is still a strong need for development of self-regulated injectable hydrogel-based drug delivery systems for continuous delivery of the drug.

SUMMARY OF THE INVENTION [006] In an aspect of the present disclosure, there is provided a composition comprising: a) silk fibroin; and b) ethylene glycol, wherein the ethylene glycol is selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200.

[007] In another aspect of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol; wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:1:1 to 1:100:100.

[008] In an aspect of the present disclosure, there is provided a process of preparing the composition comprising: a) silk fibroin; and b) ethylene glycol, the process comprising: i) preparing a first solution comprising silk fibroin and water; ii) contacting the first solution with the ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof to obtain the composition, wherein the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200.

[009] In an aspect of the present disclosure, there is provided a process of preparing the composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and (c) monoethylene glycol; the process comprising: i) preparing a first solution comprising silk fibroin and water; ii) contacting the first solution with the tri ethylene glycol and the monoethylene glycol to obtain the composition, wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1 : 1 : 1 to 1 : 100: 100.

[0010] In another aspect of the present disclosure, there is provided an injectable hydrogel comprising: (i) the composition comprising (a) silk fibroin; and (b) ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; and (ii) at least one therapeutic agent, wherein, the composition to the at least one therapeutic agent weight percentage is in the range of 0.001% to 10% and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200. [0011] In an aspect of the present disclosure, there is provided a process of preparing the injectable hydrogel, the process comprising: contacting the composition comprising (a) silk fibroin; (b) ethylene glycol, wherein the ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof, with the at least one therapeutic agent at a temperature in the range of 25 °C to 40 °C to obtain an injectable hydrogel; and wherein the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200.

[0012] In an aspect of the present disclosure, there is provided a method of treating a condition, the method comprising: administering to a subject suffering from a condition a therapeutically effective amount of the injectable hydrogel comprising: (i) the composition comprising (a) silk fibroin; (b) ethylene glycol, wherein the ethylene glycol is selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; and (ii) at least one therapeutic agent, wherein the composition to the at least one therapeutic agent weight percentage is in the range of 0.001% to 10% and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200.

[0013] In one another aspect of the present disclosure, there is provided a method for preparing the tissue engineering scaffold, the method comprising using the composition or the injectable hydrogel.

[0014] In one another aspect of the present disclosure, there is provided use of the injectable hydrogel for controlled and sustained release or delivery of therapeutic agents in their active form.

[0015] In one another aspect of the present disclosure, there is provided a method of treating a condition, the method comprising: administering to a subject suffering from a condition a therapeutically effective amount of the injectable hydrogel. [0016] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0017] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0018] Figure 1(a) depicts sol to gel transformation of SF in presence of additives (EG and TEG) at room temperature, in accordance with an implementation of the present disclosure.

[0019] Figure 1(b) depicts the change in optical density (OD) at 550 nm during the gelation process in presence of individual additives (EG and TEG, 40%, v/v) and mixture of 20% EG, 20% TEG with different SF concentrations, in accordance with an implementation of the present disclosure.

[0020] Figure 1(c) depicts FESEM image of iSFH scaffold after the gelation process, samples are washed and freeze-dried, in accordance with an implementation of the present disclosure.

[0021] Figure 1(d)(1) depicts the image of 3.6% iSFH after injection through the 23G needle, in accordance with an implementation of the present disclosure.

[0022] Figure l(d)(ii) depicts the image of rigid SF hydrogel obtained from the 6% and higher concentration SF concentration of SF protein, in accordance with an implementation of the present disclosure.

[0023] Figure 2a depicts ATR-FTIR spectrum of SF and a freeze-dried iSFH sample, in accordance with an implementation of the present disclosure.

[0024] Figure 2b depicts de-convolution spectrum of the amide I peak of hydrogel designated as the peak at 1621 cm-1 for b-sheet and random coil conformations respectively, in accordance with an implementation of the present disclosure.

[0025] Figure 2c depicts normalised spectra of Figure 2a and Figure 2b plotted together, in accordance with an implementation of the present disclosure. [0026] Figure 2d depicts Circular dichroism (CD) spectra of SF protein and the iSFH, in accordance with an implementation of the present disclosure.

[0027] Figure 2e depicts rheological analysis of iSFH (pink) and insulin-iSFH (blue) shows the storage modulus (G', lines with solid symbols) and loss modulus (G", lines with hollow symbols), in accordance with an implementation of the present disclosure.

[0028] Figure 2f depicts injectable property of 6% iSFH loaded with rhodamine B dye using 23 G needle, in accordance with an implementation of the present disclosure. [0029] Figure 3a(i') depicts release of FITC-insulin from the iSFH at different time intervals; Figure 3a(i") depicts fluorescence spectra of released FITC -insulin from hydrogel, in accordance with an implementation of the present disclosure.

[0030] Figure 3a(ii) depicts the linear fitting of in vitro FITC-insulin release profile from iSFH matrix at 37 °C, in accordance with an implementation of the present disclosure.

[0031] Figure 3a(iii) depicts FITC-insulin release profiled from the 2.4% iSFH(red) and 3.6% iSFH (green) matrix, in accordance with an implementation of the present disclosure.

[0032] Figure 3a(iv) depicts the image of rigid SF hydrogel obtained from the 6% and higher concentration of SF protein, in accordance with an implementation of the present disclosure.

[0033] Figure 3a(v) depicts FITC-insulin release profiled from the 50% of 6% iSFH(3% iSFH, red) and 3.6% (green) matrix in different time intervals, in accordance with an implementation of the present disclosure. [0034] Figure 3b depicts MALDI-TOF mass data of FITC-insulin, in accordance with an implementation of the present disclosure.

[0035] Figure 3c depicts CD spectra of insulin released from iSFH at different time intervals, in accordance with an implementation of the present disclosure.

[0036] Figure 4a depicts In vitro cytotoxicity of iSFH in mouse fibroblast cell line (L929) at different time of incubation (24, 48 and 72 h), in accordance with an implementation of the present disclosure. [0037] Figure 4b depicts toxicity profile of iSFH towards human red blood cells, in accordance with an implementation of the present disclosure.

[0038] Figure 5 depicts in vitro functional activity study of insulin released from the insulin-iSFH in HEK 293 T cells, in accordance with an implementation of the present disclosure.

[0039] Figure 6a depicts in vivo delivery and therapeutic efficacy of insulin-iSFH in STZ injected Wistar rat model through a Schematic show creation of diabetes rat model by injection with STZ, administration of insulin-iSFH and monitoring the glucose levels at different time intervals (in days/weeks), in accordance with an implementation of the present disclosure.

[0040] Figure 6b depicts blood glucose levels of Wistar rats after subcutaneous injection of free insulin, PBS, iSFH and insulin-iSFH. Values represent mean ± SD (n = 5 per group), in accordance with an implementation of the present disclosure. [0041] Figure 6c depicts initial change of blood glucose level after injection, data from Figure 6b, in accordance with an implementation of the present disclosure. [0042] Figure 7 a depicts glycol release % from hydrogel at different time interval, in accordance with an implementation of the present disclosure.

[0043] Figure 7b depicts the swelling study of hydrogel in PBS (10 mM, pH = 7.4), in accordance with an implementation of the present disclosure.

[0044] Figure 8 depicts schematic illustration of iSFH formation with the help of additives ethylene glycol and tri-ethylene glycol which is utilised for in vivo delivery in diabetic wistar rat through a subcutaneous injection leading to insulin depot within the pore structure and release over a prolonged period leading to achieving normal glucose level for 96 hours (4 days), in accordance with an implementation of the present disclosure.

[0045] Figure 9 (a) represent the SF protein gel in presence of glycols; (b) the injectability of the SF protein hydrogel in presence of PEG200 through 23 G needle, in accordance with an implementation of the present disclosure.

[0046] Figure 10 depicts the release kinetics of doxorubicin (a) and berberine (b) from PEG200 induced SF protein hydrogel, in accordance with an implementation of the present disclosure. [0047] Figure 11 depicts CD spectrum of released insulin and only insulin, in accordance with an implementation of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION [0048] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features. Definitions

[0049] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0050] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0051] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0052] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps. [0053] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably. [0054] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a temperature range of 25 °C-40 °C should be interpreted to include not only the explicitly recited limits of about 25 °C to 40 °C, but also to include sub-ranges, such as 25 °C-35 °C, 30 °C-40 °C, and so forth, as well as individual amounts, including fractional amounts, within the specified ranges, such as 25.5 °C, and 35.5 °C, for example.

[0055] The term “hydrogel” refers to a colloidal gel in which water is the dispersion medium and comprises a network of crosslinked polymeric chains that are hydrophilic. Hydrogels are capable of holding a large amount of water. In the present disclosure, the hydrogel composition comprises silk fibroin, and ethylene glycol is selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof. The hydrogel is injectable, and the injectable hydrogel comprises the hydrogel composition with at least one therapeutic agent.

[0056] The term “ethylene glycol” refers to a group of compounds with monoethylene glycol as the basic unit and has molecular formula of H-(OCH2CH2) _n-OH, wherein n = 1-50. For monoethylene glycol n=l and in tri-ethylene glycol n=3. When n=4 -50 it is referred to as oligoethylene glycol or polyethylene glycols. The molecular weight of ethylene glycol of the present disclosure ranges between 62 to 2000g/mol. In the present disclosure, ethylene glycol is selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof. The term “polyethylene glycol” refers to polymers of ethylene glycol with low or medium molecular weight. Examples of polyethylene glycol includes but not limited to PEG200, PEG300, PEG400, etc. In the present disclosure monoethylene glycol is referred as EG; tri-ethylene glycol is referred as TEG and polyethylene glycol is referred to as PEG. [0057] The term “therapeutic agent” refers to any agent administered to a subject to treat a condition, including but not limited to a molecule such as a small molecule drugs, a moiety, a peptide, a protein, a lipid, a polysaccharide, a nucleic acid, an antibody, a cell, a hormone, a growth factor, bioactive compounds, etc.

[0058] The term “b-sheet conformation” refers to secondary structure of proteins in which two or more polypeptide chains are joined together through hydrogen bonds. This means that hydrogen bonding occurs between carboxylic oxygens and amide hydrogens of two or more adjacent extended polypeptide chains. The adjacent chain in b-sheet structures are either parallel or anti-parallel.

[0059] The term “secondary structure” refers to spatial arrangement of polypeptide chains as the C-C and C-N single bonds are free to rotate in amino acid side chains. [0060] The term "at least one therapeutic agent" as used herein refers to any molecule which exerts at least one biological effect in vivo. For example, the therapeutic agent can be a biologically active molecule to treat or prevent a disease state or condition in a subject. Examples of at least one therapeutic agent include, without limitation, bioactive compounds, peptides, peptidomimetics, aptamers, antibodies or a portion thereof, antibody- like molecules, nucleic acids (DNA, RNA, siRNA, shRNA), polysaccharides, enzymes, receptor antagonists or agonists, hormones, growth factors, autogenous bone marrow, antibiotics, antimicrobial agents, small molecules and therapeutic agents. The at least one therapeutic agent can also include, without limitations, anti-inflammatory agents, anesthetics, active agents that stimulate issue formation, and/or healing and regrowth of natural tissues, and any combinations thereof

[0061] The term “small molecule drugs” refers organic, inorganic molecules or natural products of molecular weight less than lkDa. Some examples are doxorubicin, cisplatin, berberine, metformin, curcumin, paclitaxel, vancomycin, enzalutamide, etc. The drug because of its low molecular weight can enter cells easily and affect other molecules.

[0062] The term “fibroin” refers to the protein present in silk which is produced by silkworms. [0063] The term “therapeutic peptide” refers to small chains of amino acids that are administered to a subject to treat a condition. The term therapeutic peptides includes but not limited to insulin, pramlintide, teriparatide, etc.

[0064] The term “peptide” refers to small chains of amino acids wherein the amino acids are joined together by peptide bonds.

[0065] The term “N-Terminal” refers to amine group of an amino acid.

[0066] The term “C-Terminal” refers to carboxylic acid group of an amino acid. [0067] The term “proteins” refers to molecules comprising several amino acid units joined together by peptide bond.

[0068] The term “antibodies” refers to protein produced response to counteracting a substance which a body recognizes as alien such as virus, bacteria or other foreign substances in the blood.

[0069] The term “nucleic acids” refers to linear chains of nucleotides.

[0070] The term “nucleotides” refers to chemical compounds containing three components, purine or pyrimidine nucleobase (sometimes termed as nitrogenous base or simply base), a pentose sugar, and a phosphate group.

[0071] The term “volume of silk fibroin” refers to particular amount of silk fibroin dissolved in a solvent not limited to water. For example in the present disclosure, a stock solution of silk fibroin was prepared as 6% solution (W/V) in water and is considered as volume of 100. To this 6% SF, varying ratios of EG and TEG were added. By adding EG and TEG, the volume of SF decreases and volume of EG and TEG in the composition increases. However for effective gelation the volume ratio of SF:EG:TEG is maintained in the range of 60:5:5 to 90:30:30. Similarly, for effective gelation the volume ratio of SF:PEG was maintained in the range of 60: 10 to 90:40. The corresponding weight percentages and weight ratios of silk fibroin, EG and TEG are correlated and calculated by regular methods.

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0073] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0074] The present formulation is an injectable silk fibroin hydrogel (iSFH) to realize sustained insulin delivery over a prolonged period under diabetic conditions. Additionally, the injectable hydrogels have an advantage of minimally invasive delivery without surgical implantation and infection risk. In this context, silk fibroin (SF) protein extracted from the cocoons of Bombyx Mori is an excellent choice as a biomaterial owing to its biological origin, biocompatibility, biodegradability and low immunogenic property. SF, the major structural protein component of cocoon can be processed into fibers, particles, films, sponges, hydrogels and electrospun mats depending on the intended biomaterial application. In particular, silk hydrogels are excellent soft material platforms for drug delivery, tissue engineering, and regenerative medicine. However, SF takes a relatively longer gelation time (4 days) in aqueous media due to slow conformational transition rate from the random coil to the b-sheet structure and not suitable for the iSFH preparation. These drawbacks of SF have been overcome by fine-tuning the interaction between the b- sheet chains using various physical and chemical methods such as vortexing, sonication, pH and electric field or with the help of additives such as surfactant, acids, salts and polymers. The morphological architecture of SF hydrogel is tunable and potent for materials and biological applications like controlled delivery of bone morphogenetic proteins in tissue engineering among other applications. However, alternation of material properties and maintaining sterile condition are difficult to achieve through reported methods.

[0075] The present disclosure discloses a composition comprising silk fibroin with at least one ethylene glycol or mixture of ethylene glycols. Further the present disclosure discloses a silk fibroin hydrogel composition comprising silk fibroin with two biocompatible additives, monoethylene glycol (EG) and tri-ethylene glycol (TEG), contacted at room temperature to prepare an injectable hydrogel for insulin delivery in diabetic conditions. Silk fibroin : TEG : EG are present in the volume ratio of 60:20:20. EG and TEG restrict the rotation of b-sheet emerging units in SF and facilitate the conformational transition from the random coil to the b-sheet structure. The standardized the ratios of additives and SF in the formulation to ensure quick gelation with retention of injectable property. Thus, prepared iSFH was mesoporous in nature and capable of encapsulating the human recombinant insulin without affecting its structural and functional integrity. The subcutaneous injection of iSFH encapsulated insulin (insulin-iSFH) in streptozotocin (STZ) induced T1DM Wistar diabetic rats generated active insulin depot under the skin and slowly releases the entrapped insulin to maintain normal glucose level for up to 4 days under diabetic conditions (Figure 8). Surprisingly it was also found that injectable composition comprising silk fibroin and polyethylene glycol resulted in a hydrogel with lowered gelation time. The present disclosure also provides an injectable hydrogel comprising the injectable composition and at least one therapeutic agent selected from small molecule drugs, therapeutic peptides, proteins, nucleic acids, bioactive compounds, or antibodies.

[0076] In an embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200.

[0077] In an embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:100. In another embodiment of the present disclosure, there is provided a composition as disclosed herein, wherein the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:20.

[0078] In an embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; wherein the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200 and the composition is injectable.

[0079] In an embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, polyethylene glycol, or combinations thereof; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200. [0080] In another embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, polyethylene glycol, or combinations thereof; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1 : 1 to 1:200, wherein the composition is injectable.

[0081] In an embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is monoethylene glycol; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200. In another embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is monoethylene glycol, wherein the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:100. In yet another embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is monoethylene glycol, wherein the silk fibroin to the ethylene glycol is in the weight ratio range of 1 : 1 to 1 :20

[0082] In an embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is tri-ethylene glycol; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200. In another embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is tri-ethylene glycol; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1 : 1 to 1 : 100. In yet another embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is tri-ethylene glycol; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:20.

[0083] In an embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is oligoethylene glycol; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200. In another embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is oligoethylene glycol; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:100 . In another embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is oligoethylene glycol; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:20.

[0084] In an embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) polyethylene glycol; and the silk fibroin to the polyethylene glycol is in the weight ratio range of 1:1 to 1:200. In another embodiment of the present disclosure, there is provided a composition comprising

(a) silk fibroin; and (b) polyethylene glycol; and the silk fibroin to the polyethylene glycol is in the weight ratio range of 1 : 1 to 1 : 100. In yet another embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and

(b) polyethylene glycol; and the silk fibroin to the polyethylene glycol is in the weight ratio range of 1:1 to 1:20.

[0085] In an embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; and (b) polyethylene glycol; wherein the silk fibroin to the polyethylene glycol is in the weight ratio range of 1:1 to 1:200 and the composition is injectable.

[0086] In an embodiment of the present disclosure, there is provided a composition comprising (a) silk fibroin; (b) monoethylene glycol; (c) tri-ethylene glycol; and (d) polyethylene glycol.

[0087] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol; wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:1:1 to 1:100:100.

[0088] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol; wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:1:1 to 1:100:100, and the composition is injectable.

[0089] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol, wherein the silk fibroin to the tri-ethylene glycol weight ratio is in the range of 1 : 1 to 1 : 100. In another embodiment of the present disclosure, the silk fibroin to the tri ethylene glycol weight ratio is in the range of 1:1 to 1:10.

[0090] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol, wherein the silk fibroin to the monoethylene glycol weight ratio is in the range of 1 : 1 to 1 : 100. In another embodiment of the present disclosure, the silk fibroin to the monoethylene glycol weight ratio is in the range of 1:1 to 1:10.

[0091] In an embodiment of the present disclosure, the silk fibroin to the tri ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:1:1 to 1:10:10.

[0092] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol, wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:5:5 to 1:10:10.

[0093] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol, wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is of 1:6.2:6.1.

[0094] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol, wherein the silk fibroin to the monoethylene glycol weight ratio is in the range of 1:1 to 1:100; and the silk fibroin to the tri-ethylene glycol weight ratio is in the range of 1:1 to 1:100.

[0095] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; and (b) ethylene glycol; wherein the silk fibroin to the ethylene glycol volume ratio is in the range of 60:10 to 90:40. In another embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; and (b) ethylene glycol; wherein the silk fibroin to the ethylene glycol volume ratio is in the range of 60:20 to 80:40. In yet another embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; and (b) ethylene glycol; wherein the silk fibroin to the ethylene glycol volume ratio is of 60:40. In one another embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; and (b) polyethylene glycol; wherein the silk fibroin to the polyethylene glycol volume ratio is of 60:40.

[0096] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol, wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol volume ratio is in the range of 60:5:5 to 90:30:30. In another embodiment of the present disclosure, wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol volume ratio is in the range of 60:10:10 to 90:25:25.

[0097] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol, wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol volume ratio is in the range of 60:20:20 to 80:25:25.

[0098] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol, wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol volume ratio is 60:20:20.

[0099] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1 : 1 : 1 to 1 : 100: 100 and, the silk fibroin to the tri-ethylene glycol to the monoethylene glycol volume ratio is in the range of 60:5:5 to 90:30:30.

[00100] In an embodiment of the present disclosure, there is provided a composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol, wherein the silk fibroin to the tri-ethylene glycol to the ethylene glycol weight ratio is of 1:6.2:6.1 and, the silk fibroin to the tri-ethylene glycol to the monoethylene glycol volume ratio is 60:20:20.

[00101] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the silk fibroin has a weight percentage in the range of 0.06% to 10% with respect to the composition; the tri-ethylene glycol has a weight percentage in the range of 0.12% to 44.88% with respect to the composition; the monoethylene glycol has a weight percentage in the range of 0.11% to 44.40% with respect to the composition. In another embodiment of the present disclosure, the silk fibroin has a weight percentage in the range of 1% to 5% with respect to the composition; the tri-ethylene glycol has a weight percentage in the range of 0.5% to 25% with respect to the composition; the monoethylene glycol has a weight percentage in the range of 0.5% to 25% with respect to the composition.

[00102] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the silk fibroin has a weight percentage in the range of 0.06% to 5.9% with respect to the composition; the tri-ethylene glycol has a weight percentage in the range of 0.12% to 44.88% with respect to the composition; the monoethylene glycol has a weight percentage in the range of 0.11% to 44.40% with respect to the composition.

[00103] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the silk fibroin has a weight percentage in the range of 1% to 4% with respect to the composition; the tri-ethylene glycol has a weight percentage in the range of 10% to 30% with respect to the composition; the monoethylene glycol has a weight percentage in the range of 10% to 30% with respect to the composition. In another embodiment of the present disclosure, there is provided a composition as described herein, wherein the silk fibroin has a weight percentage in the range of 2% to 3.8% with respect to the composition; the tri ethylene glycol has a weight percentage in the range of 15% to 25% with respect to the composition; the monoethylene glycol has a weight percentage in the range of 15% to 25% with respect to the composition.

[00104] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the silk fibroin has a weight percentage of 3.6% with respect to the composition; the tri-ethylene glycol has a weight percentage of 22.51% with respect to the composition; the monoethylene glycol has a weight percentage of 22.26% with respect to the composition.

[00105] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition is a hydrogel with a gelation time of 0.3 hours to 24 hours. In another embodiment of the present disclosure, the composition is a hydrogel with a gelation time of 0.4 hours to 24 hours. In yet another embodiment of the present disclosure, the composition is a hydrogel with a gelation time of 0.8 hours to 24 hours.

[00106] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition has porous cross-linked structure with a pore size in the range of 60 pm to 200 pm. In another embodiment of the present disclosure, wherein the composition has porous cross-linked structure with a pore size in the range of 61 pm to 199 pm.

[00107] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition has a b-sheet conformation.

[00108] In an embodiment of the present disclosure, there is provided a process of preparing the composition comprising (a) silk fibroin; and (b) ethylene glycol, the process comprising: (i) preparing a first solution comprising silk fibroin and water; (ii) contacting the first solution with the ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof to obtain the composition, wherein the silk fibroin to the ethylene glycol weight ratio is in the range of 1:1 to 1:200. [00109] In an embodiment of the present disclosure, there is provided a process of preparing the injectable composition comprising (a) silk fibroin; (b) tri ethylene glycol and (c) monoethylene glycol, the process comprising: (i) preparing a first solution comprising silk fibroin and water; (ii) contacting the first solution with the tri-ethylene glycol and the monoethylene glycol to obtain the composition, wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:1:1 to 1:100:100.

[00110] In an embodiment of the present disclosure, there is provided a process of preparing the composition comprising (a) silk fibroin; and (b) polyethylene glycol, the process comprising: (i) preparing a first solution comprising silk fibroin and water; (ii) contacting the first solution with the polyethylene glycol to obtain the composition, wherein the silk fibroin to the polyethylene glycol weight ratio is in the range of 1:1 to 1:200.

[00111] In an embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (i) the composition comprising: (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; with the silk fibroin to the ethylene glycol weight ratio is in the range of 1:1 to 1:100; and (ii) at least one therapeutic agent, wherein the composition to the at least one therapeutic agent weight percentage is in the range of 0.001% to 10%.

[00112] In an embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (i) the composition comprising: (a) silk fibroin; and (b) ethylene glycol with the silk fibroin to the ethylene glycol weight ratio is in the range of 1:1 to 1:100; and (ii) at least one therapeutic agent, wherein the composition to the at least one therapeutic agent weight percentage is in the range of 0.001% to 2% and the ethylene glycol is selected from monoethylene glycol, tri ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof. [00113] In an embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (i) the composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol with the silk fibroin to the tri- ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:1:1 to 1:100:100; and (ii) at least one therapeutic agent, wherein the composition to the at least one therapeutic agent weight percentage is in the range of 0.001% to 10%. [00114] In an embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (i) the composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol with the silk fibroin to the tri ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:1:1 to 1:10:10 and (ii) at least one therapeutic agent, wherein the composition to the at least one therapeutic agent weight percentage is 2%.

[00115] In an embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (i) the composition comprising: (a) silk fibroin; and (b) polyethylene glycol with the silk fibroin to the polyethylene glycol weight ratio is in the range of 1:1 to 1:200; and (ii) at least one therapeutic agent, wherein the composition to the at least one therapeutic agent weight percentage is in the range of 0.001% to 10%.

[00116] In an embodiment of the present disclosure, there is provided an injectable hydrogel as described herein, wherein the at least one therapeutic agent is selected from small molecule drugs, therapeutic peptides, proteins, nucleic acids or antibodies.

[00117] In an embodiment of the present disclosure, there is provided an injectable hydrogel as described herein, wherein the at least one therapeutic agent is selected from doxorubicin, cisplatin, berberine, metformin, curcumin, paclitaxel, vancomycin, enzalutamide, or insulin.

[00118] In an embodiment of the present disclosure, there is provided an injectable hydrogel as described herein, wherein the at least one therapeutic agent is insulin. In another embodiment of the present disclosure, there is provided an injectable hydrogel as described herein, wherein the at least one therapeutic agent is doxorubicin. In yet another embodiment of the present disclosure, there is provided an injectable hydrogel as described herein, wherein the at least one therapeutic agent is berberine. In one another embodiment of the present disclosure, there is provided an injectable hydrogel as described herein, wherein the at least one therapeutic agent is metformin.

[00119] In an embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (a) silk fibroin; (b) ethylene glycol; and c) insulin wherein the ethylene glycol is selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof. In another embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (a) silk fibroin; (b) tri-ethylene glycol; (c) monoethylene glycol; and d) insulin.

[00120] In an embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (a) silk fibroin; (b) polyethylene glycol; and (c) doxorubicin. In another embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (a) silk fibroin; (b) polyethylene glycol; and (c) berberine. In yet another embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (a) silk fibroin; (b) polyethylene glycol; and (c) insulin. In one another embodiment of the present disclosure, there is provided an injectable hydrogel comprising: (a) silk fibroin; (b) polyethylene glycol; and (c) metformin.

[00121] In an embodiment of the present disclosure, there is provided an injectable hydrogel as described herein, wherein the injectable hydrogel provides controlled release of the at least one therapeutic agent.

[00122] In an embodiment of the present disclosure, there is provided a composition or the hydrogel as disclosed herein, wherein the composition or the hydrogel acts as a platform to develop oral delivery formulations for the delivery of insulin, drugs and other bioactive molecules.

[00123] In an embodiment of the present disclosure, there is provided an injectable hydrogel which acts as a platform which can protect the encapsulated drug from external changes of pH.

[00124] In an embodiment of the present disclosure, there is provided an injectable hydrogel as described herein, wherein the at least one therapeutic agent is released in a controlled way over a period of about 0.1 to 30 days. In another embodiment of present disclosure, the at least one therapeutic agent is released in a controlled way over a period of about 1 to 10 days.

[00125] In an embodiment of the present disclosure, there is provided an injectable hydrogel as described herein, wherein the injectable hydrogel has a storage modulus in the range of 1 kPa to 258 kPa. In another embodiment of present disclosure, the injectable hydrogel has a storage modulus in the range of 10 kPa to 258 kPa. In yet another embodiment of present disclosure, the injectable hydrogel has a storage modulus 258 kPa.

[00126] In an embodiment of the present disclosure, there is provided a process of preparing the injectable hydrogel as described herein, the process comprising: contacting the composition comprising (a) silk fibroin; and (b) ethylene glycol, wherein the ethylene glycol is selected from monoethylene glycol, tri ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; wherein the silk fibroin to the ethylene glycol weight ratio is in the range of 1:1 to 1:200, and the at least one therapeutic agent at a temperature in the range of 25 °C to 40 °C to obtain an injectable hydrogel.

[00127] In an embodiment of the present disclosure, there is provided a process of preparing the injectable hydrogel as described herein, the process comprising: contacting the composition comprising (a) silk fibroin; (b) tri-ethylene glycol; and (c) monoethylene glycol wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1 : 1 : 1 to 1 : 100: 100, and the at least one therapeutic agent at a temperature in the range of 25 °C to 40 °C to obtain an injectable hydrogel.

[00128] In an embodiment of the present disclosure, there is provided a process of preparing the injectable hydrogel as described herein, the process comprising: contacting the composition comprising (a) silk fibroin; and (b) polyethylene glycol; wherein the silk fibroin to the polyethylene glycol weight ratio is in the range of 1 : 1 to 1 :200, and the at least one therapeutic agent at a temperature in the range of 25 °C to 40 °C to obtain an injectable hydrogel.

[00129] In an embodiment of the present disclosure, there is provided a process of preparing the injectable hydrogel as described herein, the process comprising: contacting the composition as described herein, and the at least one therapeutic agent at a temperature in the range of 25 °C to 40 °C to obtain an injectable hydrogel; and wherein the injectable hydrogel is used for controlled and sustained release or delivery of therapeutic agents in their active form.

[00130] In an embodiment of the present disclosure, there is provided a method of treating a condition, the method comprising: administering to a subject suffering from a condition a therapeutically effective amount of the injectable hydrogel comprising (a) silk fibroin; and (b) ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; wherein the silk fibroin to the ethylene glycol weight ratio is in the range of 1:1 to 1:200, and the at least one therapeutic agent. [00131] In an embodiment of the present disclosure, there is provided a method of treating a condition, the method comprising: administering to a subject suffering from a condition a therapeutically effective amount of the injectable hydrogel comprising (a) silk fibroin; (b) tri-ethylene glycol; and (c) monoethylene glycol wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:1:1 to 1:100:100, and the at least one therapeutic agent.

[00132] In an embodiment of the present disclosure, there is provided a method of treating a condition, the method comprising: administering to a subject suffering from a condition a therapeutically effective amount of the injectable hydrogel comprising (a) silk fibroin; and (b) polyethylene glycol; wherein the silk fibroin to the polyethylene glycol weight ratio is in the range of 1:1 to 1:200, and the at least one therapeutic agent.

[00133] In an embodiment of the present disclosure, there is provided a method of treating a condition, the method comprising: administering to a subject suffering from a condition selected from diabetes, cancer, wound repair, tissue repair, bone marrow regeneration, or regenerative medicine, the method comprising: administering to a subject suffering from a condition a therapeutically effective amount of the injectable hydrogel as described herein. [00134] In an embodiment of the present disclosure, there is provided a method for managing the blood glucose level, the method comprising: injecting the injectable hydrogel comprising (a) silk fibroin; and (b) ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; wherein the silk fibroin to the ethylene glycol weight ratio is in the range of 1:1 to 1:200, and the at least one therapeutic agent, to a patient in need.

[00135] In an embodiment of the present disclosure, there is provided a method for managing the blood glucose level, the method comprising: injecting the injectable hydrogel comprising (a) silk fibroin; (b) tri-ethylene glycol; and (c) monoethylene glycol wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:1:1 to 1:100:100, and the at least one therapeutic agent, to a patient in need.

[00136] In an embodiment of the present disclosure, there is provided a method for managing the blood glucose level, the method comprising: injecting the injectable hydrogel comprising (a) silk fibroin; and (b) polyethylene glycol; wherein the silk fibroin to the polyethylene glycol weight ratio is in the range of 1 : 1 to 1:200, and the at least one therapeutic agent, to a patient in need.

[00137] In an embodiment of the present disclosure, there is provided a method for preparing the tissue engineering scaffold, the method comprising using the composition comprising: (a) silk fibroin; and (b) ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof or the injectable hydrogel comprising: (i) the composition as described herein; and (ii) at least one therapeutic agent, wherein the composition to the at least one therapeutic agent weight percentage is in the range of 0.001% to 10%.

[00138] In an embodiment of the present disclosure, there is provided a method for preparing the tissue engineering scaffold, the method comprising using the composition comprising: (a) silk fibroin; (b) tri-ethylene glycol; and, (c) monoethylene glycol or the injectable hydrogel comprising: (i) the composition as described herein; and (ii) at least one therapeutic agent, wherein the composition to the at least one therapeutic agent weight percentage is in the range of 0.001% to 10%.

[00139] In an embodiment of the present disclosure, there is provided a method for preparing the tissue engineering scaffold, the method comprising using the composition comprising: (a) silk fibroin; and (b) polyethylene glycol or the injectable hydrogel comprising: (i) the composition as described herein; and (ii) at least one therapeutic agent, wherein the composition to the at least one therapeutic agent weight percentage is in the range of 0.001% to 10%.

[00140] In an embodiment of the present disclosure, there is provided a method of developing oral delivery of insulin and other bioactive molecules using the composition or the hydrogel as disclosed herein.

[00141] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible.

EXAMPLES

[00142] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply. [00143] The working and non-working examples as depicted in the forthcoming sections highlight the criticality of the working percentages of different components namely silk fibroin, tri-ethylene glycol, ethylene glycol, and therapeutic agent in achieving composition of the present disclosure. It is further specified that the presence of all the four components is critical so as to achieve the desired properties of the hydrogel. The absence of any of the components specified above or replacement of the same with any other component substantially affects properties of the hydrogel.

[00144] The present disclosure provides an injectable silk fibroin hydrogel (iSFH) comprising silk fibroin and two glycol additives tri-ethylene glycol and ethylene glycol to form the injectable hydrogel within 50 min. The hydrogel has a mesoporous structure appropriate for insulin encapsulation in its active form. A convenient process for preparing the composition involved preparing the silk fibroin and contacting it with tri-ethylene glycol and ethylene glycol. Another process to prepare the iSFH encapsulated insulin involved encapsulating insulin in the mesoporous structured hydrogel. iSFH encapsulated insulin is biocompatible and biodegradable in nature, which makes it a potential drug delivery system for active storage, controlled and sustained delivery of insulin in diabetic conditions to maintain the physiological glucose levels. The process of the present disclosure can be industrially scaled as well.

Materials and Methods

[00145] The mulberry silkworm Bombyx Mori cocoons of CB gold variety was purchased from the Ramanagara silk cocoon market, Karnataka, India. Lithium bromide (purity >99%, MW: 86.84 g mol ¹), EG (purity > 99%, MW: 62.068 g mol- 1) and TEG (purity > 99%, MW: 150.174 g mol ¹) were purchased from Spectrochem India. Human recombinant insulin (MW: 5807.57 g mol ¹) was obtained from HiMedia (Cas No: 11061-68-0). MTT (3-(4,5-dimethythiazol-2-yl)- 2,5-diphenyltetrazolium bromide) dye (MW: 413.03 g mol ¹) was purchased from Merck. Dulbeccos modified eagle medium (DMEM) and fetal bovine serum were obtained from Thermo Fischer Scientific. L929 and HEK 293 cells were obtained from National Centre for Cell Science, Pune, India. The deionized water used for experiment (resistivity: 18.2 MQ.cm at 25 °C) was obtained from Bamstead GenPure water purifier system. High-performance liquid chromatography (Prep- Prominence UFPLC, Shimadzu LC-IOA) was used to purify the FITC-labeled insulin, and the purity was monitored by the absorbance study at 215 nm and 495 nm. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (Autoflex max, Bruker) was used to characterize the integrity of FITC-labeled insulin. Absorbance and fluorescence measurements were performed using Agilent Cary series UV-Vis NIR Spectrophotometer, Agilent Cary Eclipse Fluorescence Spectrophotometer and SpectraMax i3x Microplate Reader (Molecular Devices). Zeiss Gemini SEM 500 was used to characterize the morphology of iSFH. Circular dichroism (CD) spectra of SF, iSFH, and insulin were measured using Jasco-815 Spectropolarimeter (Japan). ATR-FTIR spectra were recorded using GladiATR, PerkinElmer. The mechanical properties were measured using Physica MCR 101 Rheometer (Anton Paar). Male Wistar rats were obtained from the JNCASR animal facility, which is maintained under 12 h light and dark cycles. Animal experiments were performed according to the guidelines of the Institutional Animal Ethics committee (IAEC), JNCASR. The protocol (TG 002) was approved by the IAEC and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

EXAMPLE 1

Silk Fibroin Extraction and Purification

[00146] Raw silk comprising the cocoon of the silkworm consists of 2 proteins. One is sericin which is soluble in hot water and other is fibroin which insoluble in hot water. Silk fibroin refers to the fibroin protein of silk which has been obtained after heat treating the raw silk with either acidic or basic solution to separate out sericin protein. Silk Fibroin (SF) was extracted from Bombyx Mori cocoon. The silkworm was removed, and the cocoon was cut into an appropriate size, washed with water and boiled for 1 h in 20 mM sodium carbonate solution. During this process, the residual sericin protein was removed. The SF was washed with plenty of water to completely remove the sericin protein and dried to obtain white fibroin fibers. It was then dissolved in 9.3 M lithium bromide as a denaturant at 60 °C for 4 h. The resulting solution was dialyzed using activated 14 kDa cellulose dialysis membrane in deionized water having a resistivity of 18.2 MQ.cm at 25 °C with water changes (for six times) at a regular time interval. The solution was centrifuged to remove residual insoluble fibrous debris and stored in -20 °C or freeze-dried to get the SF powder. The final concentration of SF solution was close to 7% (w/v), which was further diluted with water to get different lower concentrations. A higher concentration of SF solution, i.e., silk fibroin of the present disclosure was prepared by solubilizing lyophilized solid powder in water.

EXAMPLE 2

Process of preparation of the composition ( iSFH Preparation)

[00147] The process of preparation of the composition comprised preparing a first solution followed by contacting the first solution with the suitable proportions of tri-ethylene glycol and ethylene glycol. Ethylene Glycol (EG) and Tri-ethylene Glycol (TEG) were added to induce a faster gelation of SF and mixed thoroughly at room temperature. For the gelation experiments, stock solutions of SF (6% W/V ; first solution) in water and variable ratios of EG, TEG (10 - 40%) were mixed in a glass vial at room temperature. The gelation propensity was checked by vial inversion procedure at 25 °C (Figure la) and sol to gel transition was found to occur by self-assembly of the SF protein to SF hydrogel. The kinetics of gelation was monitored through optical density measurements at 550 nm at 25 °C (Figure lb).

Optical Density (OD) Measurement

[00148] The gelation time for SF in water was determined by measuring the change in optical density (OD) at 550 nm due to self-assembling network formation. For this, 6% SF solution in water was mixed thoroughly with varied concentrations of EG and TEG and transferred into 24-well plates. The optical density was measured at 5 min intervals using a microplate reader. The gelation process resulted in the formation of cross-linked structures that can change the light diffraction from the solution, which results the change in optical density. The maximum optical density change was observed at the gelation point due to complete assembly of the SF. Transparent solutions of SF protein and additives individually showed weak absorption at 550 nm. Upon mixing, SF undergo self-assembly in presence of additives to form aggregates which increased the absorption intensity (550 nm) with time and reached maximum and this saturation is indicative of the completion of gel formation (Figure lb). Further, the concentration of SF and additives were tuned to optimize the gelation process to form iSFH. Table 1 shows Gelation times of 6 % SF protein solution with different ratios of TEG and EG.

TABLE 1:

[00149] It can be observed from Table 1 that the ratio of 6%SF with 20% (v/v) EG and TEG to form iSFH took the least time for gelation i.e. 50 min. In contrast, SF (6%) transformed into gel in the presence of individual additive (EG or TEG, 20%, v/v) over a period of 18 h and 4.5 h, respectively. The glycols (combination of EG and TEG) increased the viscosity of the medium and breaks the hydration layers of SF protein, which facilitate the effective collision between the b-sheet emerging units of SF proteins triggering rapid gelation. Further, the effect of SF concentration to optimize the gelation process was assessed and the results showed that increasing concentration (6, 10 and 15%) of SF decreased the gelation time at 50, 32 and 18 min, respectively (Figure lb). Although, higher SF concentration reduced the gelation time, the injectable properties of SFH were compromised due to the hardness of the hydrogel. Thus, the ratio of SF, EG and TEG to 60:20:20 was selected, which effectively form iSFH within 50 min for further applications. The respective weight percentage of SF, TEG and EG in optimized iSFH (3.6% iSFH) were 3.6%, 22.51 % and 22.26% (W/W).

[00150] Studies were also carried out with other additives like glycerol and tri ethylene glycol monomethyl ether in combination with EG. However, gelation time was quite high in presence of glycerol, EG and tri-ethylene glycol monomethyl ether. Table 2 shows gelation time of 3.6 % SF protein(silk) solution with glycerol and tri-ethylene glycol monomethyl ether and ethylene glycol (EG).

Table 2

[00151] It can be observed from Table 2 that when EG is combined with glycerol and tri-ethylene glycol monomethyl ether in same ratios, it gives drastically high rates of gelation of 3 hr 10 min and 2 hr 30 min respectively as compared to the 50 minutes gelation time when Silk:EG:TEG volume ratio is maintained at 60:20:20. Thus including glycerol works as a negative example for present composition and further insists that Silk:EG:TEG volume ratio 60:20:20 be used for further applications.

EXAMPLE 3 iSFH encapsulated insulin characterization Field emission scanning electron microscope (FESEM) studies [00152] The surface morphology and porosity of the SF hydrogel were analyzed by

FESEM (Zeiss GeminiSEM 500) at 3 kV. The hydrogel was washed with de- ionised water having resistivity of 18.2 MQ.cm at 25 °C several times to remove the EG and TEG additives, and the sample was dried through the freeze-drying process. Samples were mounted onto FESEM stubs using double-sided carbon tape, and gold sputtering were carried out on the sample prior to the imaging. The morphology of freeze-dried iSFH was evaluated through field emission scanning microscope (FESEM), which displayed a mesoporous structure with elongated pores of 60-200 pm (variable size and shape) surrounded by laminar SF layer (Figure lc). Insulin was trapped inside these pores and retains its structural and functional activity inside these pores, which was confirmed through the circular dichroism spectra and in vitro and in vivo studies. These pores are surrounded by the cross-linked layer of SF nanofiber which was revealed by very high magnified image. These nanofibers are responsible for the slow and controlled release of insulin from the pores. This satisfied the design strategy and was thus used to encapsulate drug molecule (insulin) to validate the therapeutic potency. Attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR)

[00153] The secondary structure of SF and iSFH was analyzed using FT-IR spectroscopy with attenuated total reflectance sampling technique. In case of SF, the dried protein sample was placed on a diamond crystal cell, and measurements were performed. For iSFH, the sample was prepared by freeze-drying after washing the glycols using water. The measurement was carried out in the wavenumber range 4000-400 cm ¹, and the data was plotted as absorbance against wavenumber (cm ¹). Pure SF protein has absorption band at 1648cm ¹ (Figure 2a). due to random coil amide I stretching (C=0) along with the amide II peak at 1517cm ¹ (N-H deformation). The iSFH absorb strongly at 1621 cm ¹ for amide I, indicates the presence of b-sheet conformation in the iSFH matrix. Shoulder peak at 1648 cm ¹ signified the co-existence of b-sheet and random coil structure in the iSFH. The b- sheet content in the hydrogel structure was determined through the de-convolution of amide I stretching absorption peak into b-sheet and random coil spectra (Figure 2b, 2c). The respective peak area represents the content of the b-sheet and random coil conformation. The degree of crosslinking was found in a range of 75.22-82.63

%.

Circular dichroism (CD) studies

[00154] The observed conformational transformation was further confirmed by the circular dichroism (CD) studies. The secondary structure of SF protein was studied by CD measurements. SF stock solutions (10 pL of 6% SF protein) were diluted with 200 pL of water having resistivity of 18.2 MW.ah at 25 °C, and CD spectra were recorded from 300 to 190 nm using 1 mm quartz cuvette in Jasco-815 CD spectrometer (Jasco Co., Japan). For SF hydrogel, the SF solution (600 pL) was mixed with EG (200 pL) and TEG (200 pL). In addition, 20 pL aliquot was mixed with 180 pL of water and quickly transferred into a cuvette. CD spectra were recorded at different time intervals at room temperature. SF solution showed a characteristic negative peak at 197 nm, indicating less ordered random coil structure (Figure 2d). Interestingly, SF in the gelation sample exhibited a characteristic negative peak at 218 nm corresponding to b-sheet conformation while the negative peak at 197 nm disappeared. Thus, the conformational transformation of SF from random coil to b-sheet structure and the coexistence of minor random coil with the major b-sheet structure is the driving force for the rapid and injectable gelation property.

Rheometric analysis

[00155] The mechanical properties of hydrogels were investigated using Physica MCR 101 Rheometer (Anton Paar) on a 25 mm parallel plate. Initially, the dynamic strain sweep was carried out to check the range of viscoelasticity. The dynamic frequency sweep was performed at 0.1% strain over a frequency range of 0.1 to 100 Hz at 25 °C. The storage modulus (G') and loss modulus were plotted against the angular frequency (co) (Figure 2e). The energy stored in the iSFH (storage modulus, G") was ~70 kPa, which is higher than the dissipated energy ~ 12 kPa (loss modulus, G'O, as shown in Figure 2e. The higher G" and G" values indicate highly solid-like behavior inside the gel network. The high storage modulus allowed iSFH to encapsulate the drug molecules inside the pores and aid the controlled release from its matrix. Incorporation of insulin within iSFH matrix increased the storage modulus of the iSFH drastically to -258 kPa. This signifies the co-operative interaction and alignment inside the hydrogel matrix which enhanced the elastic property of iSFH without affecting the SF gelation behaviour.

Hydrogel iniectabilitv studies

[00156] The injectability of hydrogel was investigated by extruding it from the syringe needle (Figure 2f). The optimized SF solution (300 pL, 6% SF) was mixed with EG (100 pL), TEG (100 pL) and rhodamine B dye and transferred into the syringe with 23 G needle. The hydrogel formulation (500 pL) was extruded typically by applying pressure with syringe-piston, which confirmed that the entire sample was extruded easily through the 23 G.

[00157] The injectable property of 3.6 % iSFH Figure ld(i) was compared to 6% SF hydrogel Figure ld(ii) which shows that 6% SF hydrogel formed was hard and non-injectable while 3.6 % iSFH hydrogel was injectable through a 23 G needle. Figure ld(i) and Figure ld(ii) suggests that higher SF protein concentration increased the rigidity of hydrogel and compromised the injectability.

Glycol release studies

[00158] The interaction of additives and SF in the iSFH matrix was determined by monitoring its release from iSFH. The glycols are used for the tuning the gelation time of the silk fibroin. The EG and TEG are hydrophilic molecules with very low partition co-efficient. Therefore, they are released from the hydrogel quickly after swelling of the gel. However, the presences of highly self-assemble nano fiber layers possibly decrease the release rate of insulin from the pores that result in prolong/sustained release over time. The hydrogel was prepared by mixing 150 pF of SF solution, EG (50 pF) and TEG (50 pF) in 1.5 mF tubes with a known weight. After gelation, the hydrogel was dried at 60 °C for 12 h to remove the residual water, and the weight of dried gel was measured (Wi). The tubes were kept in a glass beaker with water and slowly stirred. At different time intervals, the tubes were taken out and dried at 60 °C to determine the weight of the dried gel (W2). The percentage of glycol (EG and TEG) released was calculated as follows: W_rei (%) = (W _{I -} Wij/W _I ^x100. The addition of water to iSFH showed -23 and 60 release of glycols at time intervals of 1 and 6 h respectively while complete release was observed at 24h (Figure 2a). This indicated a weak van der Walls interaction between the glycol and silk fibroin hydrogel matrix.

Hydrogel swelling studies through gravimetric analysis [00159] The swelling property of the hydrogel samples was measured by gravimetric analysis. Dried samples of hydrogels were dipped into PBS, and the samples were removed at different time intervals. The surface water was removed using filter paper. The samples were weighed on an analytical balance. The swelling ratio was calculated from the following equation.

Swollen weight of sample

Swelling ratio = Dry weight of sample * 100

[00160] In addition, the swelling behavior influences the drug release from the hydrogel. The swelling ratio of iSFH in PBS buffer (10 mM, pH = 7.4) was investigated at 25 °C, and found to reach the equilibrium in 4 h (Figure 2b). The maximum swelling ratio observed was -41% which remained constant even after 12 h and this swelling ratio is suitable for release of entrapped drug molecule. The lesser swelling ratio and highly cross-linked structure signify its usefulness for the delivery of drug molecules. The faster gelation, excellent mechanical strength and injectable nature of iSFH make it an excellent drug carrier. EXAMPLE 4

Encapsulation and Sustained Release of Insulin

[00161] The potential of iSFH for the encapsulation and sustained release of insulin was investigated. The insulin was labelled with FITC dye using the previously reported protocols and characterized by MALDI-TOF mass spectrometry. FITC labeling of insulin

[00162] To monitor the drug release from iSFH, insulin was labeled with the fluorescein isothiocyanate (FITC) dye. Human recombinant insulin was dissolved in water, and FITC solution in DMSO was added dropwise under basic condition. The nucleophilic amino groups of insulin react with the isothiocyanate group of FITC. Due to the presence of several amino groups on insulin, different extents of labeling were observed. The mono FITC labeled insulin was purified by UFPLC and characterized by MALDI-TOF mass spectroscopy. The fluorophore (fluorescein isothiocyanate, FITC) labeled human recombinant insulin was encapsulated into the iSFH matrix and its release kinetics was monitored by the absorbance (l„_Iί1c= 490 nm, Figure 3a(i')) and fluorescence (l_ϋhi= 515 nm) (Figure 3a(i")) of FITC .

Insulin encapsulation into iSFH

[00163] Insulin was added during the SF gelation process to obtain insulin-iSFH. The effective porous gel network of iSFH was assessed for the encapsulation and release performance for recombinant human insulin. Insulin or FITC-insulin (50 pL, 1.0 mg/mL) was added to a mixture of SF solution (300 pL), EG (100 pL) TEG (100 pL) and incubated at room temperature for gelation. Interestingly, SF solution containing insulin efficiently formed hydrogel, which indicates effective encapsulation. FITC-insulin loaded iSFH (550 pL) was treated with 1 mL of PBS buffer (10 mM, pH = 7.4), and aliquots of 5 pL were removed at different time points. The absorbance and fluorescence spectra were recorded to assess and quantify the released insulin at different time points from the iSFH. Upon addition of PBS (10 mM, pH = 7.4), insulin-iSFH swollen and slowly released the encapsulated insulin depending on its diffusion co-efficient and interaction with the iSFH matrix. As shown in Figure 3a(i), insulin was released gradually over a period of 5 days from the iSFH matrix. Figure 3a(ii) shows the normalized fluorescence spectra of released FITC-insulin released from iSFH at different time intervals and suggests approximately 80% release over a period of 5 days. This slow and sustained release of insulin from iSFH is in good agreement with the rheological and swelling properties of iSFH. This study also signified the controlled release of insulin from the iSFH matrix over a prolonged period (up to 5 days) without the sudden or burst of high insulin release. Figure 3a(i') shows fluorescence spectra of released FITC - insulin from hydrogel at 515 nm. Fluorescence intensity increased with time due to slow release of FITC-insulin from pore of hydrogel. Such controlled release profile is necessary for long-term insulin delivery under diabetic conditions to maintain the physiological glucose homeostasis. CD spectra were recorded to assess the native insulin structure released at different time points from the insulin-iSFH.

[00164] Insulin release profile for other concentration of SF protein was also studied. The in vitro studies of the hydrogel prepared from 4%SF protein (2.4% iSFH,red) showed 50% release of insulin in 24 hour time period and a complete release in 72 hours(Figure 3a(iii)). This indicated the rapid release of insulin from the hydrogel matrix, which can lead to life-threatening hypoglycemia condition in vivo condition. These results confirmed that 2.4% iSFH is not suitable for the control and sustained insulin delivery in diabetic rats. The higher SF protein concentration increased the rigidity of hydrogel and compromised the injectability (Figure 3a(iv)). Therefore, hydrogel obtained from the high SF protein concentration was not applicable for further applications.

[00165] Release kinetics of FITC-insulin for a ratio of 50:25:25 of Silk:TEG:EG was tested. A 3% iSFH hydrogel was prepared with the 50 %(V/V) of 6% SF protein with 25% of TEG and 25% of EG (V/V). With the decrease of the SF protein solution volume the protein concentration was decreased. The release kinetics of FITC-insulin from the hydrogel matrix (Figure 3a(v)) was found to be similar to 2.4% iSFH in Figure3a(iii).

MALDI-TOF mass spectrometry

[00166] The insulin contains multiple -NH2 groups [2 N-terminal NH2 groups and a Lys), which always provide a heterogeneous FITC labelling. MALDI-TOF spectrometry was done to characterize FITC-insulin. Figure 3b shows MALDI- TOF mass data of FITC-insulin. FITC labelling of insulin (Ins) showed up to three (FITC) labels. The observed molecular mass peak at 6206.39 correspond to [Ins + FITC + 10H].

Circular dichroism (CD) studies

[00167] The conformational and structural integrity of the insulin released from the insulin-iSFH matrix was ascertained by the CD measurements. PBS (10 mM, pH = 7.4) was added to the insulin-iSFH at 37 °C, and CD measurement were performed for the insulin released at different time intervals. The insulin released from iSFH showed two negative peaks at 208 and 220 nm (Figure 3c), which are characteristic of alpha-helical secondary structure of insulin. Since the native structure of insulin correlates with the released insulin from hydrogel matrix, it suggests effective functional conformation of released insulin, besides its active storage inside the pores of iSFH. The insulin encapsulation and its slow release kinetics have showcased the effectiveness of iSFH matrix for the prolonged insulin delivery with implications for diabetes treatment.

EXAMPLE 5

Cytotoxicity and Hemolysis Studies

[00168] Prior to evaluating the efficacy of insulin-iSFH in the animal model, the cytotoxicity of iSFH in L929 fibroblast cells and human red blood cells were examined.

Cytotoxicity assay

[00169] The biocompatibility of the iSFH was assessed by measuring the cell viability in L929 fibroblast cell line following the ISO 10993-5-9-2009 protocol (MTT assay). MTT dye (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) reacts with the mitochondrial reductase enzyme in live cells and converts to purple colored (E,Z)-5-(4,5-dimethylthiazol-2-yl)-l,3-dimethylformazan. For this assay, 1.5 x 10⁴ cells were cultured in 24-well plate for 24 h using Dulbecco’s modified eagle media (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin- streptomycin (PS). The iSFH was mixed with DMEM media and incubated at 37 °C for 72 h. An aliquot of the media was collected filter and sterilized through a 0.22 mM syringe filter to obtain iSFH-DMEM extract. The culture media of cells was exchanged with an equal volume of complete DMEM and iSFH treated media. L929 cells were cultured and treated with iSFH-DMEM extract in 48 well plates for 24, 48 and 72 h under cell growth media. Cells were treated with 15% of MTT solution (stock solution 5 mg/mL) in PBS and incubated for 4 h. The purple-colored formazan crystals were formed depending on the number of viable cells. These purple crystals were dissolved in 200 pL of methanol: DMSO (1:1), and absorbance was recorded at 595 nm using a microplate reader. The iSFH-DMEM extract treated cells showed > 93% cell viability (Figure 4a) after 72 h of incubation compare to the control cells (100%). This data indicates high viability of cells presence of iSFH and can be used for in vivo animal study. Hemolysis assay

[00170] The suitability of iSFH formulation for in vivo experiments was analyzed via hemolysis assay with human red blood cells. iSFH (500 pL) was incubated with 500 pL PBS solution (10 mM, pH = 7.4) for 72 h and sterilized through the 0.22 pM syringe filter. The different aliquots of the solution were mixed with 100 pL blood sample and incubated at 37 °C. The samples collected at different time intervals were centrifuged to precipitate the red blood cells. Triton- 100 was used as a negative control and 10 mM PBS was used as a positive control to check for hemolysis. Triton-100 completely lysed the red blood cells (100%), and hemoglobin was distributed through the solution. The absorbance of the supernatant was recorded at 540 nm to detect lysed hemoglobin in the supernatant. However, iSFH or PBS (10 mM, pH = 7.4) treated red blood cells did not show any significant lysis compare to Triton- 100 (Figure 4b). These cytotoxicity results confirmed that iSFH is a suitable candidate for insulin delivery in animal model.

EXAMPLE 6

In vitro Insulin Functional Activity Study

[00171] Inactivation of insulin function is major concern and hence the ability of iSFH to maintain the native structure and activity of insulin under in vitro condition (Figure 5) was evaluated. Glucose oxidase enzyme oxidizes glucose to gluconic acid and generates hydrogen peroxide (H2O2) in the presence of oxygen. Therefore, monitoring H2O2 levels by DCFDA correlates with the oxidation of glucose present in the solution. HEK-293T cells were cultured in 24-well plates at 2 x 10⁴ cell density per well with high glucose -containing media. After 24 h, the released insulin solutions (50 pL) from insulin-iSFH were added to the cell culture. From this, an aliquot (50 pL) of culture media was collected and treated with glucose oxidase solution (50 uL of 1 mg/mL stock in 10 mM PBS to generate gluconic acid and hydrogen peroxide. The hydrogen peroxide was detected using 2 ',7'- dichlorofluorescein diacetate (DCFDA, 50 mM), which was converted into strongly fluorescence 2', 7 "-dichlorofluorescein in the presence of hydrogen peroxide. The fluorescence of 2', 1 "-dichlorofluorescein was measured at 529 nm using the microplate reader. As shown in Figure 3d, the glucose concentration of collected cell culture media in insulin-iSFH treated cells at 24 h was found to be lower than the control and untreated cells media. This indicates that high glucose uptake by HEK 293T cells in the presence of active released insulin from insulin-iSFH. These results confirmed that the functional activity of insulin encapsulated within the iSFH pore is preserved and was thus further used to evaluate the in vivo delivery and efficacy.

EXAMPLE 7

In vivo insulin delivery into diabetic rat

[00172] The therapeutic efficacy of insulin-iSFH to regulate blood glucose levels under diabetic condition was evaluated in T1DM Wistar rats. The blood glucose level in normal healthy rats was -140 mg/dL, which elevated significantly and reached > 450 mg/dL after 7 days of intraperitoneal STZ treatment due to specific damage of insulin generating pancreas b -cells (Figure 6a). Diabetic rats were randomly divided into individual groups and treated with PBS (10 mM), insulin (1 mg/mL), iSFH and insulin-iSFH (insulin dose 20 mg/kg). The blood glucose levels were monitored at different time interval (1, 6 and 12 h) using the ACCU CHEK Instant S glucometer. The blood glucose level of rats treated with PBS and iSFH did not show any significant changes compare to the control diabetic rats (480 mg/dL). The subcutaneous insulin injection reduced the high glucose level to its normal levels (140 mg/dL) for a short duration and expectedly the glucose level rose again after 6 h (Figure 6b). Remarkably, treatment with insulin-iSFH reduced the glucose level from 480 mg/dL to 150 mg/dL within 3 h of injection without leading to hypoglycemia (Figure 6c), which is a frequently observed and major problem with subcutaneous insulin injection. Subsequently, the blood glucose level was maintained in a normoglycemia condition up to 4 days (Figure 6b). These studies have validated the potential of insulin-iSFH for in vivo delivery and controlled release of insulin for prolonged period to effectively manage glucose metabolism in diabetic Wistar rats.

[00173] The interaction of additives and SF in the iSFH matrix was determined by monitoring its release from iSFH. The addition of water to iSFH showed ~23, 60 and 97% release of glycols at time intervals of 1, 6 and 24 h, respectively (Figure 7a). This indicates a weak van der Waals interaction between the glycols and SF protein in the iSFH matrix. In addition, the swelling behavior influences the drug release from the hydrogel. Also the swelling ratio of iSFH in PBS buffer (10 mM, pH= 7.4) at 25 °C was investigated and found to reach the equilibrium in 4 h (Figure 7b). The maximum swelling ratio observed was -41% which remained constant even after 12 h suggesting the possible slow release of the drugs after administration. The lesser swelling ratio and highly cross-linked structure signify its usefulness for the delivery of drug molecules. The excellent gelation kinetics and mechanical strength of iSFH reassured to evaluate its injectable potential and remarkably, iSFH was found to extrude easily through the 23 G needle. The faster gelation, excellent mechanical strength and injectable nature of iSFH make it an excellent drug carrier.

[00174] Injectable SF protein hydrogel (iSFH) is developed for controlled and sustained delivery of insulin under diabetic conditions. iSFH was standardized with SF (6%) in presence of two viscous additives, EG (20%) and TEG (20%) which transform into an injectable formulation at a faster rate (< 1 h). The viscous glycols restrict the mobility of SF protein backbones and aid the conformational transformation of random coil to ordered b-sheet structure while retaining minor quantity of random coil structure, which together results in the rapid gelation. FESEM analysis of iSFH revealed highly porous microstructures with different sizes surrounded by laminar aggregation of the SF layer. The iSFH microstructures have high mechanical strength and is injectable using 23 G needle. The porous morphology of iSFH allowed the encapsulation of human recombinant insulin in its active form. In vitro release profiles of FITC-labeled insulin suggested that iSFH is a good delivery tool for sustained insulin delivery (-5 days) into the blood due to lower swelling ratio. Subcutaneous injection of insulin-iSFH in diabetic rats (T1DM Wistar rat) form active depot under the skin from which insulin leach out slowly and restores the physiological glucose homeostasis for a prolonged period of 4 days. Interestingly, the insulin-iSFH did not cause hypoglycemia through sudden burst of a high concentration of insulin into the blood [00175] Overall, the combination of silk fibroin, tri-ethylene glycol, ethylene glycol and insulin in the disclosed weight ranges is essential to have an injectable silk injectable hydrogel with gelation time of 50 minutes and imparting prolonged in- vivo drug delivery for upto 4 days. Replacing even a single component with another component does not provide the desired effect. Also, deviating from the disclosed weight ranges does not exhibit the desired result.

EXAMPLE 8

Gelation with other ethylene glycols

[00176] The SF protein gelation in presence of various polyethylene glycol was investigated to understand the role of polyethylene glycols to provide controlled gelation through inducing the hydrogen bonding between b-sheet forming units. In presence of 40% PEG 200 gelation was observed in 40 -50 min, slightly less than EG and TEG mixture, and gel showed injectability through 23 G needle. Figure 9(a) represent the SF protein gel in presence of various ethylene glycols and Figure 9 (b) depicts the injectability of the SF protein hydrogel in presence of PEG200 through 23 G needle. Therefore, the addition of the ethylene glycols and mixture of ethylene glycols is a platform to induce controlled gelation and injectability in SF protein hydrogel for drug delivery applications. It was surprisingly found that polyethylene glycols of low and medium molecular weight resulted in a better hydrogel composition with lowered gelation time. Thus, the composition comprising silk fibroin with polyethylene glycol was further extended to study its use towards drug delivery of therapeutic agents.

Small biomolecules delivery

[00177] The effect of the hydrogel network on the cumulative release of encapsulated small molecule drug doxorubicin and berberine was investigated under physiologically relevant conditions (37 °C, PBS, pH = 7.4). The doxorubicin and berberine release rates from the hydrogel matrix were varied at different times points. During the initial 24 h time, 38% doxorubicin and 32% berberine release were observed due to surface binding and high concentration gradient which enhanced the diffusion rate of the drugs. Afterward, a steady linear release was followed until 6 days, where the only diffusion was driving force toward the berberine release. Figure 10 depicts the release kinetics of doxorubicin (a) and berberine (b) from PEG200 induced SF protein hydrogel. The strong interaction between the b-sheet crystals of the SF aggregates and small bioactive molecules has a significant role in the slow release. Therefore, hydrogel also efficient to control the delivery of the bioactive small molecule drug for longer periods. Thus, the invention is a platform patent for the delivery of various bioactive molecules in a controlled and sustained way.

Insulin stability studies

[00178] The effect of the hydrogel matrix to protect encapsulated drug molecules from an external change of pH was further investigated. The insulin- encapsulated hydrogel was treated with an acidic solution of pH 2.5 and the structural conformation of released insulin was checked through the CD. The released insulin after treatment with the highly acidic solution was shown a similar absorbance band with the controlled insulin. This indicates the hydrogel matrix can provide the stability of the encapsulated drugs (insulin) from external stimuli, including acidic conditions similar to stomach/ gastrointestinal tract. Figure 11 depicts the CD spectrum of released insulin and only insulin. Therefore, the SF hydrogel system of the present disclosure can be used as a scaffold to develop oral delivery formulations and platforms for insulin and other drugs.

[00179] Overall, the composition comprising silk fibroin with polyethylene glycol along with therapeutic agent as detailed above is essential to have an injectable hydrogel with lowered gelation time in the range of 40 to 50 minutes and imparting prolonged in-vivo drug delivery. Replacing even a single component with another component does not provide the desired effect. Also, deviating from the disclosed weight ranges does not exhibit the desired result. It was also found that low and medium molecular weight polyethylene glycol produced better hydrogel and exhibited desired properties. Any deviation in the type of ethylene glycols or the mixture of ethylene glycols resulted in less desired properties of the composition and the hydrogel.

Advantages of the present disclosure

[00180] The present disclosure has aimed to develop an injectable hydrogel-based drug delivery system to overcome several drawbacks related to conventionally used hydrogels such as long gelation period, poor hydrogel stability, high immunogenic properties, poor drug compatibility, low biocompatibility and biodegradability and poor retention of injectable property. The injectable hydrogel composition of the present disclosure comprises biocompatible additives which help in in-situ gelation of silk fibroin, do not alter the material properties of the incorporated drug and maintain sterile conditions. The enhanced elastic property of iSFH also allows to encapsulate the drug molecules inside the pores and aid the controlled release from its matrix. The hydrogel has a lower swelling ratio which is suitable for release of entrapped drug molecule. The porous morphology of iSFH allowed the encapsulation of human recombinant insulin in its active form and controlled in vivo release for upto 5 days. The excellent mechanical strength, biocompatibility, encapsulation, storage and sustained delivery of active insulin in the diabetic animal thus make the injectable hydrogel composition of present disclosure an effective insulin delivery tool and have potential implications for use in diabetic patients. The hydrogel composition of the present disclosure can be used as a scaffold to develop oral delivery formulations and platforms for insulin, drugs and other bioactive molecules.

Claims

I/We claim

1) A composition comprising:

(a) silk fibroin; and b) ethylene glycol, wherein the ethylene glycol is selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof; and the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200.

2) A composition comprising:

(a) silk fibroin;

(b) tri-ethylene glycol; and

(c) monoethylene glycol; wherein the silk fibroin to the tri-ethylene glycol to the monoethylene glycol weight ratio is in the range of 1 : 1 : 1 to 1 : 100: 100.

3) The composition as claimed in any one of the claims 1-2, wherein the composition is injectable.

4) The composition as claimed in any one of the claims 1-2, wherein the composition is a hydrogel with a gelation time of 0.3 hours to 24 hours.

5) The composition as claimed in any one of the claims 1-2, wherein the composition has porous cross-linked structure with a pore size in the range of 60 pm to 200 pm.

6) The composition as claimed in any one of the claims 1-2, wherein the composition has a b-sheet conformation.

7) A process of preparing the composition as claimed in claim 1, the process comprising: a) preparing a first solution comprising silk fibroin and water; b) contacting the first solution with the ethylene glycol selected from monoethylene glycol, tri-ethylene glycol, oligoethylene glycol, polyethylene glycol, or combinations thereof to obtain the composition, wherein the silk fibroin to the ethylene glycol is in the weight ratio range of 1:1 to 1:200.

8) A process of preparing the composition as claimed in claim 2, the process comprising:

(a) preparing a first solution comprising silk fibroin and water;

(b) contacting the first solution with monoethylene glycol and tri ethylene glycol to obtain the composition, wherein the silk fibroin to the tri ethylene glycol to the monoethylene glycol weight ratio is in the range of 1:1:1 to 1:100:100.

9) An injectable hydrogel comprising:

(i) the composition as claimed in any one of the claims 1-6; and

(ii) at least one therapeutic agent, wherein the composition to the at least one therapeutic agent weight percentage is in the range of 0.001% to 10%.

10) The injectable hydrogel as claimed in claim 9, wherein the at least one therapeutic agent is selected from small molecule drugs, therapeutic peptides, proteins, nucleic acids, bioactive compounds, or antibodies.

11) The injectable hydrogel as claimed in any of the claims 9 to 10, wherein the injectable hydrogel provides controlled release of the at least one therapeutic agent.

12) The injectable hydrogel as claimed in any of the claims 9 to 11, wherein the at least one therapeutic agent is released in a controlled way over a period of about 0.5 to 30 days.

13) The injectable hydrogel as claimed in any one of the claims 9 to 12, wherein the injectable hydrogel has a storage modulus in the range of 1 kPa to 258 kPa.

14) A process of preparing the injectable hydrogel as claimed in any one of the claims 9 to 13, the process comprising: contacting the composition as claimed in any one of the claims 1 to 6, and the at least one therapeutic agent at a temperature in the range of 25 °C to 40 °C to obtain an injectable hydrogel. 15) Use of the injectable hydrogel as claimed in any one of claims 9 to 13, for controlled and sustained release or delivery of therapeutic agents in their active form.

16) A method of treating a condition, the method comprising: administering to a subject suffering from a condition a therapeutically effective amount of the injectable hydrogel as claimed in any one of the claims 9 to 13.

17) The method as claimed in claim 16, wherein the condition is selected from diabetes, cancer, wound repair, tissue repair, bone marrow regeneration, or regenerative medicine. 18) A method for preparing the tissue engineering scaffold, the method comprising using the composition as claimed in any one of the claims 1 to 6 or the injectable hydrogel as claimed in any one of the claims 9 to 13.